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https://openalex.org/W3007191987
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https://pure.eur.nl/files/48294669/Repub_125109_O-A.pdf
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English
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Comparison of sequencing methods and data processing pipelines for whole genome sequencing and minority single nucleotide variant (mSNV) analysis during an influenza A/H5N8 outbreak
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PloS one
| 2,020
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cc-by
| 16,231
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RESEARCH ARTICLE Editor: Ming Zhang, University of Georgia, UNITED
STATES Received: January 25, 2019
Accepted: December 30, 2019
Published: February 20, 2020 Copyright: © 2020 Poen et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Data Availability Statement: All data files are
available from the ENA database (project accession
number(s) PRJEB9846, PRJEB12582,
PRJEB9687). Funding: This work was supported by the
European Union’s Horizon 2020 research and
innovation program under grant agreements No
643476 (COMPARE), and No 653316 (EVAg). This
work was also supported by the NIAID/NIH grant Comparison of sequencing methods and data
processing pipelines for whole genome
sequencing and minority single nucleotide
variant (mSNV) analysis during an influenza
A/H5N8 outbreak Marjolein J. PoenID1, Anne Pohlmann2, Clara Amid3, Theo M. Bestebroer1, Sharon
M. Brookes4, Ian H. Brown4, Helen Everett4, Claudia M. E. Schapendonk1, Rachel
D. Scheuer1, Saskia L. Smits1, Martin Beer2, Ron A. M. Fouchier1, Richard J. Ellis4* a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 1 Erasmus MC, Department of Viroscience, Rotterdam, the Netherlands, 2 Institute of Diagnostic Virology,
Friedrich-Loeffler-Institute, Insel Riems, Germany, 3 European Molecular Biology Laboratory (EMBL),
European Bioinformatics Institute (EBI), Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom,
4 Animal and Plant Health Agency (APHA) - Weybridge, Addlestone, Surrey, United Kingdom 1 Erasmus MC, Department of Viroscience, Rotterdam, the Netherlands, 2 Institute of Diagnostic Virology,
Friedrich-Loeffler-Institute, Insel Riems, Germany, 3 European Molecular Biology Laboratory (EMBL),
European Bioinformatics Institute (EBI), Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom,
4 Animal and Plant Health Agency (APHA) - Weybridge, Addlestone, Surrey, United Kingdom * richard.ellis@apha.gov.uk Abstract Citation: Poen MJ, Pohlmann A, Amid C,
Bestebroer TM, Brookes SM, Brown IH, et al. (2020) Comparison of sequencing methods and
data processing pipelines for whole genome
sequencing and minority single nucleotide variant
(mSNV) analysis during an influenza A/H5N8
outbreak. PLoS ONE 15(2): e0229326. https://doi. org/10.1371/journal.pone.0229326 As high-throughput sequencing technologies are becoming more widely adopted for analysing
pathogens in disease outbreaks there needs to be assurance that the different sequencing tech-
nologies and approaches to data analysis will yield reliable and comparable results. Conversely,
understanding where agreement cannot be achieved provides insight into the limitations of
these approaches and also allows efforts to be focused on areas of the process that need
improvement. This manuscript describes the next-generation sequencing of three closely
related viruses, each analysed using different sequencing strategies, sequencing instruments
and data processing pipelines. In order to determine the comparability of consensus sequences
and minority (sub-consensus) single nucleotide variant (mSNV) identification, the biological
samples, the sequence data from 3 sequencing platforms and the *.bam quality-trimmed align-
ment files of raw data of 3 influenza A/H5N8 viruses were shared. This analysis demonstrated
that variation in the final result could be attributed to all stages in the process, but the most critical
were the well-known homopolymer errors introduced by 454 sequencing, and the alignment pro-
cesses in the different data processing pipelines which affected the consistency of mSNV detec-
tion. However, homopolymer errors aside, there was generally a good agreement between
consensus sequences that were obtained for all combinations of sequencing platforms and data
processing pipelines. Nevertheless, minority variant analysis will need a different level of careful
standardization and awareness about the possible limitations, as shown in this study. Editor: Ming Zhang, University of Georgia, UNITED
STATES * richard.ellis@apha.gov.uk Comparison of sequencing methods and data processing pipelines HHSN272201400008C to RAMF. The funders had
no role in study design, data collection and
analysis, decision to publish, or preparation of the
manuscript. compared to traditional Sanger sequencing [1, 2]. The use of next-generation sequencing
(NGS) technologies is continuously expanding and has revolutionized the field of genomics
and molecular biology. HHSN272201400008C to RAMF. The funders had
no role in study design, data collection and
analysis, decision to publish, or preparation of the
manuscript. gy
In many fields of infectious disease research, nucleotide changes in DNA or RNA sequences
are used to monitor genetic adaptions indicative of evolution, the emergence of drug resis-
tance, immune evasion or as a tool in epidemiological tracing [3]. In clinical settings, sequenc-
ing information is used to improve diagnostics and prognosis. NGS technologies play an
increasingly important role in these processes as clinically or epidemiologically important
nucleotide changes can be present in the minority of DNA or RNA sequences only, which
might be missed with more traditional (consensus) sequencing methods which determine the
most abundant sequence variants in a population. Nucleotide variants that are present in only
a minority of the sequenced virus population are referred to as minority Single Nucleotide
Variants (mSNVs). These variants, initially occurring due to replication errors, can become
fixed in the population when they have some sort of evolutionary advantage, for instance,
mutations related to drug resistance. Furthermore, mSNVs can be also used for high-resoltion
molecular epidemiology, which becomes more and more important for outbreak assessment
[4, 5]. Traditional Sanger sequencing for instance has been described to detect minority vari-
ants provided they are present in at least 10% of the analysed DNA or RNA strands within a
sample [6, 7]. Hence, the use of traditional sequencing methods is usually restricted to obtain-
ing consensus sequences or to determine heterozygosity in diploid organisms. In contrast,
NGS technologies are able to detect low frequency mSNVs in sequence fragments or even
whole genomes. Typically, NGS sensitivity for minority sequence variant identification is
restricted to a level of variation of 0.1–1%, mainly due to sequencing related background errors
[8–10], but sensitivity can be increased using sophisticated approaches like circle sequencing
[11] or improved bioinformatic analysis workflows [10]. Introduction Over the past decade, high-throughput sequencing technologies have evolved, providing faster,
cheaper, and less laborious alternatives to obtain (whole genome) DNA and RNA sequences 1 / 28 PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 The reliability of mSNV analysis
using NGS methods is influenced by many factors, like the quantity and quality of the input
sample, the laboratory procedures, the type of sequencing platform and the software and
settings used to analyse the raw sequence data. Competing interests: The authors have declared
that no competing interests exist. Competing interests: The authors have declared
that no competing interests exist. Due to the technical improvements, NGS technologies have become more important as
diagnostic tools to characterize pathogens in outbreak situations. However, the increasing use
of these technologies to address new and important (outbreak related) research and surveil-
lance questions emphasizes the need to determine the reproducibility of, and the important
technical considerations affecting, outcomes obtained by different laboratories following dif-
ferent protocols. Given this, comparative studies focusing on different platforms and data
analysis methods are essential to cross-validate different methodologies and determine the
reliability of newly obtained data. In addition, there is a growing need (as exemplified by the
recent Ebola and Zika virus outbreaks) to share also comprehensive sequencing data as quickly
as possible to help with source attribution and developing control strategies. However, the
underlying technologies and methods used for NGS are still diverse and there is a strong
demand for harmonization of laboratory procedures and approaches for a reliable and opti-
mized analysis of the data. This study is part of the European Union’s HORIZON 2020 project “COMPARE” (http://
www.compare-europe.eu/), aiming to improve the analytical tools for emerging zoonotic
pathogens and its underpinning research. Here, the comparability of NGS output data
obtained from different sequence approaches were evaluated and demonstrated suitable shar-
ing strategies for comprehensive NGS data sets. In November 2014, a newly emerging strain
of highly pathogenic avian influenza (HPAI) virus was detected in several European countries
[12, 13]. In the United Kingdom [14], Germany [15], and The Netherlands [16–18] this sub-
type was detected in commercial poultry farms within a few days of one another. In each of PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 2 / 28 Comparison of sequencing methods and data processing pipelines those countries, NGS was used to generate whole-genome sequences rapidly after detection,
but as the laboratories in each country were working independently, different approaches were
used for both sequencing and data analysis, and the data were shared as part of a wider study
to determine the likely source of the outbreak [19]. It is important to determine whether the
different analytical approaches have any impact on the outcome. Therefore, the aim of this
study was to determine how comparable consensus and minority variant results were between
laboratories performing their standard analyses, and whether discrepancies could be attributed
to the sequence platform (SP), the data processing platform (DPP) or a combination of both. With the lack of a ground truth/gold standard, all datasets obtained were compared amongst
each other. The hypothesis we test in this study is that outputs from NGS analysis of viruses
will be comparable irrespective of laboratory, sequencing platform and data analysis platform. Therefore, virus isolates obtained in each of the three countries (United Kingdom, Germany
and the Netherlands) were shared between these three partners and subsequently sequenced
and analysed in each of the three laboratories according to local procedures. In addition, the
use of a specially designed data sharing platform, a COMPARE “Data Hub” at EMBL-EBI,
Hinxton UK, was evaluated. This study presents genome coverage data, consensus sequences,
the analysis of the comparability of mSNV identifications of the different SPs, and DPPs. Our hypothesis was confirmed at the consensus sequence level, since consensus sequences
could be reproduced independent of the combination of SP and DPP used. However, the iden-
tification of minority variants appeared to be poorly reproducible, primarily due to the well-
known errors in 454 sequencing, and due to differences induced by the alignment processes in
the different DPPs. The interpretation of minority variant analysis thus needs a different level
of careful standardization and awareness about the possible limitations as shown in this study. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 Samples All samples were obtained from outbreaks in commercial poultry holdings. Isolate A/duck/
England/36254/2014 was obtained from pooled intestinal material from index case ducks
(Anas platyrhynchos domesticus). Tissue homogenate material was inoculated into embryo-
nated chicken eggs and allantoic fluid was harvested at 1 day post-inoculation [14]. The Dutch
isolate (A/chicken/Netherlands/EMC-3/2014) was obtained by passaging lung material of a
dead commercial layer hen (Gallus gallus domesticus) in MDCK cells twice and harvesting the
supernatant after approximately 40 hours post-inoculation [23]. The German isolate (A/tur-
key/Germany/AR2485/2014) originated from lung tissue of a commercially kept turkey
(Meleagris gallopavo) and was passaged in embryonated chicken eggs [15]. (Table 1). Experimental design The experimental design is summarized in Fig 1. spreadsheet file per virus to check the reproducibility of mSNV identification when using dif-
ferent combinations of SP and DPP. The experimental design is summarized in Fig 1. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 Experimental design Three avian influenza A virus isolates that were obtained from three different avian species
during the 2014/15 outbreak of HPAI H5N8 virus in Europe were shared among three institu-
tions in the United Kingdom (Animal Plant and Health Agency [APHA]), Germany (Fried-
rich-Loeffler-Institut [FLI]) and the Netherlands (Erasmus Medical Center [EMC]), later
referred to as anonymized institutions I, II and III (Fig 1). All three institutions sequenced all
three virus isolates according to their own standard procedures. Adaptors used in the sequenc-
ing processes were trimmed off before the raw sequence data files were shared. The sequence
data files (.fastq files), alignment files (.bam files), sample metadata and experimental meta-
data were shared between the three laboratories and analysed in their own DPPs yielding
sequence datasets for each virus (Table 1). This approach enabled to separate the biological
features of the viruses from variation introduced by technical methodology. Data sharing was
facilitated via a “Data Hub” provided by the EMBL-EBI’s European Nucleotide Archive (ENA)
in the framework of the COMPARE collaborative project; all data were stored and subse-
quently published in ENA [20] (https://www.ebi.ac.uk/ena, for the accession numbers, see
Table 1). ENA is an open repository for sequence and related data and a member of the Inter-
national Nucleotide Sequence Database Collaboration (INSDC; http://www.insdc.org/) [21]. A
full description of the COMPARE Data Hub system is provided in a preprint version of Amid
et al. [22]. First, consensus sequences derived from a preliminary analysis were compared and
one overarching consensus sequence was determined for each gene segment for each virus. This custom-made consensus was used by all three institutions as the reference genome for
undertaking mSNV analysis. The resulting nine mSNV reports (originating from three whole-
genome raw data sequences times three DPPs) were combined for all three viruses in one PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 3 / 28 Comparison of sequencing methods and data processing pipelines Fig 1. Flowchart of the experimental design. SP: sequence platform; DPP: data processing pipeline. https://doi.org/10.1371/journal.pone.0229326.g001 wchart of the experimental design. SP: sequence platform; DPP: data processing pipeline. Fig 1. Flowchart of the experimental design. SP: sequence platform; DPP: data processing pipeline. Fig 1. Flowchart of the experimental design. SP: sequence platform; DPP: data processing pipeline. https://doi.org/10.1371/journal.pone.0229326.g001 https://doi.org/10.1371/journal.pone.0229326.g001 spreadsheet file per virus to check the reproducibility of mSNV identification when using dif-
ferent combinations of SP and DPP. Sequencing Institution I: SP1. RNA was extracted using a Qiagen QIAamp viral RNA mini kit (Qia-
gen, Germany) according to the manufacturers’ instructions except that carrier RNA was
omitted from the AVL lysis buffer and the sample was eluted in 50μl RNAse-free water. RNA
was then processed to double-stranded cDNA (cDNA Synthesis System, Roche) using random
hexamers and purified using magnetic beads (AmpureXP, Beckman Coulter, USA). The dou-
ble-stranded cDNA was diluted to 0.2 ng/μl and used to produce a sequencing library using
the NexteraXT kit (Illumina, USA). Libraries were then sequenced in paired-end mode on an
Illumina MiSeq (Illumina, USA), with run lengths varying from 2 x 75 bases (UKDD virus) to
2 x 150 bases (NLCH and DETU viruses) depending on whether time-constraints were imple-
mented to provide a rapid response to an outbreak. Demultiplexing and removal of sequencing
adapters was done by the MiSeq RTA software to generate raw fastq files. SP1 process included
a limited 12-cycle PCR enrichment of the library. Sequencing Post-hoc analysis showed that duplication PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 4 / 28 Comparison of sequencing methods and data processing pipelines p
UKDD
DETU
NLCH
Virus strain
A/duck/England/36254/2014
A/turkey/Germany/AR2485-L01478/2014
A/chicken/Netherlands/EMC-3/2014
Isolation source
Pooled intestines
Lung tissue
Lung tissue
Host Scientific Name
Anas platyrhynchos
Meleagris gallopavo
Gallus gallus domesticus
Host Common Name
Domestic duck
Turkey
Chicken
Collection Date
14 November 2014
04 November 2014
23 November 2014
Collection Country
United Kingdom
Germany
Netherlands
Collection Region
East Yorkshire
Mecklenburg-Western Pomerania
Ter Aar
Influenza Test
Method
MP gene RRT-PCR, H5 RRT-PCR
MP gene RRT-PCR, H5 RRT-PCR
MP gene RRT-PCR, H5 RRT-PCR
Culture Status
Sample
Egg passage 1
Egg passage 1
MDCK passage 2
Institution
I
Institution
II
Institution III
Institution
I
Institition
II
Institution III
Institution
I
Institition
II
Institution III
Study Accession
PRJEB9846
PRJEB12582
PRJEB9687
PRJEB9846
PRJEB12582
PRJEB9687
PRJEB9846
PRJEB12582
PRJEB9687
Run Accession
ERR972805
ERR1293054
ERR926712
ERR926713
ERR1354020
ERR1293053
ERR926714
ERR926715
ERR1354021
ERR1293055
ERR926717
ERR926718
DPP1 .bam file run
accession
ERR3093746
ERR3093752
ERR9033756
ERR3093744
ERR3093753
ERR3093757
ERR3093745 ERR3093754
ERR3093758
DPP2 .bam file run
accession
ERR2992676
ERR2992677
ERR2992675
ERR2992679
ERR2992680
ERR2992678
ERR2992682
ERR2992683
ERR2992681
DPP3 .bam file run
accession
ERR2985803
ERR2985804
ERR2985802
ERR2985806
ERR2985807
ERR2985805
ERR2985809
ERR2985810
ERR2985808
Experiment Accession
100k
ERX315615
ERX2986848
NA
NA
ERX315616
ERX2986847
NA
NA
ERX315617
ERX2986849
NA
NA
Run Accession 100k
ERR3090788
ERR2984276
NA
NA
ERR3090789
ERR2984275
NA
NA
ERR3090790
ERR2984277
NA
NA
Using the Study Accession numbers in the European Nucleotide Archive all related data files can be accessed, or accessed directly from https://www.ebi.ac.uk/ena/data/view/accession, e.g.: https://
www.ebi.ac.uk/ena/data/view/PRJEB9846 (Study Accession Institution I), https://www.ebi.ac.uk/ena/data/view/ERR972805 (Run Accession UKDD Institution I). PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 5 / 28 Comparison of sequencing methods and data processing pipelines levels were less than 0.02% of the total reads which were considered to have negligible impact
on the results. Institution II: SP2. RNA was extracted using a combined approach with TRIzol (Thermo
Fisher Scientific, USA) and an RNeasy Kit (Qiagen, Germany). Further concentration and
cleaning was done with Agencourt RNAClean XP magnetic beads (Beckman Coulter, USA). RNA was quantified using a Nanodrop UV spectrometer ND-1000 (Peqlab, Germany) and
used as template for cDNA synthesis with a cDNA Synthesis System (Roche, Germany) with
random hexamers. Fragmentation of the cDNA applying a target size of 300 bp was done with
a Covaris M220 ultrasonicator. Sequencing The sonicated cDNA was used for library preparation using
Illumina indices (Illumina, USA) on a SPRI-TE library system (Beckman Coulter, USA) using
a SPRIworks Fragment Library Cartridge II (for Roche FLX DNA sequencer; Beckman Coul-
ter, USA) without automatic size selection. Subsequently, upper and lower size exclusion of
the library was done with Ampure XP magnetic beads (Beckman Coulter, USA). The libraries
were quality checked using High Sensitivity DNA Chips and reagents on a Bioanalyzer 2100
(Agilent Technologies, Germany) and quantified via qPCR with a Kapa Library Quantification
Kit (Kapa Biosystems, USA) on a Bio-Rad CFX96 Real-Time System (Bio-Rad Laboratories,
USA). SP2 did not amplify sample nor library. Sequencing was done on an Illumina MiSeq
using MiSeq reagent kit v3 (Illumina, USA) resulting in paired end sequences with a read
length of 300. Demultiplexed and adapter-trimmed reads were used to generate raw fastq files. Institution III: SP3. RNA was extracted using the High Pure RNA isolation kit (Roche
Diagnostics, Germany) according to manufacturer’s instructions. RNA was converted to cDNA
using the SuperScript III Reverse Transcriptase kit (Invitrogen, Thermo Fisher, USA) as
described previously [24], and amplified by PCR using primers covering the full viral genome
(S1 Table). All 32 PCR fragments from approximately 400–600 nucleotides in length, were
sequenced using the 454/Roche GS-FLX sequencing platform. The PCR fragments were pooled
in equimolar ratio and purified using the MinElute PCR Purification kit (Qiagen, Germany)
according to the manufacturer’s instructions. Rapid Library preparation, Emulsion PCR and
Next Generation 454-sequencing were performed according to instructions of the manufacturer
(Roche Diagnostics, Germany). Protocols are described in the following manuals: Rapid Library
Preparation Method Manual (Roche; May 2010), emPCR Amplification Method Manual–Lib-L
(Roche; May 2010) and Sequencing Method Manual (Roche; May 2010). All three samples were
sequenced in one run. Samples were pooled using MID adaptors to determine which sequences
came from which sample, each sample was assigned two different MID’s. Demultiplexing and
basic trimming was done by CLC-bio software to generate raw fastq files (S1 File). PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 Data processing Institution I: DPP1. In the FluSeqID script (https://github.com/ellisrichardj/FluSeqID)
the following steps are run automatically: the mapping of raw sequence data to the host
genome (BWA v0.7.12-r1039 [25]), extracting reads that do not map to the host (Samtools
v1.2 [26]), assembling non-host reads (Velvet v1.2.10 [27]), identification of the closest match
for each genome segment (BLAST v2.2.28 [28] using the custom databases generated from the
Influenza Research Database as indicated in the GitHub repository), mapping original data to
the top reference segments (BWA), calling new consensus sequences (vcf2consensus.pl), per-
forming further iterations of the last two steps to improve new consensus (IterMap), and
finally outputting the genome consensus sequence. The data processing pipeline has in-build
defaults for k-mer and coverage cut-off for de novo assembly, and the e-value cut-off for
BLAST, which can be changed via command line options (see https://github.com/ellisrichardj/
FluSeqID). Since the aligner (BWA-MEM) used performs soft-clipping and ignores low PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 6 / 28 Comparison of sequencing methods and data processing pipelines quality data, quality trimming is unnecessary. For mSNV analysis, the reads were mapped to
the unified consensus using BWA. Samtools was used to generate a pileup file which was then
analysed using custom python and R scripts to determine the depth of coverage and basecalls
at each position (available at https://github.com/ellisrichardj/MinorVar). The combination of
BWA-MEM and samtools was shown to be accurate for SNV identification [29]. In order to be
included in the final output the minimum basecall quality was 20 and the minimum mapping
quality was 50. Institution II: DPP2. Raw sequence data were analysed and mapped using the Genome
Sequencer software suite (v. 3.0; Roche, Mannheim, Germany) and the Geneious software
suite (v. 9.0.5; Biomatters, Auckland, New Zealand). Raw reads were trimmed and subsets of
each trimmed dataset were assembled de novo to generate reference sequences for each data
set (Newbler Assembler of Genome Sequencer software suite v. 3.0; Roche, Mannheim, Ger-
many). The trimmed raw influenza virus reads were mapped to the reference sequences (New-
bler Mapper of Genome Sequencer software suite v. 3.0; Roche, Mannheim, Germany). The
output assemblies were imported into the Geneious software suite (v. 9.0.5; Biomatters, Auck-
land, New Zealand) for further analysis and processing. Data processing Regions of low and high coverage
(threshold was 2 x standard deviations from the mean for low and high coverage) and regions
of low quality (minimum quality/phred score 20) were evaluated and if necessary, excluded
from further analyses. Consensus sequences were generated and annotated using annotated
reference sequences. Sequences were compared, and annotations that matched with a similar-
ity (> 90%) were copied. This was done on nucleotide sequences and also for translation in all
six reading frames. Annotations were manually inspected and curated. Trimmed raw reads of
the datasets or subsets thereof were mapped to the consensus, mapping was fine-tuned and
mSNVs were determined using generic SNP finder of the Geneious software suite, applying
parameters of maximum p-value of 10−5 and filter for strand bias. The threshold for SNP iden-
tification was set at 1%, and variants were checked manually for accuracy. Institution III: DPP3. Raw sequence data were analysed and mapped using the CLC
Genomics software package, workbench 8 (CLC Bio). Reads obtained by 454 sequencing were
sorted by MID adaptor, quality-trimmed, and analysed using the parameters as shown in S1
File. In short, after sorting by MID, the sequence reads were trimmed at 30 nucleotides from
the 30 and 50 ends to remove all primer sequences. Data from the shared Illumina sequence
files had already been trimmed and were imported in CLC Bio without additional processing
steps (S1 File). Reads were initially aligned to their own reference sequences that were
uploaded during the H5N8 outbreak (Gisaid accession numbers EPI-ISL-169282 (NLCH),
EPI-ISL-167904 (UKDD) and EPI-ISL-169273 (DETU)). Consensus sequences were automati-
cally generated by CLC after alignment to the reference, for detailed settings see S1 File. For
the mSNV analysis the raw data were mapped to the new custom-made consensus sequences
per gene segment per sample. Fastq files of these alignments were shared with the other institu-
tions. The threshold for mSNV identification was set at 1%, and registered minority variants
were checked manually for accuracy (minimal quality/phred score 20). Determining the influence of the DPP alignment steps versus DPPs mSNV identifica-
tion methods. Data processing pipelines process raw data in several steps, roughly divided
into trimming, aligning data to a reference sequence, and variant calling (the mSNV identifica-
tion procedure). PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 Data processing In order to determine to what extent the trimming and subsequent alignment
processes contributed to the observed differences the nucleotide coverage results obtained by
the three DPPs when aligning the same SP raw datasets were compared. To study the influence
of the mSNV identification process, quality-trimmed alignment files that had been generated
by each DPP and shared as .bam files were subjected to the mSNV identification process used
in DPP3 to determine the differences in mSNV detection output when only the alignment PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 7 / 28 Comparison of sequencing methods and data processing pipelines processes differed. DPP3 was randomly picked for this analyses, mSNV detection parameters
were set to the institutions default settings for mSNV identification using CLC-bio software
and can be seen in the S1 File. Data sharing To test the applicability of real-time sequence data sharing within the COMPARE network,
all raw sequence data used in this study were uploaded to and shared via a “Data Hub” in the
environment of the European Nucleotide Archive (ENA). Each institution received its own
study accession in which all raw sequence data files and metadata files were assigned with
individual experimental accession numbers (Table 1). In addition to the sequence data, all
trimmed alignment files (.bam) have been uploaded to the ENA. Using these hubs, sharing
between institutions was facilitated and immediate access to the data prior to the public release
was possible to enable joint evaluation and comparison. All data files have been made publicly
available via the ENA (https://www.ebi.ac.uk/ena). Designing the custom-made consensus sequences Each institution produced a consensus sequence for the 8 influenza gene segments (PB2, PB1,
PA, HA, NP, NA, MP, NS) for each of the three viruses. The obtained consensus sequences
were aligned using the BioEdit sequence alignment editor (version 7.2.0) [30]. Raw sequence
data from each SP were initially aligned to their own reference sequences that were uploaded
during the H5N8 outbreak (Gisaid accession numbers EPI-ISL-169282 (NLCH), EPI-ISL-
167904 (UKDD) and xxx (DETU)). PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 Results In order to determine the comparability of consensus sequences and mSNV identification the
biological samples, the sequence data from 3 SPs and the .bam quality-trimmed alignment
files of raw data of 3 influenza A/H5N8 viruses were shared. All data sets were subsequently
analysed in 3 different DPPs. The resulting 9 mSNV reports per virus (3 SP data sets each ana-
lysed in 3 DPPs) were evaluated for comparability of mSNV identification using different com-
binations of SP and DPP. Data sharing Data sharing using the COMPARE “Data Hub” provided by ENA proved to be easy, quick and
successful. The “Data Hub” enables the File Transport Protocol (FTP) protected upload and
download of large data files and facilitates sharing between collaborators with the possibility to
evaluate and compare all data prior to their public release by generating and specifically shar-
ing accession numbers using standard ENA procedures. The Data Hub used an influenza virus
sample checklist. In addition, data sets are ultimately made publicly and through the INSDC
network globally available and accessible in real-time as required without further upload to a
different repository. Full details of the COMPARE Data Hub system are available in a submit-
ted manuscript [22]. In summary, this process was suitable for quick data sharing in an out-
break scenario. Designing the custom-made consensus sequences For each of the 8 gene segments of the 3 viruses separately, 9 initial consensus sequences (3 SPs
x 3 DPPs) were generated, resulting in 72 consensus sequences per virus. The custom-made
consensus sequence per virus and per gene segment was 1) trimmed to a length represented by
all 9 initial consensus sequences and 2) nucleotides had to be identical to at least 6/9 consensus
sequences to be included. Although some sequences contained insertions or deletions, those
could always be corrected for using the other SP sequences following the criteria mentioned
previously. This resulted in a unique custom-made consensus for each gene segment for all
three viruses. mSNV analysis comparison For the mSNV analyses the custom-made consensus for each virus isolate was used as a refer-
ence for mapping, thereby standardizing positions within the genome to make comparison
between institutions easier. To avoid unnecessary increases in analytical time and memory,
datasets were down-sampled to 100.000 reads per sample when needed. Each DPP produced
a report on the identified mSNVs in a tabulated format. The analysis output files were filtered
for mSNVs only, thereby ignoring detected nucleotide insertions and deletions (InDels). There is a current lack of data or evidence-based approaches on how to calculate the required
sequence depth (i.e. coverage) for mSNV analyses an evidence-based. In this study, a mini-
mum coverage threshold for the identification of mSNVs was applied. This minimum nucleo-
tide coverage (i.e. number of reads per nucleotide after trimming) was determined using a
basic sample size calculation method, n = log β / log p’ [31]. Here β represents the required
power (e.g. for 95% chance of detecting something β = 0.05), and p’ is 1—the proportion of
events that you want to detect. For a 95% certainty of detecting a variant at 1%, a minimum
coverage of 298 reads per position is needed. For variants that occur in 5% of reads, the
number of reads required is >58, and for variants that occur in 10% of the reads the mini-
mum coverage is >28. For the mSNV identification literature commonly uses the mSNV cut-
off frequencies of 10%, 5% and 1%. However, it needs to be noted that these cut-off
values are arbitrary. Therefore, where depth of coverage was sufficient, this study will report
mSNV detected with a frequency of 1%, but initial comparisons started with positions show-
ing mSNVs with frequencies of 10% in at least one of the SP/DPP combinations, followed by
those with mSNV of 5% -<10%, and lastly those 1%-<5%. For all those positions identi-
fied, the number of reads and number of variant nucleotides in all other SP/DPP combinations
for that position will be noted regardless of frequencies. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 8 / 28 Comparison of sequencing methods and data processing pipelines PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 Consensus sequences When ignoring insertions and deletions in the homopolymer regions of the 454 data for most
gene segments the identified consensus sequences were identical regardless of the SP and DPP
combinations used with the exemption of the differences mentioned in Table 2. However, the
number of insertions and deletions in homopolymer regions of the SP3 sequences were con-
siderable in all 3 viruses. There was no clear difference in the number of insertions and dele-
tions related to homopolymer regions between the different DPPs (20, 17 and 18 for DPP 1, 2
and 3 respectively). Nucleotide differences that were not related to homopolymer regions were
only observed for sequences obtained in SP3 and SP2 data when processed in DPP1. In summary, the homopolymer errors inherent in the 454 dataset caused problems for all
DPPs, as expected. Consensus sequences generated by DPP1 from SP3 (454) data showed
some unexpected differences, but it performed well with the SP1 data formats it was designed
for and reasonably well with SP2 data. Overall, the consensus sequences can be reproduced
by all DPPs using Illumina data but that the analysis of the 454 data from SP3 was more prob-
lematic, as it would require editing of the sequences at homopolymer regions. Consensus
sequences from this study can be found in the S2 Table. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 9 / 28 Comparison of sequencing methods and data processing pipelines Table 2. The differences in consensus sequences obtained from each SP/DPP combination, sorted per virus and
per gene segment. https://doi.org/10.1371/journal.pone.0229326.t002 mutations were detected. InDel: insertions or deletion; SP: Sequence platform; DPP: Data processing pipeline; ND:
not detected. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 The letter in brackets represents the DPP (column 5) or the SP (column 6) where the insertions/deletions or The mSNV analysis comparison Nucleotide coverage and the influence of DPP-dependent alignment. The observed
number of reads per nucleotide (referred to as nucleotide coverage) differed depending on the
SP/DPP combination. All DPPs handled both 454 and Illumina data formats, although some
modifications (settings for the bwa mapper to handle single end 454 data) were required for
DPP1, which was specifically designed for Illumina paired-end reads. The observed nucleotide
coverages showed near to identical profiles for all three viruses. The coverage results obtained
from the three different SPs and DPPs for the NLCH virus (Fig 2) and for the other two viruses
(S1 Fig) were plotted. In general, lower nucleotide coverage was observed at the termini of
each gene segment. The SP3 data showed more variation in nucleotide coverage within gene
segments compared to SP1 and SP2 data, due to the sequencing of 32 PCR amplicons. The
non-normalised number of raw sequence reads and influenza virus reads per virus per SP can
be found in the S3 Table. The differences in nucleotide coverage were visualized for the three different SP raw datasets
analysed with the same DPP (Fig 2A). Overall, SP3 data (green lines) showed a lower coverage
compared to SP1 (purple) and SP2 data (yellow). The overall coverage for SP1 and SP2 data
was similar with small variations for different viruses and DPPs. The shorter read lengths in
SP1 virus data did not appear to have influenced the overall nucleotide coverage substantially. The differences in nucleotide coverage introduced by different alignment procedures were
also assessed, by comparing the coverage results for each SP raw dataset analysed with the
three different DPPs (Fig 2B). DPP2 (orange lines) generally retained the highest nucleotide
coverage for data from the different SPs. However, DPP3 (grey lines) generally also retained
high coverage for SP3 data, for which it was optimized. The nucleotide coverage of SP3 data
showed larger variation between the three different DPPs, leading to differences in nucleotide
coverage up to 50% depending on the DPP, because DPP1 and DPP2 were not optimized for
this SP. Data from SP2 were handled very similar by all three DPPs. In conclusion, both the SP and the DPP influenced the number of reads per nucleotide
position. SP3 showed the lowest output in number of reads compared to SP1 and SP2 Illumina
data. The mSNV analysis comparison The influence of the DPP depended highly on the data input, with best DPP performance
for the SP dataset for which it was optimized. The mSNV identification. The mSNV identification thresholds were set to 1% in all
DPPs. Because of the high number of mSNVs identified, the comparison of these mSNVs
started with a manually set arbitrary threshold of 10% that was subsequently decreased to
5%, and 1%. A mSNV position was identified when at least 1 of the SP/DPP combinations
showed a variant that exceeded the frequency threshold, and when the coverage at that posi-
tion exceeded the minimum number of reads needed to detect that variant with a 95% proba-
bility, as described previously. The presence of mSNV and coverage for all SP/DPP The mSNV identification. The mSNV identification thresholds were set to 1% in all
DPPs. Because of the high number of mSNVs identified, the comparison of these mSNVs
started with a manually set arbitrary threshold of 10% that was subsequently decreased to
5%, and 1%. A mSNV position was identified when at least 1 of the SP/DPP combinations
showed a variant that exceeded the frequency threshold, and when the coverage at that posi-
tion exceeded the minimum number of reads needed to detect that variant with a 95% proba-
bility, as described previously. The presence of mSNV and coverage for all SP/DPP
combinations were compared for each of the positions in which a mSNV had been detected in
at least one of the combinations. The coverages indicated for those positions where no mSNVs
were detected were derived from the alignment files and were not subjected to possible addi-
tional read filtering parameters in the mSNV identification process. The average quality (Q-
score/phred score) was set to or exceeding 20. combinations were compared for each of the positions in which a mSNV had been detected in
at least one of the combinations. The coverages indicated for those positions where no mSNVs
were detected were derived from the alignment files and were not subjected to possible addi-
tional read filtering parameters in the mSNV identification process. The average quality (Q-
score/phred score) was set to or exceeding 20. Ten positions across the three virus genomes were identified with mSNVs occurring in 10%
of reads. Three of the mSNVs (NLCH:PB2 G1879A, NLCH:PB2 G2101A and DETU:HA T963C)
were detected in all SP/DPP combinations but with slightly different relative abundance. Consensus sequences Virus
Segment
Start
End
Number of InDels at homoplymer regions
Other nucleotide differences
NLCH
PB2
1
2280
2 (DPP1)
C506A (SP3)
2 (DPP3)
G2101R (SP3)
PB1
1
2277
1 (DPP1/DPP2/DPP3)
1 (DPP1/DPP2)
A949W (SP3)
1 (DPP2/DPP3)
2272 ins AAG (SP2)
1 (DPP3)
PA
-6#
2190
1 (DPP1/DPP2)
ND
2 (DPP1)
HA
7
1704
1 (DPP2/DPP3)
A427W (SP2)
NP
1
1497
1 (DPP1)
C420Y (SP3)
NA
4
1419
ND
ND
MP
-5#
982
ND
ND
NS
1
838
ND
ND
DETU
PB2
1
2287
1 (DPP1/DPP2/DPP3)
2272 Del A (SP3)
3 (DPP1)
PB1
1
2277
1 (DPP1/DPP2/DPP3)
T956C (SP3)
1 (DPP1)
1 (DPP2)
1 (DPP3)
PA
7
2189
1 (DPP1/DPP2)
ND
HA
1
1728
1 (DPP2/DPP3)
ND
NP
1
1497
2 (DPP3)
ND
NA
1
1413
1 (DPP1)
778 ins CCA (SP3)
MP
-1#
982
1 (DPP2)
ND
NS
2
838
ND
ND
UKDD
PB2
1
2298
1 (DPP1/DPP2/DPP3)
C504T (SP3)
1 (DPP3)
C506M (SP3)
PB1
1
2277
1 (DPP1/DPP2/DPP3)
T951W (SP3)
1 (DPP2/DPP3)
PA
1
2151
2 (DPP1)
ND
1 (DPP2)
HA
1
1704
1 (DPP2/DPP3)
ND
NP
1
1497
1 (DPP3)
T1003Y (SP2)
NA
4
1420
ND
782 del TA (SP3)
MP
-5#
982
1 (DPP2)
ND
NS
-5#
849
ND
ND
The letter in brackets represents the DPP (column 5) or the SP (column 6) where the insertions/deletions or Table 2. The differences in consensus sequences obtained from each SP/DPP combination, sorted per virus and
per gene segment. The letter in brackets represents the DPP (column 5) or the SP (column 6) where the insertions/deletions or The letter in brackets represents the DPP (column 5) or the SP (column 6) where the insertions/deletions or
mutations were detected. InDel: insertions or deletion; SP: Sequence platform; DPP: Data processing pipeline; ND:
not detected. Start is counted from the ATG start codon; Exclusively identified in SP3 sequence data, InDels related to homopolymer regions;
E
l
i
l id
tifi d i DPP1 Exclusively identified in SP3 sequence data, InDels related to homopolymer regions;
Exclusively identified in DPP1; # ’-’ indicates the number of nucleotides before the ATG start codon included in the consensus 10 / 28 PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 Comparison of sequencing methods and data processing pipelines PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 The mSNV analysis comparison The other
mSNVs were identified in only one (n = 6) or two (n = 1) of the SP/DPP combinations (Table 3). Thirty-seven positions were identified with mSNVs occurring in 5% of reads. Of those,
the same mSNV was identified in all SP/DPP combinations for 9 positions (24,3%), in seven or Thirty seven positions were identified with mSNVs occurring in 5% of reads. Of those,
the same mSNV was identified in all SP/DPP combinations for 9 positions (24,3%), in seven or PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 11 / 28 Comparison of sequencing methods and data processing pipelines Fig 2. Nucleotide coverage. The non-normalised nucleotide coverage displayed as number of nucleotides per position
for full genome sequences of the NLCH virus reads mapped to the NLCH reference sequences. Panel A shows the
coverage results for the same SP dataset in the three different DPPs (DPP1: purple; DPP2: orange; DPP3 grey) for each
of the SP datasets. Panel B shows the coverage when the same DPP is used to analyse data from the three different SPs
(SP1: lilac; SP2: yellow; SP3: green) for each of the DPPs. The X-axis represents the position in the genome, the Y-axis
represents the number of sequence reads per position. https://doi.org/10.1371/journal.pone.0229326.g002 Fig 2. Nucleotide coverage. The non-normalised nucleotide coverage displayed as number of nucleotides per position
for full genome sequences of the NLCH virus reads mapped to the NLCH reference sequences. Panel A shows the
coverage results for the same SP dataset in the three different DPPs (DPP1: purple; DPP2: orange; DPP3 grey) for each
of the SP datasets. Panel B shows the coverage when the same DPP is used to analyse data from the three different SPs
(SP1: lilac; SP2: yellow; SP3: green) for each of the DPPs. The X-axis represents the position in the genome, the Y-axis
represents the number of sequence reads per position. Fig 2. Nucleotide coverage. The non-normalised nucleotide coverage displayed as number of nucleotides per position
for full genome sequences of the NLCH virus reads mapped to the NLCH reference sequences. Panel A shows the
coverage results for the same SP dataset in the three different DPPs (DPP1: purple; DPP2: orange; DPP3 grey) for each
of the SP datasets. Panel B shows the coverage when the same DPP is used to analyse data from the three different SPs
(SP1: lilac; SP2: yellow; SP3: green) for each of the DPPs. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 The mSNV analysis comparison The X-axis represents the position in the genome, the Y-axis
represents the number of sequence reads per position. https://doi.org/10.1371/journal.pone.0229326.g002 12 / 28 PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 Comparison of sequencing methods and data processing pipelines The minority variants occurring in at least one of the sequence platform—Data processing pipelines as a 5% variant. Position
Sequence platform
Data processing pipeline
1
2
3
Minor variants
Percentage
Minor variants
Percentage
Minor variants
Percentage
PB2.1879 G!A
1
81/1301
6,2%
246/2716
9,1%
112/1203
9,3%
2
47/956
4,9%
117/1137
10,3%
114/1064
10,7%
3
49/530
9,2%
131/1341
9,8%
129/1338
9,6%
PB2.2101 G!A
1
53/1118
4,7%
261/2704
9,7%
110/897
12,3%
2
21/1578
1,3%
125/1875
6,7%
121/1463
8,3%
3
13/542
2,4%
199/1433
13,9%
199/1435
13,9%
PB2.2277 T!G
1
ND/479
<1%
86/1008
8,5%
33/190
17,4%
2
ND/557
<1%
ND/623
<1%
ND/534
<1%
3
ND/680
<1%
ND/1117
<1%
ND/1024
<1%
PB1.87 A!G
1
ND/818
<1%
ND/1754
<1%
ND/1114
<1%
2
25/230
10,9%
ND/376
<1%
ND/328
<1%
3
ND/275
<1%
ND/537
<1%
ND/537
<1%
PB1.2240 G!C
1
ND/664
<1%
54/1341
4,0%
38/418
9,1%
2
ND/1231
<1%
ND/1271
<1%
ND/1233
<1%
3
ND/161
<1%
ND/277
<1%
ND/276
<1%
PB1.2268 A!G
1
ND/336
<1%
29/641
4,5%
11/176
6,3%
2
ND/993
<1%
ND/1026
<1%
ND/1002
<1%
3
ND/53
<1%
ND/159
<1%
ND/148
<1%
HA.104 A!G
1
ND/733
<1%
ND/1761
<1%
ND/1151
<1%
2
ND/437
<1%
ND/1370
<1%
ND/1156
<1%
3
ND/1
<1%
ND/105
<1%
12/105
11,4%
HA.1689 T!C
1
ND/390
<1%
ND/694
<1%
11/217
5,1%
2
ND/2018
<1%
ND/4083
<1%
ND/3979
<1%
3
ND/937
<1%
ND/1669
<1%
ND/1680
<1%
NP.105 A!G
1
ND/182
<1%
ND/449
<1%
ND/343
<1%
2
83/1507
5,5%
ND/1890
<1%
ND/1804
<1%
3
ND/89
<1%
ND/704
<1%
ND/702
<1%
NP.1239 A!T
1
32/2428
1,3%
279/5410
5,2%
ND/3092
<1%
2
ND/2345
<1%
ND/2643
<1%
ND/2453
<1%
3
ND/1711
<1%
ND/2111
<1%
ND/2117
<1%
NP.1489 G!A
1
ND/182
<1%
26/336
7,7%
ND/172
<1%
2
ND/436
<1%
ND/452
<1%
ND/444
<1%
3
ND/1320
<1%
ND/1799
<1%
ND/1799
<1%
NS.833 A!T
1
ND/187
<1%
ND/287
<1%
5/88
5,7%
2
ND/1224
<1%
ND/1327
<1%
ND/1284
<1%
3
ND/1367
<1%
ND/2430
<1%
ND/2333
<1%
PB2.1054 T!C
1
69/1369
5,0%
168/2637
6,4%
97/1304
7,4%
2
60/1477
4,1%
115/1836
6,3%
99/1605
6,2%
3
6/392
1,5%
94/2038
4,6%
48/1054
4,6%
PB2.2257 A!C
1
ND/867
<1%
ND/1563
<1%
24/463
5,2%
2
ND/531
<1%
ND/581
<1%
ND/378
<1%
3
ND/893
<1%
ND/2286
<1%
ND/1346
<1%
PB2.2277 T!G
1
ND/644
<1%
52/1150
4,5%
27/307
8,8%
2
ND/418
<1%
ND/472
<1%
ND/284
<1%
3
ND/1208
<1%
ND/1948
<1%
ND/1209
<1%
(Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 13 / 28 Comparison of sequencing methods and data processing pipelines Table 3. (Continued)
Virus
Position
Sequence platform
Data processing pipeline
1
2
3
Minor variants
Percentage
Minor variants
Percentage
Minor variants
Percentage
UKDD
PB2.2277 T!G
1
ND/415
<1%
28/507
5,5%
ND/475
<1%
2
ND/589
<1%
ND/620
<1%
ND/601
<1%
3
ND/1140
<1%
ND/1996
<1%
ND/2065
<1%
PB1.87 A!G
1
ND/387
<1%
ND/440
<1%
ND/439
<1%
2
26/327
8,0%
32/395
8,1%
ND/351
<1%
3
ND/617
<1%
ND/1133
<1%
ND/1136
<1%
PB1.728 C!A
1
ND/750
<1%
ND/832
<1%
ND/836
<1%
2
ND/776
<1%
52/928
5,6%
ND/829
<1%
3
ND/2459
<1%
ND/4290
<1%
ND/4293
<1%
PB1.730 C!T
1
ND/742
<1%
ND/824
<1%
ND/826
<1%
2
ND/767
<1%
57/1008
5,7%
ND/832
<1%
3
ND/2339
<1%
ND//4286
<1%
ND/4289
<1%
PB1.883 G!C
1
ND/942
<1%
ND/997
<1%
ND/997
<1%
2
ND/1689
<1%
ND/1865
<1%
ND/1760
<1%
3
ND/2479
<1%
47/690
6,8%
ND/3681
<1%
PA.49 G!C
1
ND/103
<1%
6/117
5,1%
ND/115
<1%
2
ND/337
<1%
ND/435
<1%
ND/392
<1%
3
ND/111
<1%
ND/207
<1%
ND/204
<1%
PA.82 C!T
1
ND/155
<1%
ND/180
<1%
ND/177
<1%
2
ND/695
<1%
ND/809
<1%
ND/745
<1%
3
ND/64
<1%
ND/247
<1%
30/248
12,1%
NS.811 G!T
1
ND/221
<1%
17/270
6,3%
ND/249
<1%
2
ND/2452
<1%
ND/2725
<1%
ND/2557
<1%
3
ND/3117
<1%
ND/4125
<1%
ND/4139
<1%
Colours display the variant frequency with 10% (green), 5–10% (purple) and <5% (pink). ND: not detected. https://doi org/10 1371/journal pone 0229326 t003 Colours display the variant frequency with 10% (green), 5–10% (purple) and <5% (pink). ND: not detected. https://doi.org/10.1371/journal.pone.0229326.t003 eight of the SP/DPP combinations for 2 positions (5,4%) and in at least two SP/DPP combina-
tions for 19 positions (51.4%), although not always in a frequency of 5%. However, for 18
positions (48.6%) the mSNV was not reproduced at a 1% frequency in any of the other SP/
DPP combinations (Table 3). Focussing on the separate SP data analysed in the 3 DPPs, most
of the identified positions with 5% mSNVs in at least 1 SP/DPP combination were identified
in SP1 data (47%) followed by SP2 (29%) and SP3 (24%) data. Looking at the 5% mSNV reproducibility per SP dataset in all three DPPs within these
thirty-seven positions, forty-eight SP datasets showed a 5% mSNV in at least one of the DPP
outputs. Comparison of sequencing methods and data processing pipelines (Continued)
Position
Sequence platform
Data processing pipeline
1
2
3
Minor variants
Percentage
Minor variants
Percentage
Minor variants
Percentage
PB1.14 C!T
1
ND/144
<1%
48/433
11,1%
ND/239
<1%
2
ND/90
<1%
ND/355
<1%
ND/304
<1%
3
ND/562
<1%
ND/792
<1%
ND/496
<1%
PB1.23 T!G
1
ND/207
<1%
30/535
5,6%
ND/315
<1%
2
ND/103
<1%
ND/365
<1%
ND/319
<1%
3
ND/699
<1%
ND/950
<1%
ND/609
<1%
PB1.87 A!G
1
ND/744
<1%
ND/1644
<1%
ND/1076
<1%
2
49/365
13,4%
ND/677
<1%
ND/576
<1%
3
ND/721
<1%
ND/1156
<1%
ND/793
<1%
PB1.2240 G!C
1
ND/757
<1%
23/1517
1,5%
26/515
5,0%
2
ND/944
<1%
ND/985
<1%
ND/806
<1%
3
ND/274
<1%
ND/439
<1%
ND/253
<1%
PB1.2268 A!G
1
5/470
1,1%
33/928
3,6%
22/278
7,9%
2
ND/798
<1%
ND/829
<1%
ND/671
<1%
3
ND/109
<1%
ND/259
<1%
ND/123
<1%
PB1.2271 A!G
1
12/446
2,7%
59/901
6,5%
16/263
6,1%
2
ND/729
<1%
47/810
5,8%
40/649
6,2%
3
1/32
3,1%
ND/123
<1%
2/83
2,4%
HA.867 C!T
1
59/1533
3,8%
206/3183
6,5%
104/1537
6,8%
2
59/2031
2,9%
150/2525
5,9%
127/2253
5,6%
3
11/180
6,1%
48/647
7,4%
28/385
7,3%
HA.963 T!C
1
122/1401
8,7%
446/3071
14,5%
189/1419
13,3%
2
90/1517
5,9%
318/2189
14,5%
247/1828
13,5%
3
5/69
7,2%
107/606
17,7%
47/293
16,0%
NP.1491 C!A
1
ND/278
<1%
71/583
12,2%
ND/206
<1%
2
ND/723
<1%
ND/769
<1%
ND/692
<1%
3
ND/799
<1%
ND/2031
<1%
ND/1206
<1%
NA.65 T!C
1
19/503
3,8%
52/1229
4,2%
16/467
3,4%
2
20/662
3,0%
50/1104
4,5%
45/992
4,5%
3
24/557
4,3%
53/1099
4,8%
37/727
5,1%
NA.78 T!C
1
23/599
3,8%
57/1403
4,1%
20/557
3,6%
2
21/692
3,0%
55/1147
4,8%
50/1033
4,8%
3
23/580
4,0%
51/1124
4,5%
37/735
5,0%
NA.89 T!C
1
23/713
3,2%
55/1670
3,3%
22/651
3,4%
2
23/798
2,9%
56/1261
4,4%
50/1134
4,4%
3
24/580
4,1%
55/1196
4,6%
40/775
5,2%
NA.117 T!C
1
37/908
4,1%
87/2140
4,1%
36/818
4,4%
2
28/1102
2,5%
67/1631
4,1%
ND/1459
<1%
3
22/531
4,1%
57/1276
4,5%
42/812
5,2%
NA.126 T!C
1
37/983
3,8%
83/2294
3,6%
36/876
4,1%
2
31/1126
2,8%
72/1676
4,3%
65/1502
4,3%
3
26/519
5,0%
62/1395
4,4%
43/812
5,3%
(C
ti
d) (Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 14 / 28 PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 Positions with 1% variants that were identified in more than one of the SPs or DPPs
respectively are displayed in the overlapping coloured areas, the centre part representing the number of 1% variants that were detected with all three
DPPs (top row) or SPs (bottom row). The total number of positions with 1% variants detected was 250in SP1, 213 in SP2, 45 in SP3, and 50 in DPP1,
353 in SP2, and 93 in SP3. This figure was produced using Venny 2.1. Fig 3. The reproducibility of 1% variants with sufficient coverage (>298) for all sequence data combined. Each figure shows the number of 1%
variants detected per sequence platform (SP, top row) and data processing pipeline (DPP, bottom row) for SP1/DPP1 (left column), SP2/DPP2 (middle
column), and SP3/DPP3 (right column). The colours represent the different DPPs and SPs respectively, in which the >1% variants were detected: SP1/
DPP1 (purple), SP2/DPP2 (yellow) and SP3/DPP3 (green). Positions with 1% variants that were identified in more than one of the SPs or DPPs
respectively are displayed in the overlapping coloured areas, the centre part representing the number of 1% variants that were detected with all three
DPPs (top row) or SPs (bottom row). The total number of positions with 1% variants detected was 250in SP1, 213 in SP2, 45 in SP3, and 50 in DPP1,
353 in SP2, and 93 in SP3. This figure was produced using Venny 2.1. https://doi.org/10.1371/journal.pone.0229326.g003 and per DPP analysing data from the three SPs (influence of SP). The genome positions with
1% variants were listed per SP/DPP combination and entered in the program Venny 2.1 that
calculated the overlapping positions as a fraction of the total number of positions between the
SP/DPP combinations as compared to the total number of positions, resulting in Fig 3. It
needs to be noted that especially SP3 did not always reach the minimum coverage require-
ments and may therefore not be suitable to detect low-frequency variants with (see also
Table 4). Positions where the coverage in one or more of the nine SP/DPP combinations
didn’t meet the minimum required coverage of 298 were not included in the comparison in
Fig 3. The reproducibility of 1% variants using one SP dataset in all three DPPs was 10%,
9.4% and 31.1% for SP1, SP2 and SP3 sequences, respectively. Additionally, for eleven positions, all in the DETU virus, the variant was reproduced
by all DPPs, however at a <5% frequency (for instance SP3 data at PB2.1054, and SP1 and SP2
data at NA.65) In 53% (31/59) of cases the same mSNVs from 1 SP dataset was reproduced in
all three DPP’s in at least a 1% frequency, in 31% (18/59) of cases the variant was only
detected in 1 DPP even though coverage in the other DPPs was theoretically high enough to
detect variants at a 1% level. Lowering the threshold value to a mSNV frequency of 1% resulted in a large increase in
the number of positions identified with mSNVs. To investigate the reproducibility of these
mSNVs, the data for all 3 viruses was combined per SP in the three DPPs (influence of DPP), PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 15 / 28 Comparison of sequencing methods and data processing pipelines Fig 3. The reproducibility of 1% variants with sufficient coverage (>298) for all sequence data combined. Each figure shows the number of 1%
variants detected per sequence platform (SP, top row) and data processing pipeline (DPP, bottom row) for SP1/DPP1 (left column), SP2/DPP2 (middle
column), and SP3/DPP3 (right column). The colours represent the different DPPs and SPs respectively, in which the >1% variants were detected: SP1/
DPP1 (purple), SP2/DPP2 (yellow) and SP3/DPP3 (green). Positions with 1% variants that were identified in more than one of the SPs or DPPs
respectively are displayed in the overlapping coloured areas, the centre part representing the number of 1% variants that were detected with all three
DPPs (top row) or SPs (bottom row). The total number of positions with 1% variants detected was 250in SP1, 213 in SP2, 45 in SP3, and 50 in DPP1,
353 in SP2, and 93 in SP3. This figure was produced using Venny 2.1. https://doi.org/10.1371/journal.pone.0229326.g003 Fig 3. The reproducibility of 1% variants with sufficient coverage (>298) for all sequence data combined. Each figure shows the number of 1%
variants detected per sequence platform (SP, top row) and data processing pipeline (DPP, bottom row) for SP1/DPP1 (left column), SP2/DPP2 (middle
column), and SP3/DPP3 (right column). The colours represent the different DPPs and SPs respectively, in which the >1% variants were detected: SP1/
DPP1 (purple), SP2/DPP2 (yellow) and SP3/DPP3 (green). PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 The reproducibility of 1% vari-
ants using raw data of a virus sequenced in three different SPs was 20%, 7.4% and 22.6% for
DPP1, DPP2 and DPP3 respectively (Fig 3). Most 1% variants were not reproduced by any
of the other DPPs processing the same SP data (~75%) for SP1 and SP2 data. This was less for PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 16 / 28 Comparison of sequencing methods and data processing pipelines Table 4. The minority variants occurring in at least one of the sequence platform—Data processing pipelines as a
1% variant in the HA segment of the DETU sample with a minimum coverage of 298 reads at that position. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 Position
Sequence
platform
Data processing pipeline
1
2
3
Minor
variants
Percentage
Minor
variants
Percentage
Minor
variants
Percentage
HA.170
T!A
1
ND/935
<1%
ND/2191
<1%
ND/1348
<1%
2
ND/300
<1%
11/693
1,59%
ND/551
<1%
3
ND/82
<1%
ND/245
<1%
ND/210
<1%
HA.170
T!C
1
ND/935
<1%
ND/2191
<1%
ND/1348
<1%
2
ND/300
<1%
18/693
2,60%
ND/551
<1%
3
ND/82
<1%
ND/245
<1%
ND/210
<1%
HA.171
C!A
1
ND/931
<1%
ND/2184
<1%
ND/1339
<1%
2
ND/323
<1%
12/698
1,72%
ND/558
<1%
3
ND/82
<1%
ND/245
<1%
ND/210
<1%
HA.194
C!A
1
ND/991
<1%
ND/2397
<1%
ND/1455
<1%
2
ND/353
<1%
22/701
3,14%
ND/553
<1%
3
ND/58
<1%
ND/250
<1%
ND/212
<1%
HA.195
C!A
1
ND/995
<1%
ND/2390
<1%
ND/1464
<1%
2
ND/356
<1%
20/701
2,85%
ND/553
<1%
3
ND/55
<1%
ND/250
<1%
ND/212
<1%
HA.268
C!T
1
ND/1140
<1%
ND/2580
<1%
ND/1626
<1%
2
ND/1293
<1%
25/1563
1,60%
ND/1338
<1%
3
ND/88
<1%
ND/252
<1%
ND/212
<1%
HA.272
A!T
1
ND/1156
<1%
ND/2593
<1%
ND/1639
<1%
2
17/1424
1,19%
20/1563
1,28%
ND/1404
<1%
3
ND/81
<1%
ND/253
<1%
ND/213
<1%
HA.407
G!T
1
ND/1144
<1%
ND/2364
<1%
ND/1553
<1%
2
ND/1773
<1%
31/2121
1,46%
ND/1855
<1%
3
ND/74
<1%
ND/237
<1%
ND/212
<1%
HA.407
G!A
1
ND/1144
<1%
27/2364
1,14%
ND/1553
<1%
2
ND/1773
<1%
ND/2121
<1%
ND/1856
<1%
3
ND/74
<1%
ND/237
<1%
ND/212
<1%
HA.418
A!G
1
ND/1111
<1%
ND/2319
<1%
ND/1492
<1%
2
29/2195
1,32%
38/2513
1,51%
ND/2197
<1%
3
ND/69
<1%
ND/237
<1%
ND/212
<1%
HA.453
T!G
1
ND/1339
<1%
29/2736
1,06%
ND/1811
<1%
2
ND/2342
<1%
ND/2695
<1%
ND/2384
<1%
3
ND/91
<1%
ND/193
<1%
ND/179
<1%
HA.560
A!G
1
43/1587
2,71%
113/3385
3,34%
55/1517
3,63%
2
56/2397
2,34%
145/2912
4,98%
113/2495
4,53%
3
21/884
2,38%
72/1754
4,10%
43/1245
3,45%
HA.715
C!T
1
ND/1663
<1%
62/3832
1,62%
24/1582
1,52%
2
26/2283
1,14%
55/2722
2,02%
50/2420
2,07%
3
ND/531
<1%
20/1883
1,06%
15/1245
1,20%
HA.867
C!T
1
59/1533
3,85%
206/3183
6,47%
104/1537
6,77%
2
59/2031
2,90%
150/2525
5,94%
127/2253
5,64%
3
11/180
6,11%
48/647
7,42%
28/385
7,27%
(Continued) Table 4. The minority variants occurring in at least one of the sequence platform—Data processing pipelines as a
1% variant in the HA segment of the DETU sample with a minimum coverage of 298 reads at that position. (Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 17 / 28 Comparison of sequencing methods and data processing pipelines Table 4. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 (Continued) (Continued)
Position
Sequence
platform
Data processing pipeline
1
2
3
Minor
variants
Percentage
Minor
variants
Percentage
Minor
variants
Percentage
HA.963
T!C
1
122/1401
8,71%
446/3071
14,52%
189/1419
13,32%
2
90/1517
5,93%
318/2189
14,53%
247/1828
13,51%
3
5/69
7,25%
107/606
17,66%
47/293
16,04%
HA.1000
A!C
1
ND/1409
<1%
48/2962
1,62%
ND/1873
<1%
2
ND/1629
<1%
ND/1919
<1%
ND/1645
<1%
3
ND/84
<1%
ND/614
<1%
ND/293
<1%
HA.1177
G!A
1
ND/1222
<1%
ND/2224
<1%
ND/1597
<1%
2
ND/1652
<1%
34/1901
1,79%
ND/1724
<1%
3
ND/289
<1%
ND/549
<1%
ND/270
<1%
HA.1183
A!G
1
ND/1210
<1%
ND/2226
<1%
ND/1589
<1%
2
ND/1770
<1%
ND/1892
<1%
ND/1723
<1%
3
ND/280
<1%
6/547
1,10%
ND/268
<1%
HA.1199
T!G
1
ND/1182
<1%
ND/2124
<1%
ND/1518
<1%
2
ND/1615
<1%
27/1899
1,42%
ND/1732
<1%
3
ND/296
<1%
ND/545
ND/266
<1%
HA.1263
A!G
1
16/963
1,66%
57/1841
3,10%
26/954
2,73%
2
26/1924
1,35%
56/2207
2,54%
41/1967
2,08%
3
ND/1161
<1%
63/2226
2,83%
33/1350
2,44%
HA.1430
A!G
1
ND/1311
<1%
ND/2870
<1%
ND/1827
<1%
2
ND/1498
<1%
36/1924
1,87%
ND/1659
<1%
3
ND/955
<1%
ND/2391
<1%
ND/1452
<1%
HA.1455
C!T
1
ND/1333
<1%
ND/2753
<1%
14/1233
1,14%
2
ND/1846
<1%
ND/2242
<1%
ND/1895
<1%
3
ND/1093
<1%
ND/2373
<1%
ND/1449
<1%
HA.1543
A!G
1
25/1209
2,07%
94/2757
3,41%
37/1142
3,24%
2
ND/1660
<1%
56/1857
3,02%
41/1585
2,59%
3
ND/1182
<1%
ND/3324
<1%
ND/1972
<1%
HA.1624
C!A
1
ND/998
<1%
ND/2174
<1%
ND/1478
<1%
2
ND/1173
<1%
25/1291
1,94%
ND/1120
<1%
3
ND/2218
<1%
ND/3654
<1%
ND/2244
<1%
HA.1634
C!A
1
ND/930
<1%
ND/2032
<1%
ND/1388
<1%
2
ND/1091
<1%
16/1218
1,31%
ND/1048
<1%
3
ND/2616
<1%
ND/3704
<1%
ND/2269
<1%
HA.1638
C!A
1
ND/932
<1%
ND/1991
<1%
ND/1368
<1%
2
ND/1083
<1%
15/1180
1,27%
ND/1010
<1%
3
ND/2600
<1%
ND/3709
<1%
ND/2276
<1%
HA.1643
T!A
1
ND/875
<1%
ND/1892
<1%
ND/1291
<1%
2
ND/1028
<1%
13/1110
1,17%
ND/944
<1%
3
ND/2612
<1%
ND/3703
<1%
ND/2278
<1%
HA.1643
T!G
1
ND/875
<1%
ND/1892
<1%
ND/1291
<1%
2
ND/1028
<1%
12/1110
1,08%
ND/944
<1%
3
ND/2612
<1%
ND/3703
<1%
ND/2278
<1%
(Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 18 / 28 Comparison of sequencing methods and data processing pipelines Table 4. (Continued)
Position
Sequence
platform
Data processing pipeline
1
2
3
Minor
variants
Percentage
Minor
variants
Percentage
Minor
variants
Percentage
HA.1691
G!A
1
ND/596
<1%
ND/1110
<1%
7/404
1,73%
2
ND/767
<1%
ND/873
<1%
ND/696
<1%
3
ND/2499
<1%
ND/3575
<1%
ND/2222
<1%
HA.1693
A!T
1
ND/582
<1%
ND/1081
<1%
7/391
1,79%
2
ND/751
<1%
ND/864
<1%
ND/690
<1%
3
ND/2310
<1%
ND/3569
<1%
ND/2219
<1%
HA.1695
T!C
1
ND/555
<1%
ND/1030
<1%
7/366
1,91%
2
ND/779
<1%
ND/3557
<1%
ND/688
<1%
3
ND/1767
<1%
ND/3557
<1%
ND/2220
<1%
HA.1698
C!T
1
ND/537
<1%
ND/977
<1%
ND/601
<1%
2
ND/758
<1%
11/852
1,29%
ND/681
<1%
3
ND/2260
<1%
ND/3520
<1%
ND/2113
<1%
HA.1705
A!G
1
ND/492
<1%
ND/883
<1%
ND/528
<1%
2
ND/733
<1%
11/832
1,32%
ND/660
<1%
3
ND/1709
<1%
ND/3300
<1%
ND/2016
<1%
Positions with a too low coverage (<298 reads/position) to detect 1% variants are marked with an asterisk (). Numbers are displayed as [number of variants]/[number of reads on that position]. ND: not detected. https://doi.org/10.1371/journal.pone.0229326.t004 SP3 data but this might be due to the fact that many positions identified in SP3 data did not
meet the minimum coverage criteria and were therefore discarded. For brevity, the detailed results for the HA gene segment of the DETU virus are shown in
Table 4. This virus segment was chosen because it showed the best reproducibility of results
for 5% minority variants in all SP/DPP combinations. In the DETU HA segment, 33 posi-
tions containing a mSNV occurring in 1% of reads with sufficient coverage (298 reads)
were identified. Only 3 of these positions (9%) were identified in all SP/DPP combinations. The majority of the positions (25/33, 76%) were only identified in one of the nine SP/DPP
combinations. However, it needs to be noted that the SP3 data coverage was insufficient in
all three DPPs to detect 1% variants for 11 of those positions (Table 4). Although a comparison between the frequencies of the detected mSNVs might be appropri-
ate, based on these results where even absence vs. presence of the mSNVs is poorly comparable
further in-depth analyses on these frequencies is not performed because of its limited value. Determining the influence of the minor variant detection method. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 Positions with a too low coverage (<298 reads/position) to detect 1% variants are marked with an asterisk (). Numbers are displayed as [number of variants]/[number of reads on that position]. ND: not detected. Comparison of sequencing methods and data processing pipelines le 5. The reproducibility of positions with at least one 5% variant when alignment files from the respective DPPs are all uploaded into DPP3 for only the
NV identification process versus when the mSNV identifications are fully performed by the respective DPPs. To isolate the effect
of just the mSNV identification step in the DPP, independent of the alignment step, quality-
trimmed alignment files (.bam files) of the data (subdivided per virus, per SP and per DPP)
were shared and subjected to the same DPP mSNV detection process (in this case DPP3) and
compared to the original outcomes from DPP1 and DPP2 (Table 5). In the majority of posi-
tions, the different mSNV identification processes did not influence the results, as 84% (119/
142) of the mSNVs were identified regardless of the mSNV identification process. Twenty-
three mSNVs that were not reproduced by DPP3 mSNV identification analysis, were repro-
duced when the ‘Direction and position Filters’ in DPP3 were ignored (Table 5, marked with #
of ##). These parameters filter out mSNVs when the set criteria for the read direction (variant
must occur in both forward and reverse reads), relative read direction (statistical approach of PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 19 / 28 Comparison of sequencing methods and data processing pipelines le 5. s
Position
Sequence
platform
Data Processing pipeline
Bam file generating processing pipeline
1
2
3
1
2
3
Minor
variants
Percentage
Minor
variants
Percentage
Minor
variants
Percentage
Minor
variants
Percentage
Minor
variants
Percentage
Minor
variants
Percentage
H
PB2.1879
G!A
1
81/1301
6,2%
246/2716
9,1%
112/1203
9,3%
132/1375
9,6%
246/2716
9,1%
121/1301
9,3%
2
47/956
4,9%
117/1137
10,3%
114/1064
10,7%
119/1122
10,6%
117/1137
10,3%
114/1064
10,7%
3
49/530
9,2%
131/1341
9,8%
129/1338
9,6%
54/542
10,0%
131//1341
9,8%
129/1338
9,6%
PB2.2101
G!A
1
53/1118
4,7%
261/2704
9,7%
110/897
12,3%
138/1180
11,7%
261/2704
9,7%
121/1086
11,1%
2
21/1578
1,3%
125/1875
6,7%
121/1463
8,3%
ND/1856##
<1%
ND/1850#
<1%
121/1463
8,3%
3
13/542
2,4%
199/1433
13,9%
199/1435
13,9%
87/625
13,9%
199/1433
13,9%
199/1435
13,9%
PB2.2277
T!G
1
ND/479
<1%
86/1008
8,5%
33/190
17,4%
ND/849
<1%
ND/1008##
<1%
37/281
13,2%
2
ND/557
<1%
ND/623
<1%
ND/534
<1%
ND/619
<1%
ND/623
<1%
ND/534
<1%
3
ND/680
<1%
ND/1117
<1%
ND/1024
<1%
ND/708
<1%
ND/1117
<1%
ND/1027
<1%
PB1.87 A!G
1
ND/818
<1%
ND/1754
<1%
ND/1114
<1%
ND/1264
<1%
ND/1753
<1%
ND/1114
<1%
2
25/230
10,9%
ND/376
<1%
ND/328
<1%
ND/368##
<1%
ND/376
<1%
ND/328
<1%
3
ND/275
<1%
ND/537
<1%
ND/537
<1%
ND/278
<1%
ND/537
<1%
ND/537
<1%
PB1.2240
G!C
1
ND/664
<1%
54/1341
4,0%
38/418
9,1%
ND/1004
<1%
ND/1341#
<1%
46/486
9,5%
2
ND/1231
<1%
ND/1271
<1%
ND/1233
<1%
ND/1277
<1%
ND/1271
<1%
ND/1235
<1%
3
ND/161
<1%
ND/277
<1%
ND/276
<1%
ND/163
<1%
ND/277
<1%
ND/276
<1%
PB1.2268
A!G
1
ND/336
<1%
29/641
4,5%
11/176
6,3%
15/322
4,66%
37/641
5,8%
13/213
6,1%
2
ND/993
<1%
ND/1026
<1%
ND/1002
<1%
ND/1025
<1%
ND/1026
<1%
ND/1002
<1%
3
ND/53
<1%
ND/159
<1%
ND/148
<1%
ND/90
<1%
ND/159
<1%
ND/151
<1%
PA.2167
T!G
1
ND/141
<1%
ND/288
<1%
ND/154
<1%
ND/235
<1%
21/288
7,29%
ND/154
<1%
2
ND/757
<1%
ND/807
<1%
ND/773
<1%
ND/812
<1%
ND/807
<1%
ND/733
<1%
3
ND/704
<1%
ND/1070
<1%
ND/1077
<1%
ND/714
<1%
ND/1070
<1%
ND/1078
<1%
HA.104
A!G
1
ND/733
<1%
ND/1761
<1%
ND/1151
<1%
ND/1175
<1%
ND/1761
<1%
ND/1135
<1%
2
ND/437
<1%
ND/1370
<1%
ND/1156
<1%
ND/1326
<1%
ND/1369
<1%
ND/1142
<1%
3
ND/1
<1%
ND/105
<1%
12/105
11,4%
ND/6
<1%
ND/105
<1%
12/105
11,4%
HA.1689
T!C
1
ND/390
<1%
ND/694
<1%
11/217
5,1%
ND/610
<1%
ND/694
<1%
13/260
5,0%
2
ND/2018
<1%
ND/4083
<1%
ND/3979
<1%
ND/4045
<1%
ND/4081
<1%
ND/3979
<1%
3
ND/937
<1%
ND/1669
<1%
ND/1680
<1%
ND/1106
<1%
ND/1669
<1%
ND/1680
<1%
NA.3 T!C
1
ND/32
<1%
ND/105
<1%
ND/49
<1%
ND/92
<1%
7/105
6,67%
ND/49
<1%
2
ND/6
<1%
ND/313
<1%
ND/297
<1%
ND/305
<1%
ND/313
<1%
ND/297
<1%
3
ND/2
<1%
ND/25
<1%
ND/25
<1%
ND/6
<1%
ND/25
<1%
ND/25
<1%
NP.105 A!G
1
ND/182
<1%
ND/449
<1%
ND/343
<1%
ND/374
<1%
6/449
1,34%
ND/343
<1%
2
83/1507
5,5%
ND/1890
<1%
ND/1804
<1%
ND/1866##
<1%
ND/1890
<1%
ND/1805
<1%
3
ND/89
<1%
ND/704
<1%
ND/702
<1%
ND/246
<1%
ND/704
<1%
ND/703
<1%
NP.1239
A!T
1
32/2428
1,3%
279/5410
5,2%
ND/3092
<1%
ND/3372##
<1%
ND/5410#
<1%
ND/3092
<1%
2
ND/2345
<1%
ND/2643
<1%
ND/2453
<1%
ND/2626
<1%
ND/2643
<1%
ND/2453
<1%
3
ND/1711
<1%
ND/2111
<1%
ND/2117
<1%
ND/1712
<1%
ND/2111
<1%
ND/2117
<1%
NP.1489
G!A
1
ND/182
<1%
26/336
7,7%
ND/172
<1%
ND/242
<1%
26/376
6,9%
ND/172
<1%
2
ND/436
<1%
ND/452
<1%
ND/444
<1%
ND/451
<1%
ND/451
<1%
ND/444
<1%
3
ND/1320
<1%
ND/1799
<1%
ND/1799
<1%
ND/1325
<1%
ND/1799
<1%
ND/1799
<1%
NS.827 C!T
1
ND/249
<1%
19/419
4,5%
ND/205
<1%
ND/365
<1%
21/412
5,3%
ND/205
<1%
2
ND/1316
<1%
ND/1423
<1%
ND/1375
<1%
ND/1427
<1%
ND/1422
<1%
ND/1375
<1%
3
ND/2091
<1%
ND/2901
<1%
ND/2757
<1%
ND/2293
<1%
ND/2898
<1%
ND/2929
<1%
NS829 G!T
1
ND/221
<1%
19/380
5,0%
ND/179
<1%
ND/328
<1%
19/376
5,4%
ND/179
<1%
2
ND/1302
<1%
ND/1391
<1%
ND/1341
<1%
ND/1388
<1%
ND/1389
<1%
ND/1341
<1%
3
ND/2117
<1%
ND/2852
<1%
ND/2727
<1%
ND/2279
<1%
ND/2852
<1%
ND/2880
<1%
NS.833 A!T
1
ND/187
<1%
ND/287
<1%
5/88
5,7%
ND/259
<1%
11/257
4,28%
5/96
5,2%
2
ND/1224
<1%
ND/1327
<1%
ND/1284
<1%
ND/1314
<1%
ND/1322
<1%
ND/1284
<1%
3
ND/1367
<1%
ND/2430
<1%
ND/2333
<1%
ND/1779
<1%
ND/2430
<1%
ND/2360
<1%
(Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 20 / 28 (Continued)
us
Position
Sequence
platform
Data Processing pipeline
Bam file generating processing pipeline
1
2
3
1
2
3
Minor
variants
Percentage
Minor
variants
Percentage
Minor
variants
Percentage
Minor
variants
Percentage
Minor
variants
Percentage
Minor
variants
Percentage
U
PB2.900
A!G
1
38/1335
2,9%
136/2740
5,0%
61/1231
5,0%
68/1328
5,12
136/2740
4,96
65/1322
4,92
2
35/1645
2,1%
77/1800
4,3%
66/1629
4,1%
70/1775
4,0%
77/1800
4,3%
66/1629
4,1%
3
30/861
3,5%
86/2308
3,7%
47/1245
3,8%
ND/1001##
<1%
ND/2308#
<1%
47/1245
3,8%
PB2.1054
T!C
1
69/1369
5,0%
168/2637
6,4%
97/1304
7,4%
105/1393
7,5%
168/2637
6,4%
100/1376
7,3%
2
60/1477
4,1%
115/1836
6,3%
99/1605
6,2%
113/1810
6,2%
115/1836
6,3%
99/1605
6,2%
3
6/392
1,5%
94/2038
4,6%
48/1054
4,6%
32/524
6,1%
94/2038
4,6%
48/1054
4,6%
PB2.2257
A!C
1
ND/867
<1%
ND/1563
<1%
24/463
5,2%
ND/1447
<1%
ND/1562
<1%
26/472
5,5%
2
ND/531
<1%
ND/581
<1%
ND/378
<1%
ND/588
<1%
ND/580
<1%
ND/378
<1%
3
ND/893
<1%
ND/2286
<1%
ND/1346
<1%
ND/1341
<1%
ND/2185
<1%
ND/1347
<1%
PB2.2277
T!G
1
ND/644
<1%
52/1150
4,5%
27/307
8,8%
ND/1062
<1%
ND/1150#
<1%
28/381
7,4%
2
ND/418
<1%
ND/472
<1%
ND/284
<1%
ND/474
<1%
ND/472
<1%
ND/284
<1%
3
ND/1208
<1%
ND/1948
<1%
ND/1209
<1%
ND/1251
<1%
ND/1948
<1%
ND/1214
<1%
PB1.14 C!T
1
ND/144
<1%
48/433
11,1%
ND/239
<1%
ND/362
<1%
48/433
11,1%
ND/239
<1%
2
ND/90
<1%
ND/355
<1%
ND/304
<1%
ND/345
<1%
ND/351
<1%
ND/304
<1%
3
ND/562
<1%
ND/792
<1%
ND/496
<1%
ND/633
<1%
ND/655
<1%
ND/504
<1%
PB1.23 T!G
1
ND/207
<1%
30/535
5,6%
ND/315
<1%
ND/470
<1%
30/535
5,6%
ND/315
<1%
2
ND/103
<1%
ND/365
<1%
ND/319
<1%
ND/365
<1%
4/365
1,96%
ND/319
<1%
3
ND/699
<1%
ND/950
<1%
ND/609
<1%
ND/702
<1%
ND/950
<1%
ND/609
<1%
PB1.87 A!G
1
ND/744
<1%
ND/1644
<1%
ND/1076
<1%
ND/1218
<1%
ND/1644
<1%
ND/1076
<1%
2
49/365
13,4%
ND/677
<1%
ND/576
<1%
13/638
2,0%
ND/674
<1%
ND/576
<1%
3
ND/721
<1%
ND/1156
<1%
ND/793
<1%
ND/731
<1%
ND/1156
<1%
ND/793
<1%
PB1.2240
G!C
1
ND/757
<1%
23/1517
1,5%
26/515
5,0%
ND/1266
<1%
ND/1515#
<1%
28/631
4,4%
2
ND/944
<1%
ND/985
<1%
ND/806
<1%
ND/994
<1%
ND/984
<1%
ND/806
<1%
3
ND/274
<1%
ND/439
<1%
ND/253
<1%
ND/301
<1%
ND/439
<1%
ND/253
<1%
PB1.2268
A!G
1
5/470
1,1%
33/928
3,6%
22/278
7,9%
28/420
6,7%
ND/928##
<1%
23/354
6,5%
2
ND/798
<1%
ND/829
<1%
ND/671
<1%
ND/839
<1%
ND/829
<1%
ND/671
<1%
3
ND/109
<1%
ND/259
<1%
ND/123
<1%
ND/193
<1%
ND/259
<1%
ND/126
<1%
PB1.2271
A!G
1
12/446
2,7%
59/901
6,5%
16/263
6,1%
29/413
7,0%
59/901
6,6%
21/336
6,3%
2
ND/729
<1%
47/810
5,8%
40/649
6,2%
43/750
5,73%
47/810
5,8%
40/649
6,2%
3
1/32
3,1%
ND/123
<1%
2/83
2,4%
5/75
6,7%
5/124
4,03%
2/83
2,4%
HA.867
C!T
1
59/1533
3,8%
206/3183
6,5%
104/1537
6,8%
112/1584
7,1%
206/3183
6,5%
109/1573
6,9%
2
59/2031
2,9%
150/2525
5,9%
127/2253
5,6%
144/2502
5,8%
150/2525
5,9%
127/2253
5,6%
3
11/180
6,1%
48/647
7,4%
28/385
7,3%
13/182
7,1%
48/647
7,4%
28/385
7,3%
HA.963
T!C
1
122/1401
8,7%
446/3071
14,5%
189/1419
13,3%
200/1468
13,6%
446/3071
14,5%
193/1455
13,3%
2
90/1517
5,9%
318/2189
14,5%
247/1828
13,5%
308/2165
14,2%
318/2189
14,5%
247/1828
13,5%
3
5/69
7,2%
107/606
17,7%
47/293
16,0%
12/81
14,8%
107/606
17,7%
47/293
16,0%
NP.1491
C!A
1
ND/278
<1%
71/583
12,2%
ND/206
<1%
ND/390
<1%
ND/579#
<1%
ND/206
<1%
2
ND/723
<1%
ND/769
<1%
ND/692
<1%
ND/766
<1%
ND/769
<1%
ND/692
<1%
3
ND/799
<1%
ND/2031
<1%
ND/1206
<1%
ND/858
<1%
ND/2031
<1%
ND/1206
<1%
NA.65 T!C
1
19/503
3,8%
52/1229
4,2%
16/467
3,4%
22/535
4,1%
52/1229
4,2%
20/540
3,7%
2
20/662
3,0%
50/1104
4,5%
45/992
4,5%
52/1063
4,9%
50/1104
4,5%
45/992
4,5%
3
24/557
4,3%
53/1099
4,8%
37/727
5,1%
28/584
4,8%
53/1099
4,8%
37/727
5,1%
NA.78 T!C
1
23/599
3,8%
57/1403
4,1%
20/557
3,6%
23/622
3,7%
57/1403
4,1%
24/638
3,8%
2
21/692
3,0%
55/1147
4,8%
50/1033
4,8%
54/1109
4,9%
55/1147
4,8%
50/1033
4,8%
3
23/580
4,0%
51/1124
4,5%
37/735
5,0%
27/585
4,6%
ND/1124#
<1%
37/735
5,0%
NA.89 T!C
1
23/713
3,2%
55/1670
3,3%
22/651
3,4%
26/731
3,6%
55/1670
3,3%
26/751
3,5%
2
23/798
2,9%
56/1261
4,4%
50/1134
4,4%
54/1224
4,4%
56/1261
4,4%
50/1134
4,4%
3
24/580
4,1%
55/1196
4,6%
40/775
5,2%
28/587
4,8%
55/1196
4,6%
40/775
5,2%
NA.117 T!C
1
37/908
4,1%
87/2140
4,1%
36/818
4,4%
40/914
4,4%
87/2140
4,7%
43/922
4,7%
2
28/1102
2,5%
67/1631
4,1%
ND/1459
<1%
70/1586
4,4%
67/1631
4,1%
ND/1459
<1%
3
22/531
4,1%
57/1276
4,5%
42/812
5,2%
28/544
5,2%
ND/1276#
<1%
42/812
5,2%
NA.126 T!C
1
37/983
3,8%
83/2294
3,6%
36/876
4,1%
39/973
4,0%
83/2294
3,6%
43/981
4,4%
2
31/1126
2,8%
72/1676
4,3%
65/1502
4,3%
75/1616
4,6%
72/1676
4,3%
65/1502
4,3%
3
26/519
5,0%
62/1395
4,4%
43/812
5,3%
30/537
5,6%
62/1395
4,4%
43/812
5,3%
(Continued) Table 5. Comparison of sequencing methods and data processing pipelines Table 5. (Continued) Locations containing mSNV detections in the DPP3 mSNV analysis of the bam files but not in the original DPPs; Locations containing 1% mSNVs that could be
reproduced by deleting DPP3s default ‘Direction and position filters’ with those exactly reproduced (#) and those approximately reproduced but with different
coverages and/or variants (##). https://doi.org/10.1371/journal.pone.0229326.t005 forward/reverse balance) and read position (removal of systemic errors) are not met. However,
DPP1 and DPP2 contain similar quality parameters in their mSNV identification process,
indicating that different DPPs deal differently with quality parameters, and data could be
excluded or included based on the DPP used. In addition, 9 additional mSNVs were identified
in the .bam files compared to the original mSNV outputs. It needs to be noted that the cover-
age of SP data analysed by DPP1 for positions identified with mSNVs was considerably lower
compared to the coverage at that position in the input .bam files, suggesting additional quality
filtering in the mSNV detection step of DPP1. However, the influence on mSNV identification
was limited most likely due to the initial high nucleotide coverage. To better visualise the differences in coverages and allele counts a graphical display of the
data for four positions showing mSNVs in different frequencies for each SP/DPP combination
is included in S2 Fig. In general, SNVs were rarely missed due to low coverage, as also high
coverage SP/DPP combinations display discrepancies (Tables 3 and 4). (Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 21 / 28 https://doi.org/10.1371/journal.pone.0229326.t005 (Continued)
Virus
Position
Sequence
platform
Data Processing pipeline
Bam file generating processing pipeline
1
2
3
1
2
3
Minor
variants
Percentage
Minor
variants
Percentage
Minor
variants
Percentage
Minor
variants
Percentage
Minor
variants
Percentage
Minor
variants
Percentage
UKDD
PB2.2277
T!G
1
ND/415
<1%
28/507
5,5%
ND/475
<1%
ND/503
<1%
ND/507#
<1%
ND/475
<1%
2
ND/589
<1%
ND/620
<1%
ND/601
<1%
ND/627
<1%
ND/620
<1%
ND/601
<1%
3
ND/1140
<1%
ND/1996
<1%
ND/2065
<1%
ND/1186
<1%
ND/1996
<1%
ND/2071
<1%
PB2.2278
T!G
1
ND/367
<1%
ND/471
<1%
ND/464##
<1%
ND/465
<1%
ND/471
<1%
17/268
6,3%
2
ND/581
<1%
ND/613
<1%
ND/581
<1%
ND/621
<1%
ND/588
<1%
ND/581
<1%
3
ND/1141
<1%
ND/1985
<1%
ND/1993
<1%
ND/1184
<1%
ND/1975
<1%
ND/2004
<1%
PB1.87 A!G
1
ND/387
<1%
ND/440
<1%
ND/439
<1%
ND/451
<1%
ND/417
<1%
ND/439
<1%
2
26/327
8,0%
32/395
8,1%
ND/351
<1%
33/385
8,6%
ND/395#
<1%
ND/351
<1%
3
ND/617
<1%
ND/1133
<1%
ND/1136
<1%
ND/622
<1%
ND/1133
<1%
ND/1136
<1%
PB1.728
C!A
1
ND/750
<1%
ND/832
<1%
ND/836
<1%
ND/853
<1%
ND/832
<1%
ND/836
<1%
2
ND/776
<1%
52/928
5,6%
ND/829
<1%
ND/888
<1%
ND/912##
<1%
ND/829
<1%
3
ND/2459
<1%
ND/4290
<1%
ND/4293
<1%
ND/2471
<1%
ND/4287
<1%
ND/4292
<1%
PB1.730
C!T
1
ND/742
<1%
ND/824
<1%
ND/826
<1%
ND/844
<1%
ND/824
<1%
ND/826
<1%
2
ND/767
<1%
57/1008
5,7%
ND/832
<1%
ND/893
<1%
ND/1008#
<1%
ND/832
<1%
3
ND/2339
<1%
ND//4286
<1%
ND/4289
<1%
ND/2464
<1%
ND/4285
<1%
ND/4284
<1%
PB1.883
G!C
1
ND/942
<1%
ND/997
<1%
ND/997
<1%
ND/1016
<1%
ND/997
<1%
ND/997
<1%
2
ND/1689
<1%
ND/1856
<1%
ND/1760
<1%
ND/1867
<1%
ND/1856
<1%
ND/1760
<1%
3
ND/2479
<1%
47/690
6,8%
ND/3681
<1%
ND/2635
<1%
ND/690##
<1%
ND/3697
<1%
PA.49 G!C
1
ND/103
<1%
6/117
5,1%
ND/115
<1%
ND/113
<1%
ND/117#
<1%
ND/115
<1%
2
ND/337
<1%
ND/435
<1%
ND/392
<1%
ND/441
<1%
ND/434
<1%
ND/392
<1%
3
ND/111
<1%
ND/207
<1%
ND/204
<1%
ND/113
<1%
ND/206
<1%
ND/206
<1%
PA.82 C!T
1
ND/155
<1%
ND/180
<1%
ND/177
<1%
ND/179
<1%
ND/180
<1%
ND/177
<1%
2
ND/695
<1%
ND/809
<1%
ND/745
<1%
ND/797
<1%
ND/809
<1%
ND/745
<1%
3
ND/64
<1%
ND/247
<1%
30/248
12,1%
ND/74
<1%
ND/247
<1%
30/248
12,1%
NS.811 G!T
1
ND/221
<1%
17/270
6,3%
ND/249
<1%
ND/261
<1%
ND/270#
<1%
ND/249
<1%
2
ND/2452
<1%
ND/2725
<1%
ND/2557
<1%
ND/2742
<1%
ND/2725
<1%
ND/2557
<1%
3
ND/3117
<1%
ND/4125
<1%
ND/4139
<1%
ND/3188
<1%
ND/4124
<1%
ND/4142
<1% Table 5. Discussion NGS data are used for different applications. Although sequence technologies and the accom-
panying analysis tools are subjected to rapid development, a lot of follow-up research is based 22 / 28 PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 Comparison of sequencing methods and data processing pipelines on initial findings. Accuracy and repeatability are key values for proper scientific research but
the impact of NGS results also reaches beyond science to clinical settings where important
clinical management and treatment decisions are based on such results. In this study the com-
parability of NGS data analyses were analysed using identical input material per virus but dif-
ferent laboratory workflows from nucleic acid extraction and sequencing to data analysis. In
addition, the COMPARE “Data Hub” platform was tested for the purpose of sharing large raw
datafiles between institutions in an outbreak situation. Using this platform, raw sequence data
files up to the size of 8 Gigabytes, alignment files and metadata files of three influenza A/H5N8
viruses were successfully shared in real-time among 3 institutions to allow independent
sequencing and analysis procedures, including mSNV identification, to be performed. The
Data Hub is available to all institutions. The aim of this study was to determine how comparable consensus and minority variant
results were between laboratories performing their standard analyses, and whether discrepan-
cies could be attributed to the SP, DPP or a combination of both. With the lack of a ground
truth/gold standard, all data obtained were compared amongst each other. Importantly, reli-
able consensus sequences were generated independently of the SP/DPP combination used,
although the well-known artefactual InDels in homopolymer regions in SP3 (Roche 454
genome sequencer) sequence data required manual editing. Such consensus sequences rou-
tinely form the basis for a detailed characterization of the influenza strain in an outbreak situa-
tion, as they are used for the prediction of pathogenicity and pandemic potential of influenza
strains. In contrast to the reproducible generation of consensus genome sequences, the hypothesis
that minority variants could be identified reproducibly has to be rejected. The observed differ-
ences were mainly attributed to the alignment processes in the different DPPs. The interpreta-
tion of minority variant analysis thus needs a different level of careful standardization and
awareness about the possible limitations as shown in this study. PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 Discussion Reproducibility of mSNV
results appeared to be influenced by both the different SPs (resulting in different sequence
depths Fig 2) and DPPs (resulting in differences in alignment and mSNV identification of the
same input data, Fig 2 and Table 5). There was limited reproducibility of mSNV identification
data, even for relative high frequency mSNVs. As expected, the reproducibility was best (30%)
for mSNVs occurring in high frequency (10%), and least for the low frequent (1%) mSNVs
(9.4% to 31.1%). Also, the number of positions with 1–5% mSNVs (with sufficient coverage)
was much higher (250 in SP1 data, 213 in SP2 data, and 45 in SP3 data) than the number of
positions with >5–10% mSNVs (n = 27) or >10% mSNVs (n = 10). The set-up of this study allowed many variables to influence the final result. The differences
from first laboratory procedures and sample preparations up to the final analysis methods can
all have contributed to the observed differences in mSNV identification. At this level, especially
with lacking an NGS gold standard, it becomes difficult to determine which identified mSNVs
are ‘true variants’ and which could be due to systematic errors introduced by RNA isolation
methods, amplification, sequencing or manipulated by data processing pipeline settings. Unsurprisingly, the results of this study imply that the choice of SP influences the final output,
but the results from this study also indicate that the DPP, especially the alignment process,
influences coverage. The SP and DPP derived differences in coverage are of importance
because up to a certain (currently unknown, probably SP/DPP dependent) threshold, a higher
coverage will provide a more reliable result about the presence of mSNVs. Although the aim
of this study was to explicitly compare the three institutions own standard workflows, some
parameters (like the phred score and detection limit) were synchronized between the different
DPPs. Moreover, the data from each SP were re-processed in each DPP. However, all DPPs
use different underlying algorithms and interpret the set parameters differently which might PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 23 / 28 Comparison of sequencing methods and data processing pipelines all contribute to the observed differences. Discussion These results are partly in line with previous research
that showed the need of NGS result validation and concluded that only those mSNVs with a
coverage >100 and a frequency of >40% could be identified by NGS methods without second-
ary confirmation [32], however, this conclusion was based on using the same sample prepara-
tion method within a single laboratory. Another recent study sets the cut-off for intrahost
virus diversity at 3% with input of at least 1000 RNA copies and a read depth of at least 400x at
each genome position for Illumina sequencing [33]. Although some studies have been published on SP error rates [34–37] and PCR amplifica-
tion induced variants [38–41], a gold standard system for mSNV analysis is lacking. In addi-
tion, the DPPs can alter the data due to elimination or inclusion of certain sequences based
on the set quality parameters. Allowing too many low-quality reads or being too stringent
on the data will influence the coverage per position and might also influence the accuracy of
the mSNV identification rate, especially when the coverage is low [42, 43]. Although a low
comparability of mSNVs identified in the different SP and DPP combinations was observed,
it can be concluded that 454 (SP3) sequencing has approximately the same accuracy as Illu-
mina (SP1 and 2) sequencing based on the number and percentage of reproducible mSNVs
in this dataset when ignoring InDel errors in homopolymer regions. Although, Roche 454
sequencing machines are no longer in production, it added value to include 454 sequencing
as an alternative sequence platform with alternative chemistry to Illumina. In addition,
because Roche 454 was the first commercially successful next generation sequencing system,
it was used in research that served as a fundament for follow-up studies [44]. A comparison
of Illumina with newer third or fourth generation sequencing platforms (e.g. Nanopore or
Pac Bio) would be interesting in the future. However, the overall error rate remains higher
than the shorter read technologies and recent work concludes that these new platforms are
currently not suitable for the detection of minor variants [33]. In addition, it would be inter-
esting to compare mSNV results of SPs outputting small sequence reads (like Illumina, 454
and Ion Torrent) to new sequencing techniques that output full-length sequence data (e.g. Nanopore [45]). Discussion The latter might be less vulnerable to quality trimming parameters com-
pared to small reads and might provide a more consistent nucleotide coverage over complete
gene segment. Although some studies have been published on SP error rates [34–37] and PCR amplifica-
tion induced variants [38–41], a gold standard system for mSNV analysis is lacking. In addi-
tion, the DPPs can alter the data due to elimination or inclusion of certain sequences based
on the set quality parameters. Allowing too many low-quality reads or being too stringent
on the data will influence the coverage per position and might also influence the accuracy of
the mSNV identification rate, especially when the coverage is low [42, 43]. Although a low
comparability of mSNVs identified in the different SP and DPP combinations was observed,
it can be concluded that 454 (SP3) sequencing has approximately the same accuracy as Illu-
mina (SP1 and 2) sequencing based on the number and percentage of reproducible mSNVs
in this dataset when ignoring InDel errors in homopolymer regions. Although, Roche 454
sequencing machines are no longer in production, it added value to include 454 sequencing
as an alternative sequence platform with alternative chemistry to Illumina. In addition, For mSNV analyses by different labs, very stringent SP/DPP protocols need to be evalu-
ated, for instance by cross-validating results. To allow a better comparison it would be rec-
ommended to create some kind of gold standard by for instance evaluating parameters
based on sequencing of technical replicates, and controlled mixes of clones. The mSNV anal-
ysis can be valuable for epidemiological tracing, to monitor early evolutionary events, or
drug resistance, possibly host adaptation, but this would require reproducibility of study
outcomes within and between laboratories. As this is currently not that case, more under-
standing of biases and errors generated by sample processing (enrichment procedures),
sequencing strategy (amplicons, shotgun), sequencing chemistry (each of which have their
own internal error rates) and the approach to data processing and analysis is needed. Under-
standing the parameters and thresholds in the software can be difficult and a systematic
study using a pipeline where the effect of changing each of these parameters both individu-
ally and in combination is required to determine the optimal settings for minor variant
analysis. As alternate high-throughput sequencing technologies arise there will be a need to under-
stand inherent error profiles and how those are handled in data processing approaches. Acknowledgments The authors would like to thank the staff of the European Nucleotide Archive and all technical
staff involved in the supporting laboratory and avian surveillance work. S1 File. DPP3 Sequence analysis protocol.
(PDF) S1 Fig. Nucleotide coverage. The non-normalised nucleotide coverage displayed as number
of nucleotides per position for full genome sequences of the UKDD and DETU virus reads
mapped to the corresponding reference sequences. Panel A shows the coverage results for the
same SP dataset in the three different DPPs (DPP1: purple; DPP2: orange; DPP3 grey) for each
of the SP datasets. Panel B shows the coverage when the same DPP is used to analyse data from
the three different SPs (SP1: lilac; SP2: yellow; SP3:green) for each of the DPPs. The X-axis rep-
resents the position in the genome, the Y-axis represents the number of sequence reads per
position. S2 Fig. Graphical display of the coverage and allele counts for four positions, showing
mSNVs in different frequencies for each SP/DPP combination. Arrows indicate the approx-
imate percentages in which the mSNVs were detected; 1–5% (orange), 5–10% (purple) and
>10% (green). (TIF) Discussion Cross-
validation should be supported by international proficiency tests on NGS techniques including
mSNV analyses that would be instrumental in validation of results and may foster the trust in
NGS-based diagnostics. 24 / 28 PLOS ONE | https://doi.org/10.1371/journal.pone.0229326
February 20, 2020 Comparison of sequencing methods and data processing pipelines Supporting information
S1 Table. PCR primers used in SP3 to cover the influenza A H5N8 gene segments. (PDF)
S2 Table. SP/DPP overarching consensus sequences. (PDF)
S3 Table. Number of raw sequences and influenza virus reads per SP per virus. (PDF)
S1 File. DPP3 Sequence analysis protocol. (PDF)
S1 Fig. Nucleotide coverage. The non-normalised nucleotide coverage displayed as number
of nucleotides per position for full genome sequences of the UKDD and DETU virus reads
mapped to the corresponding reference sequences. Panel A shows the coverage results for the
same SP dataset in the three different DPPs (DPP1: purple; DPP2: orange; DPP3 grey) for each
of the SP datasets. Panel B shows the coverage when the same DPP is used to analyse data from
the three different SPs (SP1: lilac; SP2: yellow; SP3:green) for each of the DPPs. The X-axis rep-
resents the position in the genome, the Y-axis represents the number of sequence reads per
position. (TIF)
S2 Fig. Graphical display of the coverage and allele counts for four positions, showing
mSNVs in different frequencies for each SP/DPP combination. Arrows indicate the approx-
imate percentages in which the mSNVs were detected; 1–5% (orange), 5–10% (purple) and
>10% (green). (TIF) References 1. Heather J. and Chain B, The sequence of sequencers: The history of sequencing DNA. Genomics,
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February 20, 2020 27 / 28 Comparison of sequencing methods and data processing pipelines 43. Sims D, Sudbery I, Ilott N, Heger A, Ponting C, Sequencing depth and coverage: key considerations in
genomic analyses. Nat Rev Genet, 2014. 15(2): p. 121–32. https://doi.org/10.1038/nrg3642 PMID:
24434847 44. Liu L, Li Y, Li S, Hu N, He Y, Pong R, et al., Comparison of next-generation sequencing systems. J
Biomed Biotechnol, 2012. 2012: p. 251364. https://doi.org/10.1155/2012/251364 PMID: 22829749 45. Keller M, Rambo-Martin B, Wilson M, Ridenour C, Shepard S, Start T, et al., Direct RNA Sequencing of
the Coding Complete Influenza A Virus Genome. Sci Rep, 2018. 8(1): p. 14408. https://doi.org/10. 1038/s41598-018-32615-8 PMID: 30258076 45. Keller M, Rambo-Martin B, Wilson M, Ridenour C, Shepard S, Start T, et al., Direct RNA Sequencing of
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Практические рекомендации по общим принципам проведения противоопухолевой лекарственной терапии
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ПРАКТИЧЕСКИЕ РЕКОМЕНДАЦИИ
ПО ОБЩИМ ПРИНЦИПАМ ПРОВЕДЕНИЯ
ПРОТИВООПУХОЛЕВОЙ ЛЕКАРСТВЕННОЙ
ТЕРАПИИ
Коллектив авторов: Трякин А. А., Бесова Н. С., Волков Н. М., Гладков О. А., Карасева В.
Сакаева Д. Д., Фадеева Н. В., Федянин М. Ю. Коллектив авторов: Трякин А. А., Бесова Н. С., Волков Н. М., Гладков О. А., Карасева В. В.,
Сакаева Д. Д., Фадеева Н. В., Федянин М. Ю. 1. ВВЕДЕНИЕ Противоопухолевая лекарственная терапия является самостоятельным разделом
онкологии, объединяющим химиотерапию, гормонотерапию, антиангиогенную, таргет-
ную, иммуно- и поддерживающую терапию, и играет важную роль в лечении пациентов
со злокачественными новообразованиями. Противоопухолевые препараты назначаются
в соответствии с показаниями, указанными в инструкции к препарату. Допустимо назна-
чение препарата по незарегистрированным показаниям или в незарегистрированных
в инструкции дозировках при наличии информированного согласия пациента и соблю-
дении следующих условий: a) данная опция указана в клинических рекомендациях; a) данная опция указана в клинических рекомендациях; б) данная опция не указана в клинических рекомендациях: •
по жизненным показаниям по решению врачебной комиссии или онкологического
консилиума; •
по жизненным показаниям по решению врачебной комиссии или онкологического
консилиума; •
наличие научных обоснований эффективности данной терапии (научные пуб-
ликации или регистрационные показания в других странах); •
наличие научных обоснований эффективности данной терапии (научные пуб-
ликации или регистрационные показания в других странах); •
отсутствие более эффективных и (или) менее токсичных методов лечения. •
отсутствие более эффективных и (или) менее токсичных методов лечени DOI: 10.18027 / 2224-5057-2022-12-3s2-27-40 DOI: 10.18027 / 2224-5057-2022-12-3s2-27-40 Цитирование: Трякин А. А., Бесова Н. С., Волков Н. М., Гладков О. А., Карасева В. В., Сакаева Д. Д. и соавт. Практические рекомендации по общим принципам проведения противоопухолевой лекар-
ственной терапии. Злокачественные опухоли : Практические рекомендации RUSSCO #3s2, 2022
(том 12). 27–40. ОБЩИЕ ПРИНЦИПЫ ПРОТИВООПУХОЛЕВОЙ ТЕРАПИИ ОБЩИЕ ПРИНЦИПЫ ПРОТИВООПУХОЛЕВОЙ ТЕРАПИИ 27 Версия 2022 1.2. Виды противоопухолевой лекарственной терапии Адъювантная: назначается после радикального хирургического лечения или радикальной
ЛТ с целью профилактики рецидива заболевания. Неоадъювантная: назначается при исходно операбельных опухолях до радикального
хирургического лечения или до радикальной ЛТ с целью уменьшения объема опухоли
и достижения патоморфологического ответа. Лечебная / индукционная: лекарственная терапия при метастатическом или местно-
распространенном опухолевом процессе, направлена на увеличение продолжительности
жизни пациента, в отдельных случаях может быть достигнуто выздоровление. Лечебная / индукционная: лекарственная терапия при метастатическом или местно-
распространенном опухолевом процессе, направлена на увеличение продолжительности
жизни пациента, в отдельных случаях может быть достигнуто выздоровление. Паллиативная: преследует цель устранения симптомов заболевания за счет уменьшения
опухолевой массы, не преследует цель увеличения продолжительности жизни. Паллиативная: преследует цель устранения симптомов заболевания за счет уменьшения
опухолевой массы, не преследует цель увеличения продолжительности жизни. Поддерживающая лекарственная терапия: направлена на поддержание эффекта,
достигнутого при лечебной / индукционной лекарственной терапии. Противоопухолевые лекарственные препараты могут быть использованы отдельно
в виде монотерапии или в составе режимов комбинированной лекарственной терапии,
а также могут сочетаться с другими видами терапии (лучевая, операция и др.). Противоопухолевые лекарственные препараты могут быть использованы отдельно
в виде монотерапии или в составе режимов комбинированной лекарственной терапии,
а также могут сочетаться с другими видами терапии (лучевая, операция и др.). Химиолучевая терапия — одновременное или последовательное комбинированное
применение лекарственной терапии (возможна ХТ, таргетная терапия, иммунотерапия)
и ЛТ. Химиолучевая терапия — одновременное или последовательное комбинированное
применение лекарственной терапии (возможна ХТ, таргетная терапия, иммунотерапия)
и ЛТ. Химиоиммунотерапия — одновременное комбинированное применение химиопрепаратов
и иммунопрепаратов — моноклональных антител, интерферонов. Химиогормонотерапия — одновременное комбинированное применение химиопрепаратов
и гормональных препаратов. Химиогормонотерапия — одновременное комбинированное применение химиопрепаратов
и гормональных препаратов. 1.1. Цели лекарственного лечения злокачественных опухолей 1.1. Цели лекарственного лечения злокачественных опухолей •
Излечение пациента •
Излечение пациента •
Контроль заболевания и увеличение продолжительности жизни
•
Устранение симптомов болезни и улучшение качества жизни •
Контроль заболевания и увеличение продолжительности жизни •
Устранение симптомов болезни и улучшение качества жизни том / vol. 12 № 3s2 • 2022 MALIGNANT TUMOURS
Russian Society of Clinical Oncology MALIGNANT TUMOURS
Russian Society of Clinical Oncology Практические рекомендации 28 Практические рекомендации 1.3. Способы применения противоопухолевых лекарственных
препаратов Системный: рассчитан на резорбтивный противоопухолевый эффект при приеме
противоопухолевого препарата внутрь, введении п / к, в / в, в / м, ректально. Регионарный: лекарственный препарат вводится в сосуды, питающие новообразование,
при этом системное воздействие, т. е. поступление цитостатика в другие органы
минимально. Локальный метод: препараты наносятся на поверхностные очаги в виде мазей или растворов,
вводятся непосредственно в опухолевый очаг, в серозные полости (брюшную — с целью
купирования асцита, грудную — с целью купирования плеврита), спинномозговой канал
(интратекально), применяются внутрипузырно. MALIGNANT TUMOURS
Russian Society of Clinical Oncology ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии MALIGNANT TUMOURS
Russian Society of Clinical Oncology 29 ОБЩИЕ ПРИНЦИПЫ ПРОТИВООПУХОЛЕВОЙ ТЕРАПИИ Версия 2022 2. ОБЩИЕ ПРИНЦИПЫ ЛЕКАРСТВЕННОЙ ТЕРАПИИ
ЗЛОКАЧЕСТВЕННЫХ НОВООБРАЗОВАНИЙ Выбор терапии основывается на локализации и морфологической форме опухоли,
ее молекулярном профиле, распространенности опухолевого процесса, эффективности
ранее проводимой терапии, состоянии пациента, цели лечения в конкретной клинической
ситуации, соотношении потенциальной пользы и токсичности лечения. Если целью является излечение, то даже значительные побочные эффекты являются
приемлемыми. Когда целью лечения является продление жизни, то следует тщательно
рассмотреть соотношение ожидаемой пользы и выраженности побочных эффектов
терапии. При паллиативной терапии лечение следует выбирать так, чтобы вероятность
выраженной токсичности была небольшой. В этом случае побочные эффекты не должны
быть тяжелее, чем симптомы заболевания. При лечении метастатических форм опухолей может быть использовано несколько
линий терапии. Переход от одной линии к другой производится при прогрессировании
заболевания или при непереносимости терапии из‑за выраженных нежелательных явле-
ний. При большинстве опухолей в случае непрерывного прогрессирования заболевания
на фоне трех последовательных линий терапии (при раке молочной железы — до 5)
дальнейшее лекарственное лечение не имеет смысла. том / vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии Российское общество клинической онкологии 2.1. Характеристики опухоли, учитываемые
при планировании лекарственной терапии При постановке первичного диагноза предпочтительна его гистологическая вери-
фикация. При тяжелом общем состоянии пациента, технических трудностях или невозможности
проведения биопсии опухоли, можно основываться на результатах цитологического
исследования. В ряде ситуаций, например, при герминогенных опухолях, хориокарциноме
матки, гепатоцеллюлярном раке допускается начало терапии при наличии характерной
клинической картины, данных инструментальных методов обследования и повышенного
уровня специфических маркеров. При развитии рецидива заболевания (местный рецидив опухоли или появление
метастазов) необходимо стремиться к его гистологической или цитологической вери-
фикации, если она технически выполнима без существенного риска серьезного вреда
для пациента, с целью исключения метахронных, в том числе, индуцированных зло-
качественных новообразований, изменения морфологической формы (трансформации)
и / или молекулярного профиля опухоли, особенно в следующих ситуациях: •
при поздних рецидивах; •
в ситуациях, когда возможное изменение молекулярного профиля опухоли влечет
за собой коррекцию терапевтических подходов; •
в ситуациях, когда возможное изменение молекулярного профиля опухоли влечет
за собой коррекцию терапевтических подходов; рр
р
•
в случае нетипичного варианта метастазирования опухоли; рр
р
•
в случае нетипичного варианта метастазирования опухоли; •
при наличии солитарного метастаза как единственного проявления заболевания. MALIGNANT TUMOURS
Russian Society of Clinical Oncology том / vol. 12 № 3s2 • 2022 Практические рекомендации 30 Практические рекомендации При невозможности верификации рецидива заболевания, тактика лечения опре-
деляется исходя из данных анамнеза, клинической картины, данных дополнительных
инструментальных методов исследований, определения опухолевых маркеров. 2.3. Противопоказания к проведению лекарственной терапии Противопоказания к проведению лекарственной терапии могут носить абсолютный
и относительный характер. 1.
Мнение пациента и его выбор. 1. Мнение пациента и его выбор. 2. Возраст. 3. Общее состояние (оценивается в баллах по шкале ECOG). При статусе ECOG ≥ 3 баллов
ХТ, как правило, не показана (за исключением больных, не получавших ранее терапии,
с высокочувствительными к данному виду лечения опухолями, при которых высока
вероятность достижения выраженного противоопухолевого эффекта); решение
о назначении таргетной терапии, гормональной и иммунотерапии принимается
индивидуально. 3. Общее состояние (оценивается в баллах по шкале ECOG). При статусе ECOG ≥ 3 баллов
ХТ, как правило, не показана (за исключением больных, не получавших ранее терапии,
с высокочувствительными к данному виду лечения опухолями, при которых высока
вероятность достижения выраженного противоопухолевого эффекта); решение
о назначении таргетной терапии, гормональной и иммунотерапии принимается
индивидуально. 4. Сопутствующие заболевания. Необходима оценка спектра сопутствующей пато-
логии, тяжести и прогноза для каждого заболевания. Выраженные сопутствующие
заболевания и их лечение могут являться противопоказанием для проведения
противоопухолевой лекарственной терапии. В случаях, когда продолжительность
жизни пациента определяется не опухолевым, а сопутствующим заболеванием,
проведение противоопухолевой лекарственной терапии нецелесообразно. 4. Сопутствующие заболевания. Необходима оценка спектра сопутствующей пато-
логии, тяжести и прогноза для каждого заболевания. Выраженные сопутствующие
заболевания и их лечение могут являться противопоказанием для проведения
противоопухолевой лекарственной терапии. В случаях, когда продолжительность
жизни пациента определяется не опухолевым, а сопутствующим заболеванием,
проведение противоопухолевой лекарственной терапии нецелесообразно. 5. Предшествующее противоопухолевое лечение (количество курсов лечения, ис-
пользованные лекарственные препараты, в случае применения антрациклинов —
их суммарные дозы, достигнутый ранее ответ опухоли на терапию, переносимость
терапии), остаточные проявления токсичности. 6. Психосоциальный статус пациента. Важной составляющей успешной терапии явля-
ется комплаентность пациента — способность и готовность пациента соблюдать
все предписания лечащего врача, график лечения и обследования. 6. Психосоциальный статус пациента. Важной составляющей успешной терапии явля-
ется комплаентность пациента — способность и готовность пациента соблюдать
все предписания лечащего врача, график лечения и обследования. 2.2. Характеристики пациента, учитываемые
при планировании лекарственной терапии 1. Мнение пациента и его выбор. ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии том / vol. 12 № 3s2 • 2022 2.3.1. Абсолютные противопоказания Абсолютным противопоказанием является крайне тяжелое (статус по шкале
ECOG 4 балла) общее состояние пациента. В каждой конкретной ситуации должно оцениваться соотношение потенциальных
пользы и вреда противоопухолевой лекарственной терапии. Например, при излечимых
опухолях (герминогенные опухоли, хориокарцинома матки, лимфомы) или при исполь-
зовании малотоксичных высокоэффективных препаратов (ингибиторы EGFR при раке
легкого, ГТ) терапия первой линии может проводиться пациентам и в крайне тяжелом том / vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии MALIGNANT TUMOURS
Russian Society of Clinical Oncology MALIGNANT TUMOURS
Russian Society of Clinical Oncology ОБЩИЕ ПРИНЦИПЫ ПРОТИВООПУХОЛЕВОЙ ТЕРАПИИ 31 Версия 2022 состоянии. Однако при этих же опухолях проведение терапии поздних линий, когда
шанс на излечение или значимое продление жизни минимален, задачей терапии ста-
новится контроль симптомов заболевания при минимизации токсичности и риска
тяжелых осложнений. том / vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии 2.3.2. Относительные противопоказания Относительными противопоказаниями к проведению терапии является активная
инфекция, ранее перенесенный инфаркт миокарда, острое нарушение мозгового
кровообращения давностью менее 6 мес., острый (в срок до 5–7 дней) тромбоз
глубоких вен. Для многих препаратов существуют специфические относительные противо-
показания. Для цисплатина: состояние по шкале ECOG > 1 балла; клиренс креати-
нина < 50–60 мл / мин.; сенсоневральная тугоухость > 1ст. и периферическая нейро-
патия > 1 ст.; сердечная недостаточность III–IV класса по NYHA. Для антиангиогенных
препаратов — кровотечение, наличие свища, перфорации полых органов ЖКТ в ана-
мнезе, артериальные тромбоэмболические события в анамнезе, неконтролируемая
артериальная гипертензия. Острые инфузионные реакции 1–2 степени тяжести, купирующиеся введением
антигистаминных препаратов и системных ГКС, не являются абсолютным противопо-
казанием к повторному применению вызвавшего их препарата, но требуют усиления
премедикации: 20 мг дексаметазона (или его эквивалента) внутрь приблизительно
за 12 и 6 часов до введения препарата и / или 20 мг дексаметазона внутривенно примерно
за 30–60 мин. до введения препарата, 50 мг дифенгидрамина (или его эквивалента)
внутривенно и 50 мг ранитидина (или 20 мг фамотидина) внутривенно за 30–60 ми-
нут до введения препарата, удлинения времени инфузии или применения того же
препарата другой фирмы-производителя, проведения протокола десенсибилизации
(например, при лечении карбоплатином, оксалиплатином) (см. возможный вариант
десенсибилизации: Тюляндина А. С., Коломиец Л. А., Морхов К. Ю., и соавт. Практические
рекомендации по лекарственному лечению рака яичников, первичного рака брюшины
и рака маточных труб. Злокачественные опухоли: Практические рекомендации RUSSCO
#3s2, 2021 (том 11). 10). В случае развития клинически значимых аллергических реакций, а также при отсут-
ствии в медицинской организации необходимого противоопухолевого лекарственного
препарата может быть произведена замена его на другой, схожий по эффективности
препарат, в рамках одной группы: например, при меланоме пембролизумаб может
быть заменен на ниволумаб в монотерапии; паклитаксел на доцетаксел и, наобо-
рот, — при большинстве опухолей; карбоплатина на цисплатин — при раке яич-
ников, раке молочной железы, раке легкого; оксалиплатина на цисплатин — при раке
желудка, капецитабин на инфузионный фторурацил — при большинстве опухолей. Равноэффективными и взаимозаменяемыми также являются ингибиторы ароматазы,
цетуксимаб и панитумумаб при колоректальном раке, пембролизумаб и ниволумаб
при меланоме и т. д. том / vol. 12 № 3s2 • 2022 MALIGNANT TUMOURS
Russian Society of Clinical Oncology ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии Практические рекомендации 32 2.4. Поддержание дозовой интенсивности
противоопухолевой терапии Интенсивность цитотоксической ХТ определяется как доза, вводимая в единицу
времени. Она может быть повышена за счет увеличения разовой дозы (высокодоз-
ные режимы) или уменьшения интервалов между введениями (уплотненные режимы). При производственной необходимости или при клинических показаниях допускается
раздельное применение цитостатических препаратов и противоопухолевых моно-
клональных антител, интервал между которыми может достигать 7 дней. Например,
комбинация доцетаксел + трастузумаб + пертузумаб (все препараты в день 1‑й, курсы
каждые 3 недели) может быть применена как доцетаксел (день 1‑й), пертузумаб + трасту-
зумаб (день 4‑й, курсы каждые 3 недели). С целью поддержания дозовой интенсивности
терапии, а также профилактики развития фебрильной нейтропении после завершения
цитостатической терапии возможно применение Г-КСФ, наиболее распространенным
представителем которого является филграстим и его пролонгированные формы. Доза
и продолжительность терапии филграстимом индивидуальны и определяются его
клинической эффективностью, оцениваемой по динамике уровня нейтрофилов. Вме-
сто дозы 5 мкг / кг допустимо применение филграстима в фиксированных суточных
дозах 300 мкг или 480 мкг. Возможно досрочное прекращение терапии филграстимом
в ситуациях, когда после первоначального падения уровня нейтрофилов отмечается
их рост до ≥ 10 × 109 / л либо, если их снижение после ХТ не наблюдалось, при достижении
уровня нейтрофилов ≥ 40–50 × 109 / л. ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии Режимы лечения в зависимости от дозы цитотоксического препарата В зависимости от интенсивности дозы цитотоксического препарата выделяют: 1) низкодозную (низкоинтенсивную) ХТ: часто рекомендуется больным пожилого и стар-
ческого возраста, пациентам с серьезной сопутствующей патологией, при тяжелом
и среднетяжелом общем состоянии, при проведении терапии с паллиативной целью;
2) ХТ стандартной дозовой интенсивности; 1) низкодозную (низкоинтенсивную) ХТ: часто рекомендуется больным пожилого и стар-
ческого возраста, пациентам с серьезной сопутствующей патологией, при тяжелом
и среднетяжелом общем состоянии, при проведении терапии с паллиативной целью;
2) ХТ стандартной дозовой интенсивности; 3) высокодозную (высокоинтенсивную) ХТ, как правило, характеризующуюся высокой
миело-и иммуносупрессивностью, вплоть до полной миелоаблативности; 3) высокодозную (высокоинтенсивную) ХТ, как правило, характеризующуюся высокой
миело-и иммуносупрессивностью, вплоть до полной миелоаблативности; 4) сверхвысокодозную ХТ или ХТ сверхвысокой интенсивности, которая, как правило,
представляет собой модификации уже существующих режимов высокодозной ХТ
с добавлением дополнительных химиотерапевтических агентов или с дополнитель-
ной эскалацией доз одного или нескольких химиотерапевтических агентов; 5) метрономную ХТ — регулярное назначение цитостатических препаратов в дозах,
значительно меньших максимально переносимых (в низких дозах), с небольшими
(значительно меньшими, чем в стандартных режимах) интервалами времени; 5) метрономную ХТ — регулярное назначение цитостатических препаратов в дозах,
значительно меньших максимально переносимых (в низких дозах), с небольшими
(значительно меньшими, чем в стандартных режимах) интервалами времени; 6) режим с уплотненным введением химиопрепаратов (доз-денсный режим): рас-
сматривается при быстро пролиферирующих опухолях; характеризуется меньшим
интервалом (2 недели вместо 3‑недельного интервала) между введениями цито-
статических препаратов и требует профилактического использования Г-КСФ. 6) режим с уплотненным введением химиопрепаратов (доз-денсный режим): рас-
сматривается при быстро пролиферирующих опухолях; характеризуется меньшим
интервалом (2 недели вместо 3‑недельного интервала) между введениями цито-
статических препаратов и требует профилактического использования Г-КСФ. ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии MALIGNANT TUMOURS
Russian Society of Clinical Oncology ОБЩИЕ ПРИНЦИПЫ ПРОТИВООПУХОЛЕВОЙ ТЕРАПИИ 33 Многие виды противоопухолевой лекарственной терапии (прежде всего, цитотокси-
ческой) обладают узким терапевтическим окном, в связи с чем, важно придерживаться
рекомендованных доз препаратов, а также интервалов между их введениями. Расчет дозы цитотоксического средства для большинства препаратов выполняется
на площадь поверхности тела, определить которую можно по формуле Мостеллера,
Дюбуа и Дюбуа в модификации, а также при помощи номограмм и калькуляторов. Рекомендуется округление расчетной площади поверхности тела до первого знака
после запятой. Формула Мостеллера (Mosteller, 1987): Формула Дюбуа и Дюбуа (модификация): Формула Дюбуа и Дюбуа (модификация): том / vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии Практические рекомендации 34 Практические рекомендации переносимости редуцированных доз допускается их эскалация, вплоть до полных доз,
на последующих курсах. Клинически значимая токсичность ранее проведенного курса
терапии также является основанием для редукции разовых доз препаратов, реэскалация
дозы на последующих курсах, как правило, не производится. ХТ часто носит цикловой характер. Цикл ХТ — период времени, исчисляемый с пер-
вого дня первого введения цитостатиков до планируемого первого дня следующего
курса, включает в себя дни введения препаратов и плановый перерыв между курсами. Примером 21‑дневного цикла является режим XELOX: продолжительность курса 14 дней,
перерыв между курсами 7 дней; примером 14‑дневного цикла являются режимы типа
FOLFOX или FLOT, повторное введение препаратов в которых запланировано на 15 день,
считая с 1‑го дня введения препаратов предыдущего курса. Примером 6‑недельного
цикла являются режимы, содержащие некоторые производные нитрозометилмочевины
или митомицина С. Допускается смещение даты начала следующего лечебного цикла на 3 дня в мень-
шую и большую стороны от запланированной даты без причин медицинского характера
(производственная необходимость, государственные праздники, семейные обстоятель-
ства и т. п.). Начало очередного цикла может быть отложено по медицинским показаниям
в следующих ситуациях: •
побочные эффекты лечения, не разрешившиеся до степени 0–1 к началу следующего
цикла (за исключением алопеции); •
побочные эффекты лечения, не разрешившиеся до степени 0–1 к началу следующего
цикла (за исключением алопеции); •
обострение сопутствующих заболеваний; •
любое заболевание в острой фазе. Для большинства режимов ХТ стандартными требованиями к показателям гемо-
граммы, позволяющим начать очередной курс ХТ, являются АЧН ≥ 1,5 × 109 / л и количе-
ство тромбоцитов ≥ 100 × 109 / л. Для отдельных комбинаций и клинических ситуаций
(лечение онкогематологических заболеваний, еженедельное введение паклитак-
села, гемцитабина, комбинация гемцитабина с наб-паклитакселом, режим ВЕР или ЕР
при герминогенных опухолях) применяются особые критерии гемограммы, достаточ-
ные для начала курса / введения химиопрепарата и учитываемые при редукции доз
препаратов на 8‑й и 15‑й дни. Уровень гемоглобина не должен служить поводом к отсрочке цикла терапии; в слу-
чаях выраженной анемии требуется проведение гемотрансфузии с последующим про-
должением лечения. ППТ (м2) = вес [кг] 0,425 × рост [см] 0,725 / 139,2 У больных с ожирением при отсутствии сопутствующей патологии расчет дозы
препарата производится исходя из фактической массы тела, особенно если целью
терапии является излечение. При наличии сопутствующей патологии, а также в случае
паллиативных целей терапии при площади поверхности тела свыше 2 м 2 возможен
расчет дозы на площадь 2 м2 на первом курсе терапии. При хорошей переносимости
лечения на последующих курсах рекомендуется одномоментная или последовательная
(в течение двух-трех курсов терапии) эскалация доз препаратов с расчетом на акту-
альные вес и рост. Для некоторых химиотерапевтических препаратов установлены предельно допу-
стимые разовые дозы (например, для винкристина — 2 мг, для блеомицина — 30 мг,
для карбоплатина — 1000 мг в комбинациях и 1200 мг — в монотерапии). Для препаратов,
обладающих дозолимитирующей кумулятивной токсичностью, установлены предельно
допустимые кумулятивные дозы: например, с учетом кумулятивной кардиотоксичности
антрациклинов, кумулятивная доза для доксорубицина составляет 550 мг / м2, для эпи-
рубицина — 1000 мг / м2; для блеомицина с учетом потенциальной возможности развития
фиброза легких кумулятивная доза составляет 360 мг. В отдельных ситуациях, когда
отсутствуют другие более эффективные опции лечения и при условии тщательного
мониторинга кардиотоксичности на фоне профилактической кардиотропной терапии
возможно превышение кумулятивных доз антрациклинов. Допустимо округление реально вводимой дозы противоопухолевого лекарственного
препарата в диапазоне до 10 % от расчетной. Перерасчет разовой дозы на последующих
курсах производится по актуальным показателям роста и веса в том случае, если изме-
нение массы тела пациента превысило 10 %. Допускается исходная редукция разовой дозы препарата в случаях, когда при-
менение рекомендуемых доз представляет для пациента повышенный риск развития
осложнений. К подобным ситуациям относятся общий статус по шкале ЕСОG 2–3, стар-
ческий биологический возраст, большой объем облучения плоских костей в анамнезе,
неудовлетворительные показатели функции костного мозга, почек, печени. При хорошей ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии MALIGNANT TUMOURS
Russian Society of Clinical Oncology том / vol. 12 № 3s2 • 2022 2.4.1. Обеспечение венозного доступа Так как большинство противоопухолевых лекарственных препаратов вводится в / в,
необходимо обеспечение максимального удобства и комфорта пациентам. При хорошем
состоянии периферических вен и коротких (менее 12 часов) в / в инфузиях возможно про-
ведение лечения через периферические венозные катетеры. Для проведения режимов
при затрудненном периферическом венозном доступе, а также при длительных (> 12 часов)
инфузиях препарата необходимо обеспечение центрального венозного доступа, что может
быть достигнуто различными путями: том / vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии том / vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии MALIGNANT TUMOURS
Russian Society of Clinical Oncology ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии ОБЩИЕ ПРИНЦИПЫ ПРОТИВООПУХОЛЕВОЙ ТЕРАПИИ 35 Версия 2022 •
установкой периферически имплантируемого центрального венозного катетера
(ПИК-катетер), который подходит для краткосрочного и среднего по длительности
(от 6 дней до 6 мес.) использования; •
установкой периферически имплантируемого центрального венозного катетера
(ПИК-катетер), который подходит для краткосрочного и среднего по длительности
(от 6 дней до 6 мес.) использования; •
установкой полностью имплантируемой венозной порт системы, для последующей
работы с которой используется специальная игла Губера; является оптимальным
вариантом при длительных (от 3 мес.) курсах лечения; •
катетеризацией центральной вены на каждом курсе лечения (менее предпочтитель-
ный подход). •
катетеризацией центральной вены на каждом курсе лечения (менее предпочтитель-
ный подход). Проведение суточных и многосуточных инфузий требует специального оборудова-
ния, наиболее популярным является эластомерное инфузионное устройство одноразо-
вого применения, специально разработанное для планового введения лекарственных
препаратов с заданной скоростью и в определенный промежуток времени. Для ряда
препаратов (например, фторурацила) необходимо защищать от разрушения светом
инфузионные системы и линии. 2.5. Оценка эффективности лечения Плановая оценка эффективности ХТ выполняется каждые 6–12 недель (в зависимости
от вида опухоли и задач терапии), эндокринной и иммунотерапии — каждые 8–16 недель,
внеплановая оценка проводится по показаниям, в частности при подозрении на про-
грессирование болезни. Интервал в оценке эффективности лечения при проведении
адъювантной терапии раннего рака молочной железы может достигать 6 месяцев в связи
с низким риском развития рецидива. Ориентиром для оценки эффекта лечения могут служить международные крите-
рии оценки ответа солидных опухолей на терапию (Response Evaluation Criteria in Solid
Tumors (RECIST) последняя версия 1.1 2016 или iRECIST при использовании иммуно-
терапевтических препаратов), разработанные исходно для клинических исследований
и основанные на определении размера опухоли методами лучевой диагностики (КТ,
МРТ). На практике для оценки эффективности лечения допустимо применение методов
ультразвуковой диагностики, планарной рентгенографии, эндоскопии и объективного
физикального обследования. Для получения достоверных данных о динамике процесса
необходимо использовать один и тот же диагностический метод, выбранный до начала
терапии и позволяющий качественно визуализировать опухолевые очаги, с подробным
описанием выявленных изменений в процессе лечения. том / vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии 2.5.1. Критерии RECIST 1.1 Современными критериями ответа опухоли на терапию являются критерии
RECIST 1.1 от 2009 г. Основу оценки составляет определение размера опухоли с помощью
методов лучевой диагностики (рентгенографии, КТ, МРТ) (см. «Методические рекомен-
дации по применению критериев ответа солидных опухолей на химиотерапевтическое
лечение (RECIST 1.1). Правительство Москвы, Департамент здравоохранения г. Москвы, ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии
MALIGNANT TUMOURS
Russian Society of Clinical Oncology
том / vol. 12 № 3s2 • 2022 том / vol. 12 № 3s2 • 2022 том / vol. 12 № 3s2 • 2022 MALIGNANT TUMOURS
Russian Society of Clinical Oncology ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии MALIGNANT TUMOURS
Russian Society of Clinical Oncology Практические рекомендации 36 Практические рекомендации ГБУЗ «Научно-практический центр медицинской радиологии Департамента здраво-
охранения г. Москвы», Москва 2018 г.). Опухолевые очаги В соответствии с критериями RECIST опухолевые очаги подразделяются на два типа: 1. Измеряемые очаги — очаги, выявляемые и составляющие по наибольшему диаметру:
•
≥ 10 мм при спиральной КТ при реконструкции срезов толщиной в 5 мм;
•
≥ 20 мм при шаговой КТ с толщиной среза 10 мм;
•
≥ 20 мм при рентгенографии грудной клетки;
•
лимфатические узлы > 15 мм в измерении, перпендикулярном максимальному
диаметру (т. е. по короткой оси). 2. Неизмеряемые очаги — очаги, выявляемые и составляющие по наибольшему диа-
метру при шаговом КТ < 20 мм, а при спиральной КТ < 10 мм. К неизмеряемым очагам
относятся: мелкие очаги (< 10 мм), лептоменингеальные метастазы, плеврит, асцит,
перикардит, кисты, лимфогенный канцероматоз кожи или легких, метастазы в костях,
воспалительные изменения молочных желез, очаги с кистозной и некротической
трансформацией, увеличение размеров органов (гепатоспленомегалия), перитоне-
альные импланты. Алгоритм оценки 1. В первую очередь измеряемые очаги оцениваются количественно. 1. В первую очередь измеряемые очаги оцениваются количественно. 2. Далее нужно выбрать таргетные очаги, которые в процессе терапии будут исполь-
зоваться для контроля; таргетный очаг сохраняется на протяжении всех повтор-
ных исследований таргетным, даже если размеры очага уменьшатся до предела
погрешности метода исследования. В качестве таргетного рекомендуется выбирать не наибольший, а наиболее четко
визуализируемый и оптимальный для измерения очаг. В качестве таргетного рекомендуется выбирать не наибольший, а наиболее четко
визуализируемый и оптимальный для измерения очаг. MALIGNANT TUMOURS
Russian Society of Clinical Oncology ТА РГЕ ТНЫЕ ОЧ А ГИ •
Измеряемые очаги, максимально до 2 очагов на орган, в сумме — до 5 очагов;
очаги, отражающие пораженный орган, должны соответствовать критериям
таргетного очага; •
Измеряемые очаги, максимально до 2 очагов на орган, в сумме — до 5 очагов;
очаги, отражающие пораженный орган, должны соответствовать критериям
таргетного очага; •
выбираются, исходя из их размеров (с наибольшим диаметром) и пригодности
для точного повторного измерения; •
выбираются, исходя из их размеров (с наибольшим диаметром) и пригодности
для точного повторного измерения; •
очаги в подвижных органах (например, в органах ЖКТ, яичниках) могут быть непод-
ходящими для оценки; •
очаги в подвижных органах (например, в органах ЖКТ, яичниках) могут быть непод-
ходящими для оценки; •
указываются только аксиальные размеры. •
указываются только аксиальные размеры. том / vol. 12 № 3s2 • 2022 ОБЩИЕ ПРИНЦИПЫ ПРОТИВООПУХОЛЕВОЙ ТЕРАПИИ 37 Версия 2022 НЕ ТА РГЕ ТНЫЕ ОЧ А ГИ •
Могут включать как измеряемые, так и неизмеряемые очаги; у
р
,
р
;
•
к измеряемым нетаргетным очагам относят все измеряемые очаги в данном органе,
которые не вошли в число таргетных в связи с превышением максимально допусти-
мого количества (до 2 очагов на орган, всего — не более 5); •
нетаргетные очаги могут не соответствовать критериям измеряемых, т. е. быть
не измеряемыми; •
нетаргетные очаги могут не соответствовать критериям измеряемых, т. е. быть
не измеряемыми; р
•
в идеале должны включать все очаги, не отнесенные к категории таргетны ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии Критерии оценки рии оценки
тичный ответ — уменьшение суммы диаметров таргетных очагов на ≥ 30 %. й
(
)
б й ый ответ — уменьшение суммы диаметров таргетных очагов на ≥ 30 %. •
Частичный ответ — уменьшение суммы диаметров таргетных очагов на ≥ 3 •
Полный ответ — исчезновение всех (таргетных и нетаргетных) очагов; любой из ранее
увеличенных лимфатических узлов должен иметь размер < 10 мм по короткой оси. •
Прогрессирование заболевания — увеличение на ≥ 20 % суммы диаметров таргетных
очагов; появление одного или нескольких новых очагов; безусловная прогрессия
нетаргетных очагов. •
Прогрессирование заболевания — увеличение на ≥ 20 % суммы диаметров таргетных
очагов; появление одного или нескольких новых очагов; безусловная прогрессия
нетаргетных очагов. •
Стабилизация заболевания — признаки, не соответствующие критериям полного
или частичного ответа, а также прогрессирования заболевания. Общий ответ
Таргетные очаги
Нетаргетные очаги
Новые очаги
Полный ответ
Полный ответ
Полный ответ
Нет
Частичный ответ
Полный ответ
Частичный ответ / ста-
билизация заболева-
ния
Нет
Частичный ответ
Частичный ответ
Отсутствие
прогрессирования
заболевания
Нет
Стабилизация
заболевания
Стабилизация
заболевания
Отсутствие
прогрессирования
заболевания
Нет
Прогрессирование
заболевания
Прогрессирование
заболевания
Любой ответ
Есть или нет
Прогрессирование
заболевания
Любой ответ
Прогрессирование
заболевания
Есть или нет
Прогрессирование
заболевания
Любой ответ
Любой ответ
Нет ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии MALIGNANT TUMOURS
Russian Society of Clinical Oncology 38 Практические рекомендации 2.6. Обеспечение безопасности лекарственной
противоопухолевой терапии том / vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии 2.6.1. Минимальные лабораторные и инструментальные
обследования при проведении противоопухолевой
лекарственной терапии крови ранее, чем через 7 дней после начала курса цитостатической терапии в отсутствии
клинических показаний. Рекомендуемая периодичность выполнения ЭКГ в процессе
химио-и / или таргетной терапии составляет 3–4 мес. 2.6.2. Мониторинг безопасности противоопухолевой
лекарственной терапии Оценка побочных эффектов терапии выполняется на каждом цикле. Общеприня-
той шкалой являются «Общие терминологические критерии нежелательных явлений»
(Common Terminology Criteria for Adverse Events (CTCAE), версия 5, 2017 г.). При развитии
клинически значимых побочных явлений (3–4 степени, за исключением гематологиче-
ской токсичности) выполняется редукция дозы на 20–40 %, если другое не предусмо-
трено в инструкции по применению препарата. При купировании клинически значимых
побочных явлений в отдельных ситуациях возможно возобновление лечения в полных
дозах, если другой подход не предлагается в инструкции по применению препарата. Для некоторых видов кумулятивной токсичности, например, легочной, нейротоксич-
ности клинически значимой может являться ≥ 2 степень выраженности. При проведении
паллиативной химиотерапии назначение схемы каждой из линий и изменения доз пре-
паратов определяются врачом-онкологом (специалистом по лекарственной терапии)
без привлечения консилиума. Следует иметь ввиду, что для дозолимитирующих кумулятивных видов токсичности,
таких как нейротоксичность, ототоксичность, легочная токсичность, кардиотоксич-
ность отсутствуют профилактические меры с доказанной клинической активностью. Поскольку кумулятивная токсичность развивается в процессе цикловой ХТ по мере
увеличения числа курсов, необходим целенаправленный и подробный сбор жалоб
пациента перед началом каждого цикла лечения. При выявлении клинических сим-
птомов, позволяющих заподозрить развитие кумулятивной токсичности, возможно
привлечение (при наличии необходимости) профильных специалистов и назначение
необходимых методов обследования и лечения с последующим мониторингом выра-
женности клинических симптомов побочного эффекта и повторными обследованиями
при нарастании их тяжести. 2.6.1. Минимальные лабораторные и инструментальные
обследования при проведении противоопухолевой
лекарственной терапии До начала первого курса лекарственной терапии рекомендуется выполнение про-
цедур минимального уровня обследования, перечень которых может быть расширен
по клиническим показаниям: •
клинический анализ крови (с определением уровня гемоглобина, эритроцитов,
лейкоцитов с подсчетом лейкоцитарной формулы, нейтрофилов, тромбоцитов);
выполняется не более чем за 14 дней до начала лечения; •
клинический анализ крови (с определением уровня гемоглобина, эритроцитов,
лейкоцитов с подсчетом лейкоцитарной формулы, нейтрофилов, тромбоцитов);
выполняется не более чем за 14 дней до начала лечения; •
биохимический анализ крови (с определением уровня глюкозы, общего билирубина,
АЛТ, АСТ, общего белка, креатинина); выполняется не более чем за 14 дней до начала
лечения; •
коагулограмма (фибриноген, АЧТВ, протромбиновое время); выполняется не более
чем за 30 дней до начала лечения; •
общеклинический анализ мочи (выполняется не более чем за 14 дней до начала
лечения); •
ЭКГ (выполняется не более чем за 30 дней до начала лечения);
•
по показаниям: –
эхоКГ с определением ФВлж (при планировании кардиотоксичной терапии
трастузумабом, пертузумабом, трастузумабом эмтанзином, антрациклинами); –
аудиометрия (при наличии / усугублении клинических симптомов снижения слуха
при планировании / проведении терапии цисплатином, но не карбоплатином
или оксалиплатином); –
консультации специалистов — по показаниям, определяемым лечащим врачом. Например, консультация эндокринолога или кардиолога — при необходимости
назначения или коррекции проводимой терапии в случае ее недостаточной
эффективности. –
по показаниям (выраженная общая слабость, удлинение интервала QT) — опре-
деление уровня магния у пациентов, получающих цетуксимаб или панитумумаб. –
по показаниям (выраженная общая слабость, удлинение интервала QT) — опре-
деление уровня магния у пациентов, получающих цетуксимаб или панитумумаб. В дальнейшем в процессе химио- или таргетной терапии перед началом каждого
курса должен быть выполнен общеклинический (выполняется не более чем за 5 дней
до проведения очередного курса) и биохимический анализы крови в вышеуказанном
объеме (выполняется не более чем за 7 дней до проведения очередного курса), при еже-
недельных режимах биохимический анализ крови может выполняться каждые 3–4 недели,
остальные анализы — по клиническим показаниям. При проведении ГТ, обладающей
минимальной токсичностью (антиэстрогены, ингибиторы ароматазы, агонисты ГРГ)
вышеуказанный лабораторный мониторинг допускается проводить реже (до 1 раза в два
месяца). Не рекомендуется рутинно выполнять контрольный общеклинический анализ том / vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии MALIGNANT TUMOURS
Russian Society of Clinical Oncology ОБЩИЕ ПРИНЦИПЫ ПРОТИВООПУХОЛЕВОЙ ТЕРАПИИ 39 Версия 2022 крови ранее, чем через 7 дней после начала курса цитостатической терапии в отсутствии
клинических показаний. Рекомендуемая периодичность выполнения ЭКГ в процессе
химио-и / или таргетной терапии составляет 3–4 мес. том / vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологи MALIGNANT TUMOURS
Russian Society of Clinical Oncology 2.6.3. Длительность лекарственной терапии Длительность неоадъювантной и адъювантной терапии устанавливается клиниче-
скими рекомендациями. При паллиативной и лечебной ХТ в некоторых случаях также существует четко опре-
деленное рекомендациями число циклов, превышение которого не улучшает отдаленные
результаты лечения (например, 4–6 циклов ХТ при немелкоклеточном раке легкого, 4 цикла
химиотерапии по схеме BEP при герминогенных опухолях, 16–18 недель индукционной
ХТ первой линии колоректального рака). В других случаях лечение может быть продол-
жено до прогрессирования заболевания, непереносимой токсичности или достижения
максимально допустимой кумулятивной дозы используемых препаратов, обладающих
кумулятивной токсичностью. том / vol. 12 № 3s2 • 2022 том / vol. 12 № 3s2 • 2022 MALIGNANT TUMOURS
Russian Society of Clinical Oncology ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии Практические рекомендации 40 Практические рекомендации При паллиативной ХТ важно оценивать соотношение пользы (эффекта) и вреда
(токсичности) от продолжения терапии. При достижении стабилизации заболевания
или максимального эффекта без дальнейшего уменьшения опухолевых очагов при ряде
опухолей рекомендуется провести еще 2–3 цикла ХТ с последующим наблюдением
или поддерживающей терапией. Таргетная терапия, ГТ и иммунотерапия обычно при пал-
лиативном лечении проводятся до прогрессирования заболевания или непереносимой
токсичности. ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии том / vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии том / vol. 12 № 3s2 • 2022 ЗЛОКАЧЕСТВЕННЫЕ ОПУХОЛИ
Российское общество клинической онкологии MALIGNANT TUMOURS
Russian Society of Clinical Oncology
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https://openalex.org/W2995302914
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Edible Films and Coatings Functionalization by Probiotic Incorporation: A Review
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Polymers
| 2,019
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Edible Films and Coatings Functionalization by
Probiotic Incorporation: A Review
Oana L Pop, Carmen R Pop, Marie Dufrechou, Dan C Vodnar, Sonia A
Socaci, Francisc V Dulf, Fabio Minervini, Ramona Suharoschi To cite this version: Oana L Pop, Carmen R Pop, Marie Dufrechou, Dan C Vodnar, Sonia A Socaci, et al.. Edible Films and
Coatings Functionalization by Probiotic Incorporation: A Review. Polymers, 2020, 12 (1), pp.1-15. 10.3390/polym12010012. hal-03326088 Distributed under a Creative Commons Attribution 4.0 International License Edible Films and Coatings Functionalization by
Probiotic Incorporation: A Review Oana L. Pop 1, Carmen R. Pop 1
, Marie Dufrechou 2, Dan C. Vodnar 1
, Sonia A. Socaci 1
,
Francisc V. Dulf 3
, Fabio Minervini 4,*
and Ramona Suharoschi 1,* Oana L. Pop 1, Carmen R. Pop 1
, Marie Dufrechou 2, Dan C. Vodnar 1
, Sonia A. Socaci 1
,
Francisc V. Dulf 3
, Fabio Minervini 4,*
and Ramona Suharoschi 1,* 1
Department of Food Science, University of Agricultural Sciences and Veterinary Medicine,
Calea Mănă¸stur 3-5, 400372 Cluj-Napoca, Romania; oanalelia.pop@gmail.com (O.L.C.);
carmen-rodica.pop@usamvcluj.ro (C.R.P.); dan.vodnar@usamvcluj.ro (D.C.V.);
sonia.socaci@usamvcluj.ro (S.A.S.) 1
Department of Food Science, University of Agricultural Sciences and Veterinary Medicine,
Calea Mănă¸stur 3-5, 400372 Cluj-Napoca, Romania; oanalelia.pop@gmail.com (O.L.C.);
carmen-rodica.pop@usamvcluj.ro (C.R.P.); dan.vodnar@usamvcluj.ro (D.C.V.);
sonia.socaci@usamvcluj.ro (S.A.S.) j
2
USC 1422 GRAPPE, INRA, Ecole Supérieur d’Agriculture, SFR 4207 QUASAV, 55 rue Rabelais, BP 30
4900 Agnes Cedex 01, France; m.dufrechou@groupe-esa.com 3
Department of Biochemistry, University of Agricultural Sciences and Veterinary Medicine,
Calea Mănă¸stur 3-5, 400372 Cluj-Napoca, Romania; francisc_dulf@yahoo.com
4
Department of Soil, Plant and Food Science, University of Bari Aldo Moro, 70121 Bari, Italy
*
Correspondence: fabio.minervini@uniba.it (F.M.); ramona.suharoschi@usamvcluj.ro (R.S.);
Tel.: +39-0805442946 (F.M.); +40-771636781 (R.S.) Received: 1 December 2019; Accepted: 17 December 2019; Published: 19 December 2019 Abstract: Edible coatings and films represent an alternative packaging system characterized by being
more environment- and customer-friendly than conventional systems of food protection. Research
on edible coatings requires multidisciplinary efforts by food engineers, biopolymer specialists and
biotechnologists. Entrapment of probiotic cells in edible films or coatings is a favorable approach that
may overcome the limitations linked with the use of bioactive compounds in or on food products. The recognition of several health advantages associated with probiotics ingestion is worldwide
accepted and well documented. Nevertheless, due to the low stability of probiotics in the food
processing steps, in the food matrices and in the gastrointestinal tract, this kind of encapsulation is of
high relevance. The development of new and functional edible packaging may lead to new functional
foods. This review will focus on edible coatings and films containing probiotic cells (obtaining
techniques, materials, characteristics, and applications) and the innovative entrapment techniques
use to obtained such packaging. Keywords: edible films; edible coatings; probiotics; functional food; antibacterial activity HAL Id: hal-03326088
https://hal.inrae.fr/hal-03326088v1
Submitted on 25 Aug 2021 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 Polymers 2020, 12, 12; doi:10.3390/polym12010012 1. Introduction Edible films or coatings (edible packaging or EP) are defined by any material meant to be applied
(wrapping or coating) to food in order to extend the shelf life and may be consumed together with the
food. Due to the many disadvantages related to plastic films and packaging, edible films have gained
popularity in the scientific world, and drawn the attention of authorities and consumers [1] concerned
about environmental protection. Indeed, conventional synthetic packages have a very damaging effect
on the environment [2]. EP, especially those containing microorganisms, can be considered as a living ecosystem that
selectively allows for the exchanges of respiration gases (e.g., oxygen, carbon dioxide and ethylene)
between food and the atmosphere, diminishes or prevents loss of moisture and aromas and/or protects
against undesired microorganisms [3]. Depending on the exact purpose, the film/coating can totally coat the food or can be applied
between food constituents [4]. The materials that are utilized for the edible films/coatings production Polymers 2020, 12, 12; doi:10.3390/polym12010012 www.mdpi.com/journal/polymers 2 of 15 Polymers 2020, 12, 12 are biopolymers, proteins, lipids or composites. Thus, even if they are not consumed with food, they
can be more rapidly and easily degraded with respect to plastic materials [5]. are biopolymers, proteins, lipids or composites. Thus, even if they are not consumed with food, they
can be more rapidly and easily degraded with respect to plastic materials [5]. The main difference between coating and film is in their preparation and application process. Indeed, edible films are usually obtained in parallel to food and then applied to the surface, whereas
coatings are directly prepared on food surface [6]. Both coatings and films can entrap live probiotic
microorganisms. g
Due to handling and hygienic limitations, EP can be combined with ecofriendly non-EP [6–8 The utilization of films for food preservation dates back to the 12th century in China, where
wax was utilized to delay moisture loss from fruits. Sixteen centuries ago, the first edible films made
from soymilk were used in Japan for fruits preservation and in order to obtain a shiny surface [9,10]. Due to the narrow variety of materials used to protect fruits and vegetables at that time, no big interest
was shown to this type of package. Refrigeration, controlled/modified atmosphere, heat or radiation
sterilization, smoking have ever received stronger attention than edible packaging. 1. Introduction Of course, food
conservation methods have considerably increased and have offered unlimited opportunities to prepare,
store and consume any type of food in any season. However, EP can currently be applied to a large
variety of food products, with unique, tailored and innovative ways of action than conventional food
preservation techniques [1]. Among various roles played by EP, physical protection [11] amplification and protection of food
properties, carriers of food additives and prolongation of shelf life are the most important ones. EP may be categorized according to the class of their constituent material. Hydrocolloids
(polysaccharides and proteins) and lipids are the most used materials. Among these, polysaccharides
are the easiest to purchase and more suitable to form films or coatings. The presence of a large number of
hydroxyl groups and hydrogen bonds favor the formation of film. Different properties can be observed
between films and coatings made of negatively charged gums (i.e., alginate, pectin, or carboxymethyl
cellulose) [7]. Proteins used for EP have mostly animal origin (gelatin, casein, whey proteins, collagen or egg
albumin). However, plant-derived proteins (e.g., corn, soybean, wheat, cottonseed, peanut, and rice)
are also appreciated and compatible with the vegetarian diet. The film/coating forming process is
started, in most of the cases, with protein denaturation using heat or pH adjustment, followed by a
conglomeration of peptide chains through new intermolecular interactions [12]. These type of films
are suitable mainly for meat products, due to their affinity to hydrophilic surfaces [13–15]. Lipids do not form cohesive films, unlike hydrocolloids. For this reason, they are used especially
for coatings or in mixture with polysaccharides in order to obtain an optimized water vapor barrier [16]. Lipids do not form cohesive films, unlike hydrocolloids. For this reason, they are used especially
for coatings or in mixture with polysaccharides in order to obtain an optimized water vapor barrier [16]. The integration of different additives (i.e., probiotic microorganisms but also organic acids [17],
essential oils [18], plant extracts [19], and antibacterial compounds [20]) into the EP has the benefit The integration of different additives (i.e., probiotic microorganisms but also organic acids [17],
essential oils [18], plant extracts [19], and antibacterial compounds [20]) into the EP has the benefit
of ensuring slower release of these compounds to food [21]. The aim of this paper is to review the
application of various types of natural EP that incorporates live probiotic microorganisms. 3. Probiotics in Food and Human Health Nowadays, probiotics are associated with a world that lives on and inside humans and animals,
modulating the host’s health [28,29]. The amount of bacteria that can be found in the human body
exceeds the number of human cells by more than ten times. Due to the great impact of the gut microbiota
on the human body and its health modulation, the human gastrointestinal network is also called “the
other brain” [30]. The human gut hosts about 1500 bacterial species, of which about 500 species have
pathogenic or probiotic traits. The gut’s influence on health, exerted by microorganisms inhabiting human body (especially
some sections of the gut), starts in the womb of mother, depends on the child’s delivery (C-section or
vaginal) modality, milk (breast-fed or artificial milk) ingested by newborn, and afterwards, is mostly
modulated by diet. Other genetic and epigenetic factors, as well as environmental drivers (geographic
location, stress, physical activity, and drug intake), further modulate the balance in the gut microbiota. While being relatively stable in adulthood, during aging, the gut microbiota composition continuously
changes [31]. In elderly individuals, the frequently observed decrease in the Bacteroidetes/Firmicutes
ratio is correlated with functionality decline of the immune system. Any modification in the diversity of the gut microbiota (dysbiosis) may result in the onset of
certain illnesses and dysfunctions. The use of probiotic supplements is a possible, cost-effective and
easy-to-use solution to counteract dysbiosis and face the pressing issue of microorganisms capable of
resisting multiple antibiotics [32]. The current definition of probiotic microorganism underlines it as a viable, single or mixed,
culture of bacteria or yeast which beneficially impact animal or human health when ingested in the
adequate amount [33]. Members of the genera Bifidobacterium and Lactobacillus are consistently used
for their probiotic effect, whereas members of the genera Streptococcus and Enterococcus contain
several opportunistic pathogens [34,35]. Some yeasts, mostly Saccharomyces boulardii, are accepted for
use as probiotics. Probiotics help to prevent or, in some cases, treat diarrhea, ulcerative colitis, irritable bowel
syndrome, allergies, obesity, and diabetes [36,37]. Several modes of action are well-known for probiotics;
for instance, they are able to modulate nutrients absorption [38], act as a barrier against pathogenic
bacteria at the level of intestinal mucosa [39], have an impact on the immune system [40], and
influence the gut–brain axis [41]. 3. Probiotics in Food and Human Health Some mechanisms of action exerted by probiotic microorganisms
are mediated by their metabolites, such as molecules with antimicrobial activity (e.g., organic acids,
ethanol, hydrogen peroxide, and bacteriocins) and short chain fatty acids that are used by enterocytes
as nutrients [42]. 2. Bioactive Molecules in EP and Perspectives in Food Industry EP may be used not only for their protective effect but as carriers of bioactive substances too. Some
examples of bioactive molecules are: antimicrobial compounds, probiotics, anti-browning compounds,
omega-3 fatty acids, and other nutraceuticals [22]. Active food packaging that incorporates bioactive
molecules not only acts as a traditional protective system for the food product, but also promotes the
health of consumers. Even more, the utilization of byproducts in order to obtain the edible package or to extract a
bioactive molecule that will be further incorporated into the package will sustain the economical
approach [23]. Obviously, when compared with fresh fruits and vegetables, the utilization of agricultural
byproducts, like fruit peels to prepare edible films, seems much more profitable from the perspective
of resource recycling and environmental protection, and needs further study [24]. 3 of 15 Polymers 2020, 12, 12 The utilization of EP that contain bioactive molecules have multiple advantages [25]. One example
of EP that can sustain human health and influence the final product are fried food products. A decrease
of oil intake in deep-fried food products represents a significant application of EP, namely coatings. Obesity and heart diseases have been linked to an excess of fat in food. The use of film made of
methylcellulose and hydroxypropyl/methylcellulose allows for a decrease in oil absorption by food,
thus helping to reduce fat intake [26]. The processing techniques used to obtain EP vary depending on the material and bioactive
compounds added in the EP [27]. Legislation, polymer types, active molecules, destination and are all
factors that influence the matrix choice. 4. Entrapped Probiotics in EP In order to benefit from the consumption of probiotics, a dose of 108–9 viable cells per day is
recommended. In many products, to reach this dose is challenging due to high sensibility of probiotics
to environmental conditions. Survival of probiotics depends on strain, food characteristics (e.g., pH),
processing technologies, storage conditions and time [43]. Biological activities of probiotic bacteria and
yeasts can be negatively affected by their loss of viability during food processing and storage. The use 4 of 15 Polymers 2020, 12, 12 of encapsulated probiotics in edible films or coatings could favor the optimal survival of beneficial
microorganisms in food. EP that incorporate probiotics display, besides those characteristics that are
peculiar to all the EP, features specifically addressed to maintain the host in good health (Figure 1). In addition, since probiotic microorganisms often showed inhibitory activity towards spoilage or
pathogenic bacteria, their incorporation in EP can increase food stability and safety. Food packaged in
coatings or films containing probiotics may be regarded as functional food, a special group of food
items that, if regularly introduced in diet, benefit health, beyond their nutritional value [44]. Polymers 2019, 11, x FOR PEER REVIEW
a special group of food items that, if regularly introduced in diet, benefit health, beyond
nutritional value [44]. Figure 1 Characteristics of edible packaging (EP) containing probiotics and some of the most st
Figure 1. Characteristics of edible packaging (EP) containing probiotics and some of the most studied
healthy effects exerted by probiotics. Figure 1 Characteristics of edible packaging (EP) containing probiotics and some of the most s
Figure 1. Characteristics of edible packaging (EP) containing probiotics and some of the most studied
healthy effects exerted by probiotics. healthy effects exerted by probiotics. E
l ti
f
bi ti
i
EP
b
bt i
d
i
d
i
[45]
ith
Encapsulation of probiotics in EP may be obtained using spray drying [45] with or without
protectants, spray freeze-drying or electrospray, and cross-linking gelation. healthy effects exerted by probiotics. E
l
i
f
bi
i
i
EP
b
b
i
d
i
d
i
[45]
i h
Encapsulation of probiotics in EP may be obtained using spray drying [45] with or without
protectants, spray freeze-drying or electrospray, and cross-linking gelation. Encapsulation of probiotics in EP may be obtained using spray drying [45] with or w
protectants, spray freeze-drying or electrospray, and cross-linking gelation. 4. Entrapped Probiotics in EP The addition of probiotics to EP is much less frequent than the addition of plant ex
Nowadays, this technology allows widening the range of probiotic-carrier food products that v
probiotics, satisfying the demand for nondairy foods, fostered by vegan consumers and la
intolerant people [40]. In one of the first research studies about the encapsulation of probiotics
Bifidobacterium animalis subsp. lactis BB-12 was entrapped in alginate and gellan-based edible co
of apple and papaya slices. The addition of BB-12 seemed to cause an increase in the space be
the polymer chains. During 10 days of storage at 2 °C, the cell density of the strain was
i i
d d (106 C l
F
i
U it /
CFU/ ) [46 47] H
th
The addition of probiotics to EP is much less frequent than the addition of plant extracts. Nowadays,
this technology allows widening the range of probiotic-carrier food products that vehicle probiotics,
satisfying the demand for nondairy foods, fostered by vegan consumers and lactose-intolerant people [40]. In one of the first research studies about the encapsulation of probiotics in EP, Bifidobacterium animalis
subsp. lactis BB-12 was entrapped in alginate and gellan-based edible coatings of apple and papaya
slices. The addition of BB-12 seemed to cause an increase in the space between the polymer chains. During 10 days of storage at 2 ◦C, the cell density of the strain was above minimum recommended
(106 Colony-Forming Units/g or CFU/g) [46,47]. However, the coating containing the probiotic strain
showed higher (50%) water vapor permeability than the control coating [46]. minimum recommended (106 Colony-Forming Units/g or CFU/g) [46, 47]. However, the c
containing the probiotic strain showed higher (50%) water vapor permeability than the c
coating [46]. EP containing probiotics could be exploited to overcome the otherwise unavoidable l
viability of beneficial microorganisms during food processes carried out at high tempera
Microcapsules containing a probiotic strain of L. acidophilus were dispersed (1% or 2%) in a
(5%) solution which covered the surface of bread dough [48] This technology allowed L acido
EP containing probiotics could be exploited to overcome the otherwise unavoidable loss of viability
of beneficial microorganisms during food processes carried out at high temperatures. Microcapsules
containing a probiotic strain of L. acidophilus were dispersed (1% or 2%) in a starch (5%) solution, which
covered the surface of bread dough [48]. This technology allowed L. 4. Entrapped Probiotics in EP acidophilus to keep its viability
after baking, without any negative impact on the taste of bread and texture properties of the crumb. In addition, the edible coating reduced bread crust crispness [48]. (
)
,
to keep its viability after baking, without an
5. Materials and Techniques Used for Probiotic EP Due to the materials
due to the incorporated active molecules, the EP are meant to protect the food or just to act as
er for the active compounds, to reduce contamination, to improve/maintain the food product
l appearance, to enhance the mechanical properties of fragile food products or to boost the
ance and flavor (Figure 2). Figure 2. Edible films and coatings components and roles. Figure 2. Edible films and coatings components and roles. 2. Edible films and coatings components and roles. Figure 2. Edible films and coatings components and roles. iverse biocompatible components, such as hydrocolloids, lipids and composites, are used in
paration [50]. According to their specific purpose, miscellaneous compounds may be exploited
trapment of probiotics in EP are miscellaneous In these cases the package is defined as
Diverse biocompatible components, such as hydrocolloids, lipids and composites, are used in EP
preparation [50]. According to their specific purpose, miscellaneous compounds may be exploited for
entrapment of probiotics in EP are miscellaneous. In these cases, the package is defined as composite [5,16]. trapment of probiotics in EP are miscellaneous. In these cases, the package is defined as
site [5, 16]. ydrocolloids include polysaccharides and proteins. Among polysaccharides, cellulose and its
tives, dextran, inulin, alginate, carrageenan, starch derivatives, pectin derivatives, chitosan,
ed extracts, and galactomannan are the most utilized for edible films and packages [14, 24, 51]. lysaccharides successfully protect food from oxygen, odor, and oil absorption; on the other
they show high water permeability [49] In subsequent paragraphs, a concise presentation of
Hydrocolloids include polysaccharides and proteins. Among polysaccharides, cellulose and its
derivatives, dextran, inulin, alginate, carrageenan, starch derivatives, pectin derivatives, chitosan,
seaweed extracts, and galactomannan are the most utilized for edible films and packages [14,24,51]. All polysaccharides successfully protect food from oxygen, odor, and oil absorption; on the other hand,
they show high water permeability [49]. In subsequent paragraphs, a concise presentation of the most
utilized materials is made: they show high water permeability [49]. In subsequent paragraphs, a concise presentation of
st utilized materials is made:
ellulose and cellulose derivate (e.g., methylcellulose and hydroxypropyl methylcellulose)
nt oil absorption from fried food items [52] and have been successfully used for EP-containing
tics [53-55]. Alginic acid, also known as alginate, may be conveniently applied to meat
cts, where it considerably delays lipid oxidation [56, 57] [58]. (
)
,
to keep its viability after baking, without an
5. Materials and Techniques Used for Probiotic EP properties of the crumb. In addition, the edible coating reduced bread crust crispness [48]. 5. Materials and Techniques Used for Probiotic EP
Commonly, EP are expected to be transparent, flavorless and unable of modifying the se
properties of food products. However, some applications (e.g., sushi wraps) may require sp
e
o y
o e tie
a
la k of e olutio
of
e ati e o
a ole ti
ha a te i ti
EP a e u
Commonly, EP are expected to be transparent, flavorless and unable of modifying the sensory
properties of food products. However, some applications (e.g., sushi wraps) may require specific sensory
properties as lack of evolution of negative organoleptic characteristics. EP are usually composed of two
major components: (i) a macromolecule-based substance, biopolymers, (ii) additives as plasticizers,
cross-linkers, nanoreinforcements and (iii) precursors as proteins, polysaccharides, lipids or resins 5 of 15 Polymers 2020, 12, 12 (Figure 2). The macromolecule-based substance represents the base that, dissolved in a solvent (usually
water), forms a cohesive assembly. The plasticizer is added in order to improve mechanical properties
of package (e.g., elasticity, toughness, resistance to tearing), so that the package gains flexibility and
higher stability [14]. Plasticizers, such as sorbitol, polyethylene glycol, glycerol and sucrose, are
commonly needed when the package is composed of proteins and polysaccharides. In some cases,
emulsifiers are used, instead of plasticizers, in order to increase the stability of film/coatings, made of
lipids and polysaccharides [49]. Due to the materials and/or due to the incorporated active molecules,
the EP are meant to protect the food or just to act as a carrier for the active compounds, to reduce
contamination, to improve/maintain the food product natural appearance, to enhance the mechanical
properties of fragile food products or to boost the appearance and flavor (Figure 2). 2019, 11, x FOR PEER REVIEW
5 of 18
hylene glycol, glycerol and sucrose, are commonly needed when the package is composed of
ns and polysaccharides. In some cases, emulsifiers are used, instead of plasticizers, in order to
se the stability of film/coatings, made of lipids and polysaccharides [49]. (
)
,
to keep its viability after baking, without an
5. Materials and Techniques Used for Probiotic EP Chitosan is obtained from chitin
ylation and is usually obtained from the exoskeleton of crustaceans and fungal cell walls [59]. acetylation process influences the chitosan molecular weight and, in turns its properties (i.e.,
linity, hydrophobicity, degradation, tensile strength and moisture content) [60, 61]. Chitosan
antimicrobial properties [62, 63]. Starch and its derivatives are cost-effective and easy to
. In addition, they are typically clear, inodorous and insipid [64, 65]. The starch films and
g characteristics are strongly influenced by the amylose/amilopectin ratio. A strong and flexible
obtained from a starch rich in amylose content [66]. Pectin, frequently utilized in jams and
Cellulose and cellulose derivate (e.g., methylcellulose and hydroxypropyl methylcellulose)
prevent oil absorption from fried food items [52] and have been successfully used for EP-containing
probiotics [53–55]. Alginic acid, also known as alginate, may be conveniently applied to meat
products, where it considerably delays lipid oxidation [56–58]. Chitosan is obtained from chitin
deacetylation and is usually obtained from the exoskeleton of crustaceans and fungal cell walls [59]. The deacetylation process influences the chitosan molecular weight and, in turns its properties
(i.e., crystallinity, hydrophobicity, degradation, tensile strength and moisture content) [60,61]. Chitosan
shows antimicrobial properties [62,63]. Starch and its derivatives are cost-effective and easy to handle. In addition, they are typically clear, inodorous and insipid [64,65]. The starch films and coating
characteristics are strongly influenced by the amylose/amilopectin ratio. A strong and flexible film is
obtained from a starch rich in amylose content [66]. Pectin, frequently utilized in jams and jellies, was
used to produce films and coatings containing probiotics [67,68]. Polymers 2020, 12, 12 6 of 15 Proteins are dissolved or dispersed in solvents (i.e., water or ethanol) that are further evaporated
in order to obtain the package. The protein-based structure forming process is favored by heat or acid
conditions [69,70]. Compared to polysaccharides, proteins have lower vapor permeability. Lipids may be commonly utilized in the form of waxes, oils, fats, and resins for building
EP-entrapping probiotics [71]. Since they are characterized by a high resistance to water penetration,
in most cases, the lipids are combined with polysaccharides or proteins [72]. Table 1 summarizes some of the types of composite used in order to obtain EP, the materials and
the designated food products with some generic and specific effects. (
)
,
to keep its viability after baking, without an
5. Materials and Techniques Used for Probiotic EP The materials used for EP may be derived from food industry byproducts, such as whey, corn zein
(source of proteins), mung bean or fruit pomace (source of pectin). This represents an environmentally
friendly solution and assists in cost reduction. Nevertheless, the utilization of food byproducts in EP
could signal consumer mistrust due to confusion between byproducts and wastes. Extensive applications of mentioned materials has been literally obstructed by some difficulties
in the material preparation process [73,74]. Most of these difficulties are related to the solubility of
the materials in solvents that are accepted in food industry. However, scientists innovate in order to
obtain best properties of the EP. An edible biocomposite film was proposed to be obtained directly
from psyllium seed, but it was proven that the utilization of seeds husk and husk flour was more
suitable [75]. In general, lipids are difficult to apply on the surface of some foods due to their poor
adhesion to food products with hydrophilic surfaces [76]. Chitosan can ensure many benefits, such as
excellent hydrophilicity, high porosity, big adhesion area, and can be cross-linked to avoid dissolution
in acidic solutions (pH < 2). The use of chitosan as material for the entrapment of probiotics has
been widely studied, but the too-soft texture and similarities between the density of the EP and that
of water (leading to easy float) limits its industrial function. Therefore, efforts have been made to
support the structure through the addition of activated clay and crosslinking with glutaraldehyde,
which has been demonstrated to permit superior operational stability. However, these alternatives
are not suitable for the food industry. Nevertheless, more studies regarding the challenges in the
materials preparation process need to be conducted in order to smooth the processes and sustain this
environmentally friendly method [77]. In order to sustain the applicability of probiotic EP in the food industry at an industrial scale, new
and innovative techniques need to be developed. Nanotechnology and the utilization of nanomaterials
is a promising area that can broaden the use of probiotic EP. Formulation of non-nanomaterials in
nanosized structures can bring enormous benefits due to the new and unique obtained bioactive
properties [78]. The utilization of electrospinning in the preparation of EP materials can be a suitable
technique for the restructuration of biopolymers in nanoscale. 7 of 15 Polymers 2020, 12, 12 Table 1. (
)
,
to keep its viability after baking, without an
5. Materials and Techniques Used for Probiotic EP Some polysaccharides, proteins, lipids, and composites-based EP for different food products with their generic and specific functions. Materials/Methods
Generic Effects
Specific Composition
Type of Food
Specific Effects
Reference
Polysaccharides
Starch
+ colorless
+ oil-free appearance
+ reduced caloric content
+ prolong shelf life
+ suitable for fruits, vegetables, meat
+ control oxygen transmission
+ reduce darkening of the surface
- no moisture barrier
- hydrophilic nature
Starch-based coatings with D-glucose, silver nitrate. Chicken Sausages
Antimicrobial activity. [79]
Cellulose and derivatives
Hydroxypropyl methylcellulose (HPMC) and
beeswax coatings. Cherry tomatoes
Prevent weight loss, sustain fruit firmness,
improved sensory attributes. [80]
Pectin
Pectin and sodium alginate coatings with citral and eugenol
essential oils. Raspberries
Maintain the color, prevent weight loss, trolox
equivalent antioxidant capacity, prevent
microbial growth. [81]
Pullulan
Pullulan-based coatings with sweet basil extract. Apples
Sustain color, appearance and sensory attributes
during hypothermia storage. [82]
Alginates
Alginate-chitosan and ZnO nanoparticle
Guavas
Increase the shelf-life of the fruit. [83]
Chitosan
Chitosan-based coatings with vacuum packaging. Beef
Effects on color preservation and lipid oxidation
during retail presentation. [84]
Proteins
Vegetable-based proteins
+ provide mechanical stability
+ good transparency
- not suitable for some diets (vegan)
Whey proteins coatings with lysozyme. Salmon
Overall quality of salmon. [85]
Gluten and zein coatings with potassium caseinate, rennet
casein, xanthan gum, locust bean additives. Trout Fillets
Sensorial attributes and the physical
biochemical qualities. [86]
Animal-based proteins
Caseinate-based coatings with ascorbic acid additives. Beef
Effect of gamma irradiation on microbiological
characteristics of ground beef. [87]
Furcellaran-gelatin-based edible coating. Salmon sushi
Exhibit good transparency, mechanical and
barrier properties and can be manufactured by
extrusion or casting processes. [88]
Fats
Oils
+ reduce water transmission
Lipid-based (sunflower oil and chocolate) coating with
stearic acid, polyglycerol. Apple slices
Moisture barrier. [89]
Waxes
Candelilla wax coating with ellagic acid. Avocado
Antifungal characteristics to enhance shelf life. [90]
Carnauba wax coating. Eggplant
Increase in the water vapor resistance and
reduction in weight loss. [91]
Candelilla wax coatings with mineral oil. Guava fruit
Weight loss ethylene emission, gloss, retention of
the color, firmness. [92]
Chitosan-Beeswax coating. Strawberries
Reduction in weight loss. [93]
Multicomponents/Composites
+ special tailored for specific
characteristics
+ enhance the permeability or
mechanical properties
- may get expensive
Composites of carrageenan and whey protein coatings with
CMC sodium salt, polyethylene glycol, calcium chloride,
glycerol and oxalic acid additives. Apples
Reduce brownness. [94]
Composite of chitosan and gelatin coatings. 6. Probiotics Viability in EP Probiotics may be used in pharmaceutical or food-based products [99,100]. The edible coating
or films may be regarded as a carrier of probiotics. The major challenge to be faced by probiotic
microorganisms is their resistance to entrapment, an essential prerequisite for their viability in the
final product. Only viable probiotics at adequate cell numbers can successfully colonize the colon. Some studies were specifically devoted to investigating the viability of probiotics entrapped in edible
coatings/films. Composition and storage temperature affect viability of probiotics in edible coatings/films. Pullulan is a polysaccharide that can be used as a base for EP. A pullulan-based film embedding probiotic
lactobacilli (L. reuteri ATCC (American Type Culture Collection) 55730, L. rhamnosus GG ATCC 53103,
and L. acidophilus DSM 20079) proved to sustain the viability of probiotics, during 10 and 20 days’
storage at room temperature at levels of 10.3 and 4.5 log CFU/mL, respectively. A similar film, but
containing a mixture of pullulan and potato starch, was less effective in maintaining the viability of
probiotic lactobacilli. In detail, the higher the starch content, the lower the probiotic viability. However,
when lower storage temperature (4 ◦C) was applied, no differences were found in terms of viability
loss between the pullulan- and pullulan/starch-based film. The viability loss did not exceed 10%
even after 30 days of storage [101]. Entrapment of L. rhamnosus GG in a film based on a mixture of
starch (from rice and corn) and proteins (bovine gelatin, sodium caseinate, and soy protein) resulted
in higher viability of the probiotic strains at refrigeration than at room temperature. The presence of
proteins increased viability of L. rhamnosus GG during the film formation process [102]. L. plantarum and
Kluyveromyces marxianus incorporated in a film composed of kefiran (a polysaccharide secreted by lactic
acid bacteria) and glycerol did not negatively affect the film optical and physical properties. Compared
to a suspension, both microorganisms showed better tolerance to acid conditions in the film and
maintained their viability through storage at room temperature. In addition, the yeast showed higher
resistance to the film-forming procedure than the lactic acid bacterium [103]. B. animalis subsp. lactis
BB-12 was incorporated in alginate and gellan (2% solutions) edible coatings and applied on fresh-cut
apple and papaya. Although a viability decrease of the probiotic higher than 85% was observed, BB-12
was maintained above the minimum recommended (106 CFU/g) [46]. (
)
,
to keep its viability after baking, without an
5. Materials and Techniques Used for Probiotic EP Red bell peppers
Improve firmness, diminish weight loss, and
ethanol concentration. [95]
Composite of hydroxypropyl methyl cellulose (HPMC) and
lipid coating with potassium sorbate, sodium benzoate,
sodium propionate, stearic acid, glycerol additives. Oranges
Antifungal properties improved during long-term
cold storage. [96]
Shellac, gelatin and Persian gum. Orange
Improve permeability characteristics. [97]
Hydroxypropyl methylcellulose-lipid composite
edible coatings. Citrus fruits
Maintain postharvest quality. [98] es-based EP for different food products with their generic and specific functions. 8 of 15 Polymers 2020, 12, 12 8. Antimicrobial Effects of Probiotics Incorporated in EP Besides their positive effects on human health, probiotic microorganisms incorporated in EP could
protect food from pathogenic bacteria, leading to increased food safety. They could also inhibit spoilage
microorganisms, thus extend the shelf-life of food. A probiotic strain belonging to Lactobacillus sakei
was embedded in a sodium caseinate-based film through either direct incorporation in the film-forming
suspension or by spraying on an already-formed film. The film, and its counterpart not containing
probiotic lactobacilli, were applied on plates of tryptic soy agar on fresh beef slices, which were
inoculated with Listeria monocytogenes. During four days of incubation on plates, the probiotic strain
increased of one log cycle its cell density. L. monocytogenes decreased (3.0–3.6 log cycles) during the
12 days of storage. In the beef slices stored at 4 ◦C for 21 days, L. sakei cell density was higher than
6 log CFU/cm2. In addition, the cell density of the pathogenic bacterium was two log cycles lower
than in the probiotic-free film [106]. In the presented study, it can be observed that the presence pf
probiotics from lactobacillus species negatively influenced the multiplication of L. monocytogenes on
the beef slices by producing bacteriocin-like substance. Thus, the production of this substance was
nonexistent after a long period of time. This fact can be explained by the death of lactobacillus as an
effect of the environmental conditions and lack of nutrients. A similar study that echoes the above-presented results is an alginate-based film containing
Carnobacterium maltaromaticum, a potential probiotic bacterium normally found as commensal of
various fish species [107,108], was applied on smoked salmon, inoculated with L. monocytogenes at
4 log CFU/cm2. This film had a bacteriostatic effect towards L. monocytogenes during 28 days of storage
at 4 ◦C [109]. The authors of the study declare that the antibacterial effect can be explained due to the
neutralized supernatant and therefore was not due to acidity or pH. A gelatin-based coating containing probiotic strains of L. acidophilus and Bifidobacterium bifidum was
applied to hake (Merluccius merluccius). Both probiotic strains maintained their initial level (109 CFU/mL)
of viability for 6 days of storage at 2 ◦C. The spoilage agent Shewanella putrefaciens, typically producer of
H2S, was found in coated hake at significantly lower counts than the uncoated hake. However, the
antibacterial effect had no relevant link to the presence of probiotics in the edible package. 7. Synbiotics in EP Probiotics may be combined with prebiotic compounds, i.e., substances capable of favoring
beneficial microbes in the human gut. The term “synbiotics” is used for indicating products containing
at least one probiotic microorganism and one prebiotic substance. Such products may help to maintain
the cell viability of probiotics and have been experimented inside edible films. The presence of
fructooligosaccharides (FOS) as prebiotic compounds in a methylcellulose-based film containing two
probiotic strains (L. delbrueckii subsp. bulgaricus CIDCA 333 and L. plantarum CIDCA 83114) was effective
in the protection of both probiotics. However, it had a negative effect in the film forming-process,
by reducing the glass transition temperature of the film [66]. Inulin, galacto-oligosaccharide and
FOS in chitosan-based film favored viability of probiotic Bifidobacterium infantis ATCC 15697 and
Lactobacillus fermentum ATCC 9398. Besides the prebiotic effect, the oligosaccharides increased the
extensibility of the film, compared to a prebiotic-free film [104]. Viability of L. rhamnosus GG was
monitored during time in a gelatin-based film added with inulin, polydextrose, gluco-oligosaccharides
and wheat dextrin. The presence of prebiotic compounds did not impair the film structure. Viability
loss was found regardless of the type of prebiotic compound, but especially with film containing
gluco-oligosaccharides (about 40%) or polydextrose (almost 85%). Among the tested prebiotics, inulin
allowed to maintain viability of the probiotic strain at acceptable level over 100 days of storage, whereas
in the film containing the other compounds an acceptable viability was maintained for a shorter time
(63–83 days) [102]. Thus, the limitations and difficulties in the utilization of pro- and prebiotics in EP formulations
need to be addresses, despite the fact that very few scientific papers discuss this aspect. The utilization
of prebiotics, together with the probiotics may lead to serious changes in the final properties of EP. 9 of 15 Polymers 2020, 12, 12 Ensuring low production costs is the main challenge for future EP process and formulation technologies. The exploitation of food-grade raw materials such as native, and physically or enzymatically processed
biopolymers, is one example of a method that has the potential to meet the challenge of widening
the range of EP types into which probiotic and prebiotic can be favorably incorporated [105]. Novel
developments for control release systems from the EP will also provide new possibilities. 7. Synbiotics in EP Negative
changes in the EP formulation, that are not affecting the characteristics of food products and ensure the
extension of shelf life (i.e., transparency, brightness, etc.) will be accepted by the consumers only if
they realize that the benefits related to the presence of prebiotics in the probiotic EP are greater than
the characteristic related to the food appearance. 8. Antimicrobial Effects of Probiotics Incorporated in EP Treatment of
coated hake through high hydrostatic pressure (200 MPa for 10 min at 20 ◦C) proved to be effective in
decreasing the spoiling agent, but had no effect on the viability of probiotics [98]. The ability of an agar-based film, incorporating green tea extract and two probiotic strains
(Lactobacillus paracasei L26 and B. animalis subsp. lactis B94), to inhibit two spoiling bacteria was
investigated in hake fillets. The spoiling agents, S. putrefaciens and Photobacterium phosphoreum were
deliberately added (103–104 CFU/g) to hake fillets, prior to film application. The results showed that
probiotic bacteria migrated from the film to fish and that fish wrapped in the film displayed lower
values of spoilage indicators compared to untreated fish (e.g., pH, count of H2S-producing bacteria,
concentration of trimethylamine). Overall, the use of this probiotic film extended the shelf life of hake
fillets for at least one week [110]. The type of material constituting the edible package affects probiotics viability and their
antimicrobial activity. A probiotic strain of L. plantarum was embedded in an edible film based
on sodium caseinate, pea protein, methylcellulose or hydroxymethylcellulose [54]. The probiotic strain 10 of 15 Polymers 2020, 12, 12 showed higher viability in protein than in cellulose-based film. Interestingly, when applied in the
cellulose-based film, L. plantarum produced higher levels of bacteriocin, resulting in the total inactivation
of Listeria innocua during 8 days of storage at refrigerated temperature [54]. When incorporated in sodium caseinate- or methylcellulose-based film, L. acidophilus displayed
higher viability than L. reuteri. After three days of storage, higher antilisterial activity was found for
the methylcellulose-based film than for the one made of sodium caseinate. Compared to similar films
without probiotic lactobacilli, Listeria sp. decreased by about 1.5 log cycle after 12 days of storage [111]. Alginate, whey proteins, or a mixture thereof were used for forming an edible coating containing
L. rhamnosus GG and was applied to bread [43]. During the two drying processes considered (60 ◦C
for 10 min, 180 ◦C for 2 min), the composite-based coating provided L. rhamnosus GG with higher
protection, with respect to alginate- or whey proteins-based coating. However, following simulated
gastrointestinal digestion, the highest cell density of L. rhamnosus GG (106 CFU/g) was found in the
bread coated with alginate [43]. g
The antibacterial activity of probiotics embedded in EP is limited due to the specific activity of the
probiotic metabolites. 8. Antimicrobial Effects of Probiotics Incorporated in EP This fact can explain why same probiotic strain act as antimicrobials against
certain pathogens and some have no influence. Nevertheless, as seen [54], the material used for the
incorporation of probiotics has a great impact regarding the antimicrobial activity of the probiotic
strain. This activity modulation can be correlated to the permeability of the EP for the antimicrobials
metabolites produced by the probiotic cells and by the material capacity to protect the active cells. 9. Concluding Remarks Nowadays, the increasing consciousness of consumers about the link between dietary habits and
health fosters the market of food containing probiotic microorganisms. EP technologies allow us to
broaden the fields of application of probiotics to unexplored food items (e.g., baked goods). Overall,
at an industrial scale, the number of applications of edible coatings/films containing probiotics is
much lower than that of research studies carried out in the laboratory. One of the major challenges
to be faced in order to achieve a wider industrial application is to obtain the perfect combination of
materials, technologies and probiotic strains, tailored to specific foods and consumers’ needs, and at
an acceptable cost. Another challenge is in the need to maintain a high cell density of probiotics during
the formation process of EP and, especially, after ingestion. This is a prerequisite to impact human
health positively. Future research efforts should be dedicated to these two challenges. In addition, a
higher number of studies about the health benefits of EP are essential. Author Contributions: O.L.P. researched studies and wrote the manuscript; C.R.P. researched studies and collected
data, M.D., D.C.V., S.A.S., F.V.D., provided ideas, discussion, corrected the manuscript, F.M. and R.S. revised and
corrected the manuscript and supervised this project. All authors have read and agreed to the published version
of the manuscript. Funding: The publication was supported by funds from the National Research Development Projects to fina
xcellence (PFE)-37/2018–2020 granted by the Romanian Ministry of Research and Innovation. Acknowledgments: We kindly thank Marian Rodion Pop for image processing. Conflicts of Interest: The authors declare no conflicts of interest. Conflicts of Interest: The authors declare no conflicts of interest. uilbert, S.; Gontard, N.; Cuq, B. Technology and applications of edible protective films. Packag. Technol. Sci.
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article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). References López de Lacey, A.M.; López-Caballero, M.E.; Montero, P. Agar films containing green tea extract and
probiotic bacteria for extending fish shelf-life. LWT 2014, 55, 559–564. [CrossRef] 110. López de Lacey, A.M.; López-Caballero, M.E.; Montero, P. Agar films containing green tea extract and
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films containing lactic acid bacteria. Food Control 2014, 35, 200–206. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Isolated Intracranial Extramedullary Hematopoiesis in an Adult Patient
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Journal of the Belgian Society of Radiology
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RAMAZAN ORKUN ONDER
ALPTEKIN TOSUN
TÜMAY BEKCI *Author affiliations can be found in the back matter of this article CORRESPONDING AUTHOR:
Ramazan Orkun Onder CORRESPONDING AUTHOR:
Ramazan Orkun Onder This article describes the case of a 56-year-old male patient who presented with a
headache and swelling on the left side of his head. Medical examinations, including
non-contrast computed tomography (CT) and contrast-enhanced magnetic
resonance imaging (MRI), revealed the presence of an expansile soft tissue lesion
in the frontoparietal bone structures, causing compression of the brain tissue. Further immunohistochemical analysis confirmed the diagnosis of extramedullary
hematopoiesis (EMH) in the bone lesion, with predominant involvement of the myeloid
series. EMH is a compensatory mechanism of the body to meet the demand for
erythropoiesis. While EMH is commonly associated with thalassemia in children and
myelofibrosis in adults, it can occur in various tissues throughout the body, including
the intracranial region, although it is rare. The imaging findings of EMH on MRI typically
show iso-hypointense signals on T1-weighted images and hypointense signals on
T2-weighted images due to the presence of hemosiderin. However, in this case, the
lesions appeared hyperintense on T2-weighted images, which posed a challenge in the
differential diagnosis. The article highlights the importance of clinical evaluation and
imaging findings in diagnosing EMH, as these lesions can resemble other conditions
such as metastases, angiomatous meningioma, chloroma, or epidural hematoma. Giresun University, Faculty of
Medicine, Turkey Giresun University, Faculty of
Medicine, Turkey orkunonder535@gmail.com IMAGES IN CLINICAL
RADIOLOGY
Isolated Intracranial
Extramedullary
Hematopoiesis in an Adult
Patient
RAMAZAN ORKUN ONDER
ALPTEKIN TOSUN
TÜMAY BEKCI
*Author affiliations can be found in the back matter of this article IMAGES IN CLINICAL
RADIOLOGY
Isolated Intracranial
Extramedullary
Hematopoiesis in an Adult
Patient
RAMAZAN ORKUN ONDER
ALPTEKIN TOSUN
TÜMAY BEKCI
*Author affiliations can be found in the back matter of this article KEYWORDS: intracranial extramedullary
hematopoiesis; metastases;
meningioma; chloroma;
epidural hematoma Isolated Intracranial
Extramedullary
Hematopoiesis in an Adult
Patient IMAGES IN CLINICAL
RADIOLOGY TO CITE THIS ARTICLE: TO CITE THIS ARTICLE:
Orkun Onder R, Tosun A,
Bekci T. Isolated Intracranial
Extramedullary Hematopoiesis
in an Adult Patient. Journal
of the Belgian Society of
Radiology. 2023; 107(1):
63, 1–3. DOI: https://doi. org/10.5334/jbsr.3268 Teaching Point: Isolated cases of intracranial EMH should be considered as a
differential diagnosis in adult patients as they may be radiologically confused with
malignant conditions such as metastases, angiomatous meningioma, chloroma or
epidural hematoma. Orkun Onder et al. Journal of the Belgian Society of Radiology DOI: 10.5334/jbsr.3268 2 CASE HISTORY dominance of the myeloid series. The patient underwent
a whole body CT scan and no other foci were found. A 56-year-old male patient presented with headache and
swelling on the left side of the head. His medical history
was unremarkable. Hemoglobin was 11.7 g/dL. Non-
contrast computed tomography (CT) of the brain showed
an expansile soft tissue lesion eroding the frontoparietal
bone structures extending subcutaneously from the
epidural space, and compressing the brain parenchyma
on the left (Figure 1). Magnetic resonance imaging
(MRI) showed that the lesion was hyperintense on T2-
weighted images (WI) (Figure 2A), iso- to hypointense
on T1WI (Figure 2B) and homogenously enhancing on
contrast imges (Figure 3; red arrow). Smaller similar
lesions were also observed in the ethmoid bone, orbit
and other calvarial bone structures. (Figure 3; green
arrows). Immunohistochemical examination confirmed
the diagnosis of extramedullary hematopoiesis (EMH) in
the frontoparietal bone lesion, characterized by diffuse
staining with myeloperoxidase and CD33, indicating Figure 3. in our case. These lesions are defined as extra-axial
masses that can potentially compress the underlying
neuroparenchyma and resemble conditions such as
metastases, angiomatous meningioma, chloroma or
epidural hematoma [1]. Alptekin Tosun
orcid.org/0000-0003-1783-9171
Giresun University, Faculty of Medicine, Department of
Radiology, Giresun, Turkey
Tümay Bekci
orcid.org/0000-0002-3147-2786
Giresun University, Faculty of Medicine, Department of
Radiology, Giresun, Turkey Alptekin Tosun
orcid.org/0000-0003-1783-9171
Giresun University, Faculty of Medicine, Department of
Radiology, Giresun, Turkey Alptekin Tosun
orcid.org/0000-0003-1783-9171
Giresun University, Faculty of Medicine, Department of
Radiology, Giresun, Turkey Tümay Bekci
orcid.org/0000-0002-3147-2786
Giresun University, Faculty of Medicine, Department of
Radiology, Giresun, Turkey AUTHOR AFFILIATIONS Ramazan Orkun Onder
orcid.org/0000-0003-0616-2564
Giresun University, Faculty of Medicine, Turkey Ramazan Orkun Onder
orcid.org/0000-0003-0616-2564
Giresun University, Faculty of Medicine, Turkey s12098-018-2618-z The authors have no competing interests to declare. 1. Merchant R, Choudhari AJ, Verma M, Patkar DP, Doctor
P. Intracranial Hematopoiesis in Beta Thalassemia: A Case
Series. Indian J Pediatr. 2018; 85(8): 679–681. Epub 2018
Jan 25. PMID: 29368108. DOI: https://doi.org/10.1007/ 1. Merchant R, Choudhari AJ, Verma M, Patkar DP, Doctor
P. Intracranial Hematopoiesis in Beta Thalassemia: A Case
Series. Indian J Pediatr. 2018; 85(8): 679–681. Epub 2018
Jan 25. PMID: 29368108. DOI: https://doi.org/10.1007/ TO CITE THIS ARTICLE: Orkun Onder R, Tosun A, Bekci T. Isolated Intracranial Extramedullary Hematopoiesis in an Adult Patient. Journal of the Belgian Society
of Radiology. 2023; 107(1): 63, 1–3. DOI: https://doi.org/10.5334/jbsr.3268 Submitted: 11 July 2023 Accepted: 28 July 2023 Published: 25 August 2023 COMMENTS EMH acts as the body’s compensatory mechanism to
meet the demand for erythropoiesis. Thalassaemia
primarily causes EMH in children, while myelofibrosis
is more common in adults. In addition to the bone
marrow, EMH can occur in a wide variety of tissue
and organss, including the liver, spleen, lymph nodes,
adrenal glands, thymus, pleura, pulmonary interstitium,
skin, gastrointestinal tract, paranasal sinuses, and even
the dura mater. Intracranial EMH is particularly rare. Clinical evaluation with imaging findings is typically
used for the diagnosis of EMH. On MRI, lesions exhibit
iso-hypointense signal on T1WI and hypointense
signals on T2WI due to the presence of hemosiderin. However, the lesions were slightly hyperintense on T2WI EMH acts as the body’s compensatory mechanism to
meet the demand for erythropoiesis. Thalassaemia
primarily causes EMH in children, while myelofibrosis
is more common in adults. In addition to the bone
marrow, EMH can occur in a wide variety of tissue
and organss, including the liver, spleen, lymph nodes,
adrenal glands, thymus, pleura, pulmonary interstitium,
skin, gastrointestinal tract, paranasal sinuses, and even
the dura mater. Intracranial EMH is particularly rare. Clinical evaluation with imaging findings is typically
used for the diagnosis of EMH. On MRI, lesions exhibit
iso-hypointense signal on T1WI and hypointense
signals on T2WI due to the presence of hemosiderin. However, the lesions were slightly hyperintense on T2WI Figure 1. Figure 2. Figure 1. Figure 1. Figure 2. Figure 2. Figure 2. 3 Orkun Onder et al. Journal of the Belgian Society of Radiology DOI: 10.5334/jbsr.3268 3 Figure 3. REFERENCE The authors have no competing interests to declare. The authors have no competing interests to declare. COPYRIGHT: © 2023 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0
International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original
author and source are credited. See http://creativecommons.org/licenses/by/4.0/. Journal of the Belgian Society of Radiology is a peer-reviewed open access journal published by Ubiquity Press.
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English
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A comparative analysis of the effects of sevoflurane and propofol on cerebral oxygenation during steep Trendelenburg position and pneumoperitoneum for robotic-assisted laparoscopic prostatectomy
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Journal of anesthesia
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J Anesth (2016) 30:949–955
DOI 10.1007/s00540-016-2241-y ORIGINAL ARTICLE ORIGINAL ARTICLE Keywords Robotic-assisted laparoscopic prostatectomy
(RALP) · Steep Trendelenburg position · Cerebral
oxygenation · Sevoflurane · Propofol Keywords Robotic-assisted laparoscopic prostatectomy
(RALP) · Steep Trendelenburg position · Cerebral
oxygenation · Sevoflurane · Propofol Keywords Robotic-assisted laparoscopic prostatectomy
(RALP) · Steep Trendelenburg position · Cerebral
oxygenation · Sevoflurane · Propofol 1
Department of Anesthesiology, School of Medicine, Iwate
Medical University, 19‑1 Uchimaru, Morioka 020‑0023,
Japan A comparative analysis of the effects of sevoflurane and propofol
on cerebral oxygenation during steep Trendelenburg position
and pneumoperitoneum for robotic‑assisted laparoscopic
prostatectomy Aya Doe1 · Motoi Kumagai1 · Yuichiro Tamura1 · Akira Sakai1 · Kenji Suzuki1 Received: 14 April 2016 / Accepted: 13 August 2016 / Published online: 26 August 2016
© The Author(s) 2016. This article is published with open access at Springerlink.com r2 = 0.065; p = 0.004) shows a weak relationship betw
SjO2 and SctO2. Conclusions Sevoflurane maintains higher SjO2 levels
propofol during RALP. SctO2 does not accurately re
SjO2. r2 = 0.065; p = 0.004) shows a weak relationship between
SjO2 and SctO2.l * Motoi Kumagai
bear@kxd.biglobe.ne.jp Abstract Purpose Steep Trendelenburg position and pneumoperito-
neum during robotic-assisted laparoscopic prostatectomy
(RALP) increase intracranial pressure (ICP) and may alter
cerebral blood flow (CBF) and oxygenation. Volatile anes-
thetics and propofol have different effects on ICP, CBF, and
cerebral metabolic rate and may have different impact on
cerebral oxygenation during RALP. In this study, we meas-
ured jugular venous bulb oxygenation (SjO2) and regional
oxygen saturation (SctO2) in patients undergoing RALP to
evaluate cerebral oxygenation and compared the effects of
sevoflurane and propofol. We also verified whether SctO2
may be an alternative to SjO2. Conclusions Sevoflurane maintains higher SjO2 levels than
propofol during RALP. SctO2 does not accurately reflect
SjO2. Introduction Robotic-assisted
laparoscopic
prostatectomy
(RALP)
requires a steep Trendelenburg position and pneumoperi-
toneum, both of which can increase intracranial pressure
(ICP) [1, 2]. Increased ICP, in turn, may decrease cerebral
perfusion pressure and cerebral oxygenation [3, 4]. Methods Fifty patients scheduled for RALP were ran-
domly assigned to undergo sevoflurane (group S) or propo-
fol (group P) anesthesia. SjO2, SctO2, mean arterial pres-
sure (MAP), heart rate (HR), cardiac index (CI), central
venous pressure (CVP), partial pressures of arterial oxygen
(PaO2) and carbon dioxide (PaCO2), hemoglobin concen-
tration (Hb), Bispectral Index (BIS) and nasopharyngeal
temperature (BT) were recorded 5 min before surgery com-
mencement, 5 min after pneumoperitoneum, 5, 30, 60, 90,
and 120 min after pneumoperitoneum in a Trendelenburg
position, and after desufflation in a supine position. Although sevoflurane and propofol are commonly used
anesthetics, they have different effects on cerebral blood
flow (CBF) and the cerebral metabolic rate for oxygen
(CMRO2). Volatile anesthetics primarily increase CBF via
local vasodilatation and reduce CMRO2. At low concentra-
tions, where the effect of CMRO2 is dominant, these anes-
thetics constrict cerebral vessels by suppressing CMRO2
and prevent an increase in the CBF and CBF/CMRO2 ratio. At the medium-to-high concentrations commonly used in
clinical settings, the direct vasodilatory effect becomes
dominant, causing CBF and the CBF/CMRO2 ratio to
increase. With sevoflurane, this effect is accompanied by an
increase in cerebral blood volume (CBV) and an elevated
ICP [5]. In RALP, where ICP is already increased, it is con-
ceivable that the CBV increase caused by volatile anesthet-
ics could further increase ICP and in turn lower the CBF/
CMRO2 ratio and cerebral oxygenation. Supporting this Results SjO2 was significantly higher in group S than in
group P at all measurement points [group S vs. group P:
77 % (11) vs. 65 % (13), mean of all measurement points
(1SD); p < 0.01]. Linear regression analysis (β = 0.106; 1 J Anesth (2016) 30:949–955 950 kg) with an oxygen fraction (FiO2) of 0.45. The respira-
tory rate was adjusted to maintain an end-tidal carbon diox-
ide tension (ETCO2) of 35–40 mmHg. In both groups, the
concentrations of sedative drugs were adjusted to achieve
a Bispectral Index (BIS) of 40–60. Sevoflurane was initi-
ated at concentrations of 2.0 % and adjusted above 1.7 %
(one minimum alveolar concentration; MAC), and propo-
fol was maintained by TCI. Introduction Also, remifentanil was adjusted
to maintain a mean arterial pressure (MAP) within 20 %
of the preinduction value in both groups. When the MAP
decreased to 80 % of the preinduction value, it was treated
with an 8-mg bolus of ephedrine. Patients not anesthe-
tized under these conditions were excluded from the final
analysis. hypothesis, one study showed cerebral oxygen desatura-
tion in a steep Trendelenburg position and pneumoperito-
neum with isoflurane anesthesia [4]. Propofol reduces CBF
by cerebral vasoconstriction and suppresses CMRO2. The
relationship between the vasoconstriction and CMRO2 sup-
pression effects of propofol is unclear; some studies have
reported approximate equivalency [6], and others have
reported that vasoconstriction is slightly dominant [7]. Propofol at least does not increase the CBF/CMRO2 ratio. Volatile anesthetics and propofol each have the potential to
decrease cerebral oxygenation during RALP. Patients undergoing RALP are typically elderly and
often have cerebrovascular disease that is associated with
impaired cerebral oxygenation. These are not only asso-
ciated with cerebral vasoconstriction or vasospasm dur-
ing surgery but also with postoperative cognitive decline
(POCD) [8]. Some studies suggest that the choice of anes-
thetic affects cerebral oxygenation and POCD [9, 10]. Thus, it is important to monitor cerebral oxygenation and
choose anesthetics that best preserve cerebral oxygenation. Routine monitoring including electrocardiography,
pulse oximetry, and non-invasive automated blood pres-
sure measurement, started on arrival in the operating room. After the induction of anesthesia, a radial artery catheter
was inserted for direct arterial blood pressure measurement
and blood sampling. The arterial line was also connected
to a FloTrac sensor and a third-generation Vigileo moni-
toring system (Edwards Lifesciences, Irvine, CA, USA) to
monitor the cardiac index (CI) data. The ETCO2 and end-
tidal concentration of sevoflurane were measured using
a Vamos anesthetic gas monitor (Dräger Japan, Tokyo,
Japan). Central venous pressure (CVP) was measured by a
single-lumen central venous catheter that was inserted into
the right internal jugular vein. A BIS electrode was applied
to the forehead and was measured continuously using an
Aspect BIS A2000 monitor v3.31 (Aspect Medical Sys-
tems, Natick, MA, USA). Nasopharyngeal temperature
(BT) was monitored continuously. Jugular venous bulb oxygen saturation (SjO2) accurately
reflects the CBF/CMRO2 ratio but is invasive and complex
[11]. By contrast, near-infrared spectroscopy (NIRS) can
non-invasively assess SctO2, which can reflect the CBF/
CMRO2 ratio [12]. However, it has also been suggested that
SctO2 and SjO2 are poorly correlated [12, 13]. Introduction To date, the difference in SjO2 between common anes-
thetic agents has not been investigated during RALP. In this
study, we compared the effects of sevoflurane and propofol
on SjO2, and verified whether SctO2 could be used as an
alternative to SjO2 during RALP. For SctO2 measurement, the cerebral oximeter probes
were placed bilaterally ≥1 cm above each eyebrow. SctO2
was monitored with an INVOS5100B cerebral oximeter
(Somanetics, Troy, MI, USA), and the values from each
side were averaged. For the continuous monitoring of
SjO2, a Pre-Sep Oximetry Catheter (Edwards Lifesciences,
Irvine, CA, USA) was connected to the Vigileo system and
placed in the left jugular venous bulb. The catheter position
was verified radiographically. 1 3 Methods The correlation between
SjO2 and SctO2 was evaluated by simple linear regression. Significance was determined at p < 0.05, and we used
XLSTAT 2015 for Windows (Addinsoft, New York, USA)
for all analyses. Data are expressed as mean (standard
deviation). Fig. 1 The time courses of jugular venous oxygen saturations
(SjO2) in groups that received sevoflurane (filled circle) and propo-
fol (opened circle). Values are expressed as mean (SD). Multi symbol
p < 0.05 vs. group P Fig. 2 The time courses for regional oxygen saturations (SctO2) in
groups receiving sevoflurane (filled circle) and propofol (opened cir-
cle). Values are expressed as mean (SD). *p < 0.05 vs. T0 Methods There were no statistically significant differences between the two
groups Fig. 1 The time courses of jugular venous oxygen saturations
(SjO2) in groups that received sevoflurane (filled circle) and propo-
fol (opened circle). Values are expressed as mean (SD). Multi symbol
p < 0.05 vs. group P that we needed a minimum sample size of 17 patients per
group to detect an effect size of 0.89 using independent
Student’s t tests with an α of 0.05 and a power of 0.80. Thus, we aimed to include 25 patients per each group to
allow for a 30 % drop-out rate. The Shapiro–Wilk test was
used to test the normality of the data. Demographic vari-
ables, duration of surgery and anesthesia, ephedrine and
remifentanil doses, blood loss, urine output, and intrave-
nous fluid volume between groups were compared using
unpaired Student’s t tests. A two-way repeated-measures
analysis of variance (ANOVA) with post hoc unpaired t
test and Bonferroni correction was used to compare SjO2,
SctO2, MAP, HR, CI, CVP, PaO2, PaCO2, Hb, BT, and
BIS between the groups. A one-way repeated-measures
ANOVA with Bonferroni post hoc tests was used to ana-
lyze these variables across time within the groups. All
statistical tests were two-tailed. The correlation between
SjO2 and SctO2 was evaluated by simple linear regression. Significance was determined at p < 0.05, and we used
XLSTAT 2015 for Windows (Addinsoft, New York, USA)
for all analyses. Data are expressed as mean (standard
deviation). that we needed a minimum sample size of 17 patients per
group to detect an effect size of 0.89 using independent
Student’s t tests with an α of 0.05 and a power of 0.80. Thus, we aimed to include 25 patients per each group to
allow for a 30 % drop-out rate. The Shapiro–Wilk test was
used to test the normality of the data. Demographic vari-
ables, duration of surgery and anesthesia, ephedrine and
remifentanil doses, blood loss, urine output, and intrave-
nous fluid volume between groups were compared using
unpaired Student’s t tests. A two-way repeated-measures
analysis of variance (ANOVA) with post hoc unpaired t
test and Bonferroni correction was used to compare SjO2,
SctO2, MAP, HR, CI, CVP, PaO2, PaCO2, Hb, BT, and
BIS between the groups. A one-way repeated-measures
ANOVA with Bonferroni post hoc tests was used to ana-
lyze these variables across time within the groups. All
statistical tests were two-tailed. Methods The institutional ethics committee of Iwate Medical Uni-
versity Hospital, Japan, approved this study, and it has
been registered in the UMIN Clinical Trials Registry
(UMIN000016685). After obtaining written informed con-
sent, we enrolled adult male patients who were undergo-
ing RALP with the requirement that they were American
Society of Anesthesiologists physical status class 1 or 2. Patients with known histories of cerebral ischemia or hem-
orrhage were excluded. Participants were randomized by
computer generation into groups that received either sevo-
flurane (group S) or propofol (group P). We recorded the SjO2, SctO2, MAP, heart rate (HR), CI,
CVP, PaO2, PaCO2, hemoglobin concentration (Hb), BIS,
and BT at the following points: 5 min before surgery (T0);
5 min after inducing a 15 mmHg pneumoperitoneum (T1);
5, 30, 60, 90, and 120 min after inducing the pneumoperi-
toneum in a 30° Trendelenburg position (T2, T3, T4, T5,
and T6, respectively); and after desufflation in a supine
position (T7). Anesthesia was induced in group S by a bolus injec-
tion of thiopental (5 mg/kg) and in group P by an effect-
site target-controlled infusion (TCI) of propofol of 5 µg/
ml; remifentanil (0.2–0.4 µg/kg/min) was used in both
groups. After administering rocuronium (0.8 mg/kg), the
trachea was intubated and the lungs were mechanically
ventilated in volume control mode (tidal volume 8–10 ml/ Based on a pilot study, the sample size was calculated
to detect a 9 % difference in the mean SjO2 value at T7
using G power: 68 % (±9 %) for group P compared with
77 % (±11 %) for group S. The power analysis indicated 1 3 1 3 J Anesth (2016) 30:949–955 951 Data are presented as the mean (SD). There were no statistically significant differences between the two
groups
Group S (n = 22)
Group P (n = 19)
p value
Age (years)
67 (4)
66 (4)
0.21
Weight (kg)
67 (8)
68 (6)
0.65
Height (cm)
165 (6)
166 (5)
0.22
Anesthetic time (min)
284 (39)
282 (37)
0.69
Operation time (min)
201 (40)
201 (32)
0.73
Fluid administered (ml)
1132 (362)
1103 (287)
0.78
Blood loss (ml)
94 (119)
108 (81)
0.65
Urine output (ml)
163 (124)
121 (82)
0.15
Ephedrine administered (mg)
47 (20)
40 (16)
0.11
Total dose of remifentanil administered (mg)
4.2 (1.2)
4.7 (0.8)
0.19 Table 1 Demographic and
anesthetic data Data are presented as the mean (SD). Results Of the 50 patients who were scheduled to enroll in the
study, seven (three in group S and four in group P) were
excluded. One patient in group P was found to have a his-
tory of cerebral infarction after consenting to the study and
six withdrew their consent before the operation. In group P,
an additional two patients were excluded, one because nica-
rdipine was administered to treat intraoperative hyperten-
sion, and another because the FiO2 needed to be increased
beyond the protocol limit to maintain the SaO2 >94 %. All
of the measured values were normally distributed. There
were no major complications on normal postoperative Fig. 2 The time courses for regional oxygen saturations (SctO2) in
groups receiving sevoflurane (filled circle) and propofol (opened cir-
cle). Values are expressed as mean (SD). *p < 0.05 vs. T0 rounds in either group. As shown in Table 1, there were
no significant differences in the demographic or anesthetic
data between the groups. 1 3 J Anesth (2016) 30:949–955 952 952
J Anesth (2016) 30:949 955
Fig. 3 Changes in a mean arterial pressure (MAP), b heart rate
(HR), c cardiac index (CI), d central venous pressure (CVP), e naso-
pharyngeal temperature (BT), f Bispectral Index (BIS), g partial pres-
sures of arterial oxygen (PaO2), h partial pressures of carbon dioxide
(PaCO2), and i hemoglobin concentration (Hb). Measurements were
performed: 5 min before the commencement of surgery (T0); 5 min
after a 15 mmHg pneumoperitoneum (T1); 5, 30, 60, 90 and 120 min
after the Trendelenburg position (T2, T3, T4, T5, and T6, respec-
tively); and after exsufflation in the supine position (T7). Plus symbol
p < 0.05 vs. T0 in group S. Hash symbol p < 0.05 vs. T0 in group P. Multi symbol p < 0.05 vs. group P Fig. 3 Changes in a mean arterial pressure (MAP), b heart rate
(HR), c cardiac index (CI), d central venous pressure (CVP), e naso-
pharyngeal temperature (BT), f Bispectral Index (BIS), g partial pres-
sures of arterial oxygen (PaO2), h partial pressures of carbon dioxide
(PaCO2), and i hemoglobin concentration (Hb). Measurements were
performed: 5 min before the commencement of surgery (T0); 5 min after a 15 mmHg pneumoperitoneum (T1); 5, 30, 60, 90 and 120 min
after the Trendelenburg position (T2, T3, T4, T5, and T6, respec-
tively); and after exsufflation in the supine position (T7). Results Plus symbol
p < 0.05 vs. T0 in group S. Hash symbol p < 0.05 vs. T0 in group P. Multi symbol p < 0.05 vs. group P Figure 1 shows that the SjO2 was significantly higher in
group S at all measurement points (p < 0.05) with no sig-
nificant changes throughout the procedure when compared
with T0. By contrast, Fig. 2 shows that there was no sig-
nificant difference in the SctO2 values between the groups
at each point, but that SctO2 was significantly higher at T2
than at T0 within each group. pneumoperitoneum in the Trendelenburg position (T2–T6)
compared with T0. In group P, Hb was significantly lower
at T7 than at T0. Compared with T0, there were no differ-
ences within each group for PaO2, PaCO2, CI, Hb, BT, and
BIS at each time point. Figure 4 shows the linear regression analysis for SjO2
and SctO2 during anesthesia, with evidence of only a weak
relationship (β = 0.106; r2 = 0.065; p = 0.004). Figure 3 shows that there were no differences across
the measured time points between the groups in most of
the key parameters that could affect SjO2 and SctO2. How-
ever, HR in group S was lower than that in group P at T0
and T1. Compared with T0, the MAP was significantly
higher at T1–T6 in group S, and at T1, T2, and T6 in group
P. In both groups, CVP was significantly higher during Discussion Although volatile anesthet-
ics and propofol presumably have the potential to decrease
cerebral oxygenation during RALP, we showed that neither
did so. Considering the lack of significant changes in SjO2
compared with the baseline (T0), it is conceivable that pneu-
moperitoneum and a 30° Trendelenburg position may not
affect cerebral oxygenation within 2 h. This may be because
the predicted raise in ICP was within the physiological com-
pensation, such as cerebrospinal fluid (CSF) and blood mov-
ing into the spinal canal and extracranial vasculature not to
suppress cerebral oxygenation, and also may be due to the
effect of cerebrovascular autoregulation although that might
be partly impaired in group S. Fig. 4 Linear regression showing a weak relationship between
regional oxygen saturation (SctO2) and jugular venous oxygen satura-
tion (SjO2). Data are for 328 measurement pairs from 41 patients and showed that sevoflurane had significantly higher lev-
els of SjO2 than propofol at all measurement points. We
also observed no intergroup differences in MAP, CI, CVP,
BT, BIS, PaO2, PaCO2, Hb at any time point and in the
total ephedrine and remifentanil dose, even though HR was
lower in group S at T0 and T1. The difference in HR may be
explained by the higher doses of ephedrine administered in
group P to treat hypotension during this period. Although not
statistically significant, the PaO2 of group S was higher by
about 15 mmHg at all measurement points, but this is likely
too small to affect SjO2 physiologically and also from a lit-
erature review [14]. BT and BIS have been strongly related
to cerebral metabolism [15, 16], but there were no signifi-
cant differences in these values between the groups suggest-
ing that there was no difference in CMRO2. Because we used
sevoflurane at a mid-range concentration (above 1 MAC), we
presumed that the cerebrovascular dilation effect was domi-
nant in group S and that the intergroup differences in SjO2
were due to the differences in CBF. Cerebral oxygen desaturation during surgery can predict
early POCD and has been associated with delirium and pro-
longed hospitalization among the elderly [17, 18]. Current
study might show that sevoflurane is better than propofol for
RALP, but the result of our study does not directly indicate
that sevoflurane is a superior choice for RALP. Discussion The present study is the first to compare cerebral oxygena-
tion by SjO2 between anesthetic regimens during RALP 3 1 3 953 J Anesth (2016) 30:949–955 Fig. 4 Linear regression showing a weak relationship between
regional oxygen saturation (SctO2) and jugular venous oxygen satura-
tion (SjO2). Data are for 328 measurement pairs from 41 patients SjO2 due to cerebrovascular stenosis during pneumoperito-
neum and a steep Trendelenburg position. A study compar-
ing sevoflurane and propofol anesthesia among patients with
baseline SjO2 values <50 % showed no differences in post-
operative cognitive function [20], but the study did not show
the SjO2 difference between the groups and did not control
for BP, CI, or PaCO2, so we cannot assess the true relation-
ship between the anesthetics, SjO2, and postoperative cogni-
tive function. Several studies have shown the differences in
cerebral oxygenation depending on the anesthetic used in a
variety of clinical settings, but only a few have done so for
pneumoperitoneum in the Trendelenburg position. One study
tested whether SctO2 differed between propofol and sevoflu-
rane anesthesia during laparoscopic surgery in a 20° Tren-
delenburg position [21], and concluded that propofol was
associated with significantly lower SctO2 than sevoflurane. In situations of raised ICP (e.g., brain tumors), research with
volatile anesthetics have shown that they may maintain a
significantly higher SjO2 compared with propofol [22]. Fur-
ther, in the supine position for normal surgery [23], in the
sitting position for arthroscopy [13], and during one-lung
ventilation for lung surgery [24], volatile anesthetics can
maintain higher cerebral oxygenation than propofol. One
pilot study has reported that, compared with sevoflurane,
propofol increased cerebral SctO2 only when the stimulus
of surgery was intense [25]. Although the authors specu-
lated that an increased propofol dose due to the stimulation
caused the CMRO2 to decrease, the mechanism remains
unclear because the concentrations of sevoflurane were not
disclosed. Most of the studies discussed above, including the
current study, indicate that volatile anesthetics cause higher
cerebral oxygenation levels with no differences in neurologi-
cal complications or POCD when compared with propofol
anesthesia. We also showed that SjO2 was well preserved
in both groups (>50 %; Fig. 1). Although volatile anesthet-
ics and propofol presumably have the potential to decrease
cerebral oxygenation during RALP, we showed that neither
did so. Discussion Considering the lack of significant changes in SjO2
compared with the baseline (T0), it is conceivable that pneu-
moperitoneum and a 30° Trendelenburg position may not
affect cerebral oxygenation within 2 h. This may be because
the predicted raise in ICP was within the physiological com-
pensation, such as cerebrospinal fluid (CSF) and blood mov-
ing into the spinal canal and extracranial vasculature not to
suppress cerebral oxygenation, and also may be due to the
effect of cerebrovascular autoregulation although that might
be partly impaired in group S. SjO2 due to cerebrovascular stenosis during pneumoperito-
neum and a steep Trendelenburg position. A study compar-
ing sevoflurane and propofol anesthesia among patients with
baseline SjO2 values <50 % showed no differences in post-
operative cognitive function [20], but the study did not show
the SjO2 difference between the groups and did not control
for BP, CI, or PaCO2, so we cannot assess the true relation-
ship between the anesthetics, SjO2, and postoperative cogni-
tive function. Several studies have shown the differences in
cerebral oxygenation depending on the anesthetic used in a
variety of clinical settings, but only a few have done so for
pneumoperitoneum in the Trendelenburg position. One study
tested whether SctO2 differed between propofol and sevoflu-
rane anesthesia during laparoscopic surgery in a 20° Tren-
delenburg position [21], and concluded that propofol was
associated with significantly lower SctO2 than sevoflurane. In situations of raised ICP (e.g., brain tumors), research with
volatile anesthetics have shown that they may maintain a
significantly higher SjO2 compared with propofol [22]. Fur-
ther, in the supine position for normal surgery [23], in the
sitting position for arthroscopy [13], and during one-lung
ventilation for lung surgery [24], volatile anesthetics can
maintain higher cerebral oxygenation than propofol. One
pilot study has reported that, compared with sevoflurane,
propofol increased cerebral SctO2 only when the stimulus
of surgery was intense [25]. Although the authors specu-
lated that an increased propofol dose due to the stimulation
caused the CMRO2 to decrease, the mechanism remains
unclear because the concentrations of sevoflurane were not
disclosed. Most of the studies discussed above, including the
current study, indicate that volatile anesthetics cause higher
cerebral oxygenation levels with no differences in neurologi-
cal complications or POCD when compared with propofol
anesthesia. We also showed that SjO2 was well preserved
in both groups (>50 %; Fig. 1). References veins, and capillaries combined; and is affected by extrac-
erebral blood flow, cerebrospinal fluid, and systemic blood
pressure. By contrast, SjO2 measures only the intracerebral
mixed venous saturation [27, 28]. Interestingly, in this study,
SctO2 showed similar changes to BP across time, whereas
SjO2 did not. Third, NIRS calculates SctO2 based on a fixed
artery-to-vein ratio, regardless of changes induced by the
Trendelenburg position and anesthetic agents [9, 28]. 1. Mavrocordatos P, Bissonnette B, Ravussin P. Effects of
neck position and head elevation on intracranial pressure
in anesthetized neurosurgical patients. J Neurosurg Anesth. 2000;12:10–4. 2. Halverson A, Buchanan R, Jacobs L, Shayani V, Hunt T, Riedel
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not routinely perform cognitive tests. Thus, it remains
unclear whether postoperative cognitive outcomes correlate
with differences in cerebral oxygenation by different anes-
thetic agents; further research is needed. Second, we meas-
ured SjO2 unilaterally, and although no research has shown
a clear difference between left and right SjO2, this may have
affected the results. Third, we did not include patients with
cerebrovascular disease and complications, so we cannot
state how the SjO2 or SctO2 would have responded to RALP
in patients with a cerebral pathology. Fourth, the values for
ICP, CBF, and CMRO2 were not measured directly, which
should be rectified in future research. Fifth, we limited the
time spent with a pneumoperitoneum in the Trendelenburg
position to 120 min, yet there have been reports of neuro-
logical complications due to brain edema when this proce-
dure exceeds 8 h [29]. Actually, cerebrovascular autoregula-
tion gradually changes with prolonged pneumoperitoneum
in the Trendelenburg position; one study showed that it is
decreased from 170 min [19]. Considering the inhibitory
effect of volatile anesthetics on cerebrovascular autoregula-
tion, it may be worth comparing the effects of longer sur-
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superiority of sevoflurane over propofol in a clinical setting
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tests, our study might suggest the appropriateness of sevoflu-
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and SctO2. There are several factors that could contribute to
the inconsistency between the two values. First, SjO2 is an
index of oxygenation for the entire brain, whereas SctO2 is
an index of localized oxygenation in the frontal lobe [9–11,
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ner C, Closhen D. Time course of cerebrovascular autoregulation
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tatic surgery. Anaesthesia. 2014;69:58–63. 25. Sen P, Izdes S, But A. Effects of sevoflurane and propofol anaes-
thesia on cerebral oxygenation during normocapnia and mild
hypercapnia: a pilot study. Br J Anaesth. 2013;110:318–9. g y
20. Guo JY, Fang JY, Xu SR, Wei M, Huang WQ. Effects of propo-
fol versus sevoflurane on cerebral oxygenation and cognitive
outcome in patients with impaired cerebral oxygenation. Ther
Clin Risk Manag. 2016;12:81–5. 26. Yoshitani K, Kawaguchi M, Iwata M, Sasaoka N, Inoue S, Kuru-
matani N, Furuya H. Comparison of changes in jugular venous
bulb oxygen saturation and cerebral oxygen saturation during
variations of haemoglobin concentration under propofol and
sevoflurane anaesthesia. Br J Anaesth. 2005;94:341–6. g
21. Kim SJ, Kwon JY, Cho AR, Kim HK, Kim TK. The effects
of sevoflurane and propofol anesthesia on cerebral oxygena-
tion in gynecological laparoscopic surgery. Korean J Anesth. 2011;61:225–32. 27. Yoshitani K, Kawaguchi M, Miura N, Okuno T, Kanoda T, Ohni-
shi Y, Kuro M. Effects of hemoglobin concentration, skull thick-
ness, and the area of the cerebrospinal fluid layer on near-infrared
spectroscopy measurements. Compliance with ethical standards Anesthesiology. 2007;106:458–62. ;
22. Muñoz HR, Núñez GE, de la Fuente JE, Campos MG. The
effect of nitrous oxide on jugular bulb oxygen saturation during
remifentanil plus target controlled infusion propofol or sevoflu-
rane in patients with brain tumors. Anesth Anal. 2002;94:389–92. 28. Pollard V, Prough DS, DeMelo AE, Deyo DJ, Uchida T, Widman
R. The influence of carbon dioxide and body position on near-
infrared spectroscopic assessment of cerebral hemoglobin oxy-
gen saturation. Anesth Anal. 1996;82:278–87. 23. Valencia L, Rodríguez-Pérez A, Kühlmorgen B, Santana RY. Does sevoflurane preserve regional cerebral oxygen saturation
measured by near-infrared spectroscopy better than propofol? Ann Fr Anesth Reanim. 2014;33:e59–65. 29. Pandey R, Garg R, Darlong V, Punj J, Chandralekha, Kmar A. Unpredicted neurological complications after robotic laparo-
scopic radical cystectomy and ileal conduit formation in steep
Trendelenburg position: two case reports. Acta Anaesthesiol
Belg. 2010;61:163–6. 24. Iwata M, Inoue S, Kawaguchi M, Takahama M, Tojo T, Tani-
guchi S, Furuya H. Jugular bulb venous oxygen saturation 1 3 1 3 3
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BOEKBESPREKING
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toe, liet algemeene verschooningsrecht, waarop de openbare accountant
aanspraak kan maken. toe, liet algemeene verschooningsrecht, waarop de openbare accountant
aanspraak kan maken. p
Op den beambte-accountant, de collega met de specifieke accoun-
tantskennis, maar in dienstbetrekking bij een particuliere onderneming,
rust, dit spreekt welhaast vanzelf, de zwijgplicht. Hij kan echter geen
aanspraak op verschooningsrecht maken. p
p
g
Hoe is bij het Nederlandsch Instituut van Accountants het beroeps
geheim geregeld? Het antwoord kan kort zijn. Het woord beroepsgeheim
komt nog niet in Statuten en Reglementen voor. Evenwel is thans een
wijziging van het Reglement van Arbeid in behandeling; art. 2 van het
ontwerp der herziening schrijft o.a. voor dat de leden gehouden zijn hun
arbeid te verrichten met eerlijkheid, nauwkeurigheid en onzijdigheid en
met inachtneming van de meest mogelijke geheimhouding. Onder ,,de
meest mogelijke geheimhouding” moet niet worden verstaan: „absolute
geheimhouding”. Vervolgens enkele opmerkingen over enkele Koninklijke Besluiten, wel
ke na de bevrijding zijn gepubliceerd en waarin het verschooningsrecht
wordt 'aangetast. Dit zijn het Besluit herstel rechtsverkeer (E 100) en
het Besluit vijandelijk vermogen (E 133). In deze besluiten wordt
uitdrukkelijk bepaald, dat advocaten, procureurs, notarissen, accountants,
bankiers en ambtenaren, als getuigen of deskundigen opgeroepen, zich
niet kunnen beroepen op eenig beroeps- of ambtsgeheim. Onder erkenning
van het groote gemeenschapsbelang, dat is gelegen in rechtsherstel en in
het achterhalen van vijandelijk en on-Vaderlandsch vermogen, aarzelt de
inleider toch deze aantasting van het verschooningsrecht goed te keuren,
want wordt niet aan een ander groot gemeenschapsbelang, de onbe
schroomde raadpleging van vertrouwenslieden, groote schade toege
bracht? Voorts vestigt de inleider nog de aandacht op een brief, welken het
bestuur van het Nederlandsch Instituut van Accountants schreef aan den
Raad van Ministers in verband met de opheffing van het beroepsgeheim,
zooals bepaald in het Buitengewoon Navorderingsbesluit. Tenslotte gaat de inleider nog summier na, welke opvattingen in het
buitenland heerschen met betrekking tot het beroepsgeheim van den open
baren accountant en van de wijzigingen, welke daarin gedurende of na
den oorlog zijn gekomen. BOEKBESPREKING Sociale spanningen, rede uitgesproken bij de aanvaarding van
het ambt van hoogleraar aan de Rijksuniversiteit te Gronin
gen, door Dr. P. J. Bouman. Uitgeverij H. ]. Paris, Amster
dam, 30 blz. d
D
F
H
k Sociale spanningen, rede uitgesproken bij de aanvaarding van
het ambt van hoogleraar aan de Rijksuniversiteit te Gronin
gen, door Dr. P. J. Bouman. Uitgeverij H. ]. Paris, Amster
dam, 30 blz. d
D
F
H
k door Dr. F. van Heek In vrijwel alle wetenschappen hebben sterk sprekende feiten en duide
lijk aan den dag komende verschijnselen eerder de aandacht getrokken dan
langzame veranderingen of verborgen, min of meer gelijkmatig verloo-
pende processen. Een soortgelijke ontwikkeling valt bij de geschiedenis
en de sociologie waar te nemen. De voorkeur der oudere historiografie
voor gewelddadige gebeurtenissen — met veronachtzaming der geschie- m a b blz. 27 a b blz. m a b b m a b denis van het alledaagsche leven —■ is bekend. In de sociologie vinden wij
een rijke literatuur over revoluties, terwijl de sociale spanningen vrijwel
buiten beschouwing blijven. Dat deze verwaarloozing zeer ten onrechte
geschiedt wordt door den schrijver van de te bespreken publicatie duide
lijk aangetoond. j
g
De sociale spanning kenmerkt zich volgens Bouman door een massaal
karakter. Daarbij komt een zekere mate van duurzaamheid; vluchtige
emoties mogen er niet toe worden gerekend. Een derde kenmerk van de
sociale spanning is haar latenten aard. De spanning is een phychische
gesteldheid die zich ieder oogenblik in daden kan omzetten. Noodzakelijk
is dit echter niet. In vele gevallen zullen spanningen fluctueeren en menig
maal zelfs zoodanig verzwakken, dat zij hun naam ternauwernood meer
verdienen. In dit verband gezien is het methodisch gewenscht de psy
chische gesteldheid van de spanning te onderscheiden van het handelen
dat uit de spanning resulteert. Zoo gaat de Marxistische klassenstrijd-
theorie uit van de veronderstelling dat sociale tegenstellingen in ieder
geval tot revoluties leiden. Marx verwart echter de symptomen die een
aanwezigheid van sociale spanningen verraden, met klassenstrijd als
zoodanig. g
In Bouman’s rede wordt bijzondere aandacht geschonken aan de sociale
spanningen, die hun oorsprong vinden in het ressentiment van een massa,
die zich als rechteloos beschouwt en de bestaande sociale orde veroor
deelt Deze spanningen zijn, volgens Mannheim, in de tegenwoordige
maatschappij bijzonder gevaarlijk omdat onze samenleving in steeds min
dere mate beheerscht wordt door de cultureel leiding gevende groepen. BOEKBESPREKING Groote massa’s laten zich gemakkelijk mobiliseeren door psychopaten
die de gevoelens van hun individueel ressentiment op de door hen gelei
den weten over te dragen. Zoo kan onze fragmentarisch over-gerationa-
liseerde maatschappij een plotselinge omslag vertoonen naar wilde en on-
beheerschte sociale bewegingen, waarin de maatschappelijke spanningen
zich een uitweg zoeken. Bouman stelt de vraag, welke middelen ons ten
dienste staan om dit gevaar op te heffen. Hij wijst op de noodzakelijk
heid van redelijk overleg bij tegenover elkaar staande groepen en op de
wenschelijkheid van deugdelijke voorlichting. Het is evenwel bekend,
dat een beroep op het verstand of het redelijk inzicht in zijn effect niet
mag worden overschat. Somtijds kunnen echter ongemotiveerde gevoe
lens van haat of verachting gecompenseerd worden door een gemeen
schappelijk élan b.v. in een emotioneel nationaal saamhoorigheidsgevoel. Het is beschamend te constateeren, dat een dergelijk élan gewoonlijk
slechts in oorlogstijd wordt waargenomen. Is het mogelijk onder de huidige maatschappelijke omstandigheden een
samenvattend ideaal te vinden, even krachtig als de oorlog zelf? Kan het
ideaal der sociale rechtvaardigheid daartoe dienen of moet alle heil ver
wacht worden van een herleving der christelijke moraal? Schrijver stelt
deze vragen zonder ze te beantwoorden. Een derde weg om de sociale
spanningen te doen verminderen, is het tegengaan van de massale opeen-
hoopingen van menschen, die door sociaal-cultureele omstandigheden aan
geestelijke en moreele nivelleering zijn prijsgegeven. Oordeelskracht en
persoonlijk verantwoordelijkheidsgevoel gedijen slechts in kleiner groeps
verband, waarin nog gemeenschapsgedachten leven. In verband met het bovenstaande noemt Bouman als sociaal en cultu
reel hoopgevende tendenties van dezen tijd, de bereidwilligheid der ker
ken om geestelijk leiding te geven bij groepsvorming van velerlei aard,
de toenemende belangstelling voor bedrijfssolidariteit, de opkomst van de m ab blz. 28 m ab blz. 28 z.g. „wijkgedachte” in de steden (waarin stadswijken en buurten als orga
nische eenheden tot hun recht komen) en ten slotte ook symptomen van
sociale, cultureele herleving op het platteland, o.a. het werk van de volks-
hoogescholen. g
Het door Bouman gekozen onderwerp kan wegens de veelzijdigheid
van het object moeilijk in het kader van een inaugurale rede worden
samengevat. Niettemin is hij er in geslaagd een van de belangrijkste socio
logische vraagstukken van dezen tijd op treffende wijze aan de orde te
stellen. MEDEDEELING VAN HET NEDERLANDSCH INSTITUUT VAN
ACCOUNTANTS Leden van verdienste; Bestuurssamenstelling; Nieuwe leden In de laatstgehouden Algemeene Vergadering van het Nederlandsch
Instituut van Accountants zijn tot leden van verdienste dier vereeniging
benoemd de heeren: Prof. R. A. Dijker te Wassenaar;
H. R. Reder te Amsterdam. Prof. R. A. Dijker te Wassenaar; Tot bestuursleden werden gekozen de heeren: Tot bestuursleden werden gekozen de heeren: T. Keuzenkamp te 's-Gravenhage; G. Hartog te Amsterdam; G. Hartog te Amsterdam;
K C Sl
t
t
A
t d K. C. Sluyter te Amsterdam. De bestuurssamenstelling is voor 1947 als volgt: De bestuurssamenstelling is voor 1947 als volgt: De bestuurssamenstelling is voor 1947 als volgt: J. Kraayenhof te Amsterdam, Voorzitter;
T
K
k
’ G
h
Pl T. Keuzenkamp te ’s-Gravenhage, Plaatsvervangend-voorzitter:
G
A
d
S
i G. Hartog te Amsterdam, Secretaris;
A Th d
L
d
Pl A. Th. de Lange, ec. drs., Plaatsvervangend-secretaris; K. C. Sluyter te Amsterdam, Penningmeester:
J P
d k
d
' G
h
B J. Paardekooper, ec. drs. te 's-Gravenhage, Bibliothecaris; W . C. Koppenberg te Borne; Mr. B. Moret, ec. drs. te Rotterdam; A. Nierhoff te Amsterdam. In bedoelde Algemeene Vergadering werden de volgende heeren tot
Ik! benoemd: J. G. Baak Aerdenhout Amsterdam
A
d S. C. Bakkenist, ec. drs. E B
k Amsterdam
A
f E. Beekman
J H
d J. H. van der Burg
A
C Amersfoort A. M. Cats Den Haag
d E. Driessen Amsterdam Den Haag
d J. Ferdinandusse H. Fros Amsterdam Den Haag
R
d A. C. ). de Jong
k g
Rotterdam
d R. Jonker C. F. Kamer Amsterdam J. G. Klimbie Haarlem Amsterdam A. J. König m a b blz. 29
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https://openalex.org/W3091088548
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https://link.springer.com/content/pdf/10.1007/s40572-020-00292-3.pdf
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English
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Nutrition Transition and Climate Risks in Nigeria: Moving Towards Food Systems Policy Coherence
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Current environmental health reports
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cc-by
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| ERROR: type should be string, got "https://doi.org/10.1007/s40572-020-00292-3\nCurrent Environmental Health Reports (2020) 7:392–403 https://doi.org/10.1007/s40572-020-00292-3\nCurrent Environmental Health Reports (2020) 7:392–403 CLIMATE CHANGE AND HEALTH (C GOLDEN, SECTION EDITOR) Abstract Purpose of Review The purpose of this review is to describe the combined impacts of the nutrition transition and climate change\nin Nigeria and analyze the country’s national food-related policy options that could support human and planetary health\noutcomes. Recent Findings This paper uses a food systems framework to analyze how the nutrition transition and climate change interact in\nNigeria affecting both diets and the double burden of malnutrition, resulting in what has been termed the syndemic. Interactions\nbetween climate change and the nutrition transition in Nigeria are exacerbating diet-related inequities and will continue to do so if\nfood systems continue on their current trajectory and without significant transformation. Siloed policy actions that attempt to\nmitigate one aspect of food system risk can create a negative feedback loop in another aspect of the food system. Our analysis\nfinds that Nigeria has five national policies that include actionable steps to address food system insufficiencies; however, each of\nthese policies is constrained by the boundaries of singular nutrition, climate change, and agricultural objectives. The country\nshould consider a coherent policy environment that explicitly identifies and links underlying systemic and institutional drivers\nbetween climate change and malnutrition that simultaneously and comprehensively address both human and planetary health\noutcomes of food systems. Summary The systemic and institutional outcomes of this emerging syndemic—undernutrition, obesity, and climate change—\nare inexorably linked. Nigeria lacks a coherent policy environment taking on this challenging syndemic landscape. The analysis\nin this paper highlights the need for Nigeria to prioritize their national nutrition and agricultural and climate policies that uncouple\nfeedback loops within food systems to address climate change and malnutrition in all its forms. Keywords Nigeria . Food systems . Policy coherence . Nutrition transition . Climate change . Planetary health Nutrition Transition and Climate Risks in Nigeria: Moving Towards\nFood Systems Policy Coherence Alexandra E. Morgan1 & Jessica Fanzo2 Accepted: 16 September 2020\n# The Author(s) 2020\n/ Published online: 1 October 2020 Accepted: 16 September 2020\n# The Author(s) 2020\n/ Published online: 1 October 2020 2\nThe Nitze School of Advanced International Studies, Bloomberg\nSchool of Public Health, Berman Institute of Bioethics, Johns\nHopkins University, Washington, DC, USA 1\nThe Nitze School of Advanced International Studies, Johns Hopkins\nUniversity, Washington, DC, USA This article is part of the Topical Collection on Climate Change and\nHealth Introduction staple grains and salt, and sanitation and hygiene improve-\nments are partially responsible for reductions in the worldwide\nburden of undernourishment from 14.8 to 10.8% [3]. However, urbanization, income shifts, and work and lifestyle\npatterns have simultaneously produced an increase in over-\nweight and obesity across all regions [3]. The past two decades have produced a dynamic shift in world\ndiets for low and lower middle-income countries [1•, 2]. Increased income and some significant interventions such as\nthe promotion of exclusive breastfeeding, fortification of As populations become more affluent, dietary consumption\npatterns shift. While diets become more diverse, moving away\nfrom meals composed predominantly with staple grains and/\nor tubers, there is also higher intake of refined carbohydrates\nand highly processed foods (and drinks) with higher amounts\nof added sugars, unhealthy fats, and sodium, and animal\nsourced foods. This typical dietary shift is known as the nu-\ntrition transition [4, 5]. This pattern is associated with global-\nization and urbanization factors but also an increasingly sed-\nentary lifestyle [6], and the combination of these trends leads\nto shifts away from infectious diseases towards diet-related This article is part of the Topical Collection on Climate Change and\nHealth 1\nThe Nitze School of Advanced International Studies, Johns Hopkins\nUniversity, Washington, DC, USA 1\nThe Nitze School of Advanced International Studies, Johns Hopkins\nUniversity, Washington, DC, USA 1\nThe Nitze School of Advanced International Studies, Johns Hopkins\nUniversity, Washington, DC, USA Curr Envir Health Rpt (2020) 7:392–403 393 non-communicable diseases such as cardiovascular disease,\ndiabetes, and stroke [4, 5]. undernutrition, and unsustainable food systems are associated\nwith both burdens [8•, 15••, 18]. If no action is taken to ad-\ndress climate change and its impacts on food systems, the\nhistoric progress made to reduce undernutrition could be lost,\nand overweight and obesity trends will continue to increase\n[1•, 8•, 15••]. Human and planetary health outcomes cannot be\ndecoupled, and effective policies must target the feedback\nloops linking the health of both the planet and people [9••,\n15••]. What results from this transition is a population dealing\nwith both undernutrition and overweight and obesity—what\nhas been termed the double burden of malnutrition (also\ntermed the multiple burdens of malnutrition) [1•]. The inter-\naction between the nutrition transition and the epigenetic ef-\nfects of undernutrition has fueled this trend. Introduction Early nutritional\ndeficits put certain populations at risk for overweight and obe-\nsity later into adulthood [1, 7]. Both of these trends affect\nmorbidity and mortality, and poor diets remain a high-risk\nfactor for childhood death as well as adult non-\ncommunicable disease. Nigeria is the most populous country in Africa, and its\npopulation of 196 million people [19] is expected to double\nby 2050 [20]. Within the Economic Community of West\nAfrican States, Nigeria makes up more than two thirds of\nGDP [21]. Because of its economic growth and political status\nin the region, continent, and globally, guiding Nigeria towards\na trajectory towards sustainable development is critical\n[21–23]. Food systems connect and contribute to these dietary shifts,\npoor health outcomes, and environmental degradation [8•,\n9••]. Unsustainable food production systems allocate calories\nand nutrients in inefficient ways, while also contributing to\nclimate change [8•, 9••] and natural resource degradation\n[9••, 10••, 11]. Because of this circularity, climate change will\ncontinue to compound the health effects of malnutrition in all\nits forms [8•, 12–14]. The food systems framework (Fig. 1)\nhighlights the interactions between environment, food, and\nhealth. Nigeria is already experiencing the earlier described\n“syndemic.” Average maximum temperatures and average\nnumber of hot days have increased, while precipitation has\ndecreased in the country [24]. Temperature change has led\nto increasing desertification in the north, erratic and changing\nrainfall across the country, and increased flood risk in coastal\nregions and along the major river systems [25–28]. These\nchanges make food production, distribution, and utilization\nmore vulnerable, which in turn affect dietary and nutrition\noutcomes [16••, 29]. At the same time, Nigeria is experiencing\nthe double burden of malnutrition [30–32] with malnutrition\nranked as the number one risk factor for death and disability,\nand sub-optimal diets have risen to become the 7th highest\nrisk in the past decade [33]. Both climate change and the\nnutrition transition interact with the demographic shifts Climate change, undernutrition, and obesity share underly-\ning societal causes, and feedback loops between them magni-\nfy negative health and nutrition outcomes. This “syndemic” is\na synergy of epidemics that occur at the same time and place,\ninteract, and share common drivers [15••]. These emerging\nsyndemic risks exist in most countries but are particularly\nacute for the rural poor in the global South [16••, 17]. Food Systems Framing Food systems are composed of the people and activities that\nplay a part in growing, transporting, supplying, and, ultimate-\nly, eating food. These processes also involve elements that\noften go unseen, such as food preferences and resource invest-\nments. Food systems influence diets by determining what\nkinds of foods are produced and available in markets. They\nalso influence what foods people want to eat and are able to\naccess. As shown in Fig. 1, the different parts of the food\nsystem include food supply chains, food environments, and\nconsumer behavior (not shown in this framework for simpli-\nfication purposes). Food supply chains consist of the activities\nand actors that take food from production, storage and distri-\nbution, process and packaging, retail, and waste [34, 35]. Food environments are the physical, economic, political and\nsocio-cultural surroundings, opportunities, and conditions that\ncreate everyday prompts, shaping people’s dietary preferences\nand choices as well as nutritional status [34, 36]. They can be\nmarkets, restaurants, or cafeterias. These different parts shape\nfood systems and can lead to both positive and negative out-\ncomes across a range of outcomes [34]. Both rural-urban and north-south divisions are character-\nized by different nutritional challenges in Nigeria. While the\npopulation is evenly split between rural and urban residents\n[49], currently, urban populations are more likely to experi-\nence obesity and overweight while rural populations are more\nlikely to experience undernutrition [45]. However, evidence\nsuggests that rural places are beginning to catch up with urban\nobesity trends [50]. At the same time, DHS data show that in\nthe last 20 years underweight and vitamin deficiencies have\nbeen higher in the northern arid zones of the country, while\noverweight and obesity have been higher in the southern\nzones. Although northern zones produce more food for the\ncountry, residents in the north are the most likely to be food\ninsecure [23]. Childhood stunting and undernutrition are par-\nticularly acute in northern Nigeria. Almost half of all children\nin the northeast and northwest are stunted [51]. Food systems are not static. They are in constant transition\nand are shaped and shifted by different internal and external\ndrivers that push or pull the system in different directions [37]. While Fig. Introduction An\nincreasing number of countries are experiencing a double bur-\nden of overweight and obesity alongside continued Food supply chains\nFood environments\nFood production \nsystems and \ninput supply\nStorage and distribution\nProcessing and \npackaging\nDiets\nNutrition and \nhealth \noutcomes\nOther impacts\nSocial\nEconomic\nEnvironmental\nClimate \nChange\nGlobalization \nand Trade\nIncome \nGrowth and \nDistribution\nFood availability\nFood affordability\nProduct properties\nVendor properties\nFood messaging\nUrbanization\nFig. 1 Food systems framework. Source: 35 Income \nGrowth and \nDistribution Urbanization Nutrition and \nhealth \noutcomes Processing and \npackaging Fig. 1 Food systems framework. Source: 35 Curr Envir Health Rpt (2020) 7:392–403 394 occurring in Nigeria, exacerbating dietary inequalities and af-\nfecting access to high quality, nutrient-dense, and safe food on\na reliable basis. environments. For the purposes of this paper, we are focusing\non the gray-shaded elements and relationships of Fig. 1. Climate change and natural resources shape food system\nfunctionality, efficiency, and resiliency [38]. In turn, food sys-\ntems are a cause of climate change and environmental degra-\ndation [39]. Food systems contribute to greenhouse gas emis-\nsions, depletion of freshwater resources, deforestation, and\nnutrient pollution on landscapes and waterways [12]. The di-\nversity, safety, and quality of diets very much depend on\nthriving food systems. However, evidence suggests that sub-\noptimal diets dominate across the world, contributing to both\nenvironmental and climate degradation and detrimental health\noutcomes [9••]. This paper uses an adapted food systems framework (Fig. 1) from the High-Level Panel of Experts Report on Nutrition\nand Food Systems [34] to analyze the combined effects of\nclimate change and the nutrition transition on Nigerian diets,\nexamining multiple ways climate change will affect food sup-\nply chains and environments and its dietary and nutritional\nimpacts on consumers. It also reviews the literature on two-\nway relationship of role climate change in accelerating the\nnutrition transition and vice versa in Nigeria and assesses to\nwhat degree current national food-related policies in Nigeria\naddress the syndemic. Siloed actions that attempt to mitigate\nthe effects of one issue can create potential negative feedback\nloops and impacts in another area [15••]. In order to address\nclimate change and malnutrition, Nigeria must prioritize pol-\nicies that address the syndemic—by uncoupling feedback\nloops between climate, undernutrition, and obesity by\nstrengthening food system actions. Nutrition Transition of Nigeria Malnutrition in all its forms is the number one risk factor for\ndeath and disease in Nigeria [33]. The combined effects of\nrising obesity and stagnating stunting in children indicate that\nNigeria is going through a nutrition transition. Demographic\nand Health Survey (DHS) data have shown slight declines in\nstunting for children and adult female thinness as well as in-\ncreasing adult female overweight and obesity over the last\n20 years [40–44]. Independent studies have found that urban\nobesity increased by 20% in Nigeria between 2002 and 2010\n[45]. While some populations have access to sufficient calo-\nries, stunting and thinness have decreased slowly, and under-\nnourishment overall has risen since 2000, indicating that ad-\ndressing malnutrition will require multiple strategies that span\nhealth, food, care, and the environment [46–48]. Food Systems Framing 1 does not show all the myriad drivers that shape\nfood systems, climate change, urbanization, and globalization\nare significant drivers that instigate feedback loops of food\nsystem outcomes—diets, nutrition and health, environment,\nand livelihoods—on food supply chains and food Currently, Nigeria cannot meet food requirements through\ndomestic production [52]. The major food crops produced in\nthe country include cassava, cowpea, and sorghum [31, 53]. The Government of Nigeria (GON) reports that demand ex-\nceeds supply by more than 50% for rice, wheat, fish, milk, and\ntomatoes. Nigeria also underproduces maize, chicken, yams, Curr Envir Health Rpt (2020) 7:392–403 395 to 2010, and sugar-sweetened beverages have increased. There are increases, although small, in the consumption of\nmilk and fruits. This data source does not capture fish and\nseafood consumption, but these food sources are important\ncomponents of the Nigerian diet. Overall, the health of the\ndiet has not changed much over the last 15 years. Across the\nentire country, only 56% of women meet the minimum criteria\nfor dietary diversity, a marker for dietary quality, in the pre-\nvious day [40]. and oil palm to a lesser extent [52] and has significant losses of\nfruits and vegetables as these perishable foods move along the\nsupply chain [54]. Because it cannot meet its population’s\ndemand with its own production, Nigeria imports between\n$3 and $5 billion worth of food annually [55]. Food imports both reduce Nigeria’s foreign exchange re-\nserves and make consumers vulnerable to international prices. Over half of the population lives on less than $2 per day,\nincluding the majority of rural residents [4]. While some\nsmallholder farmers produce enough food for family con-\nsumption, the average rural family only produces a quarter\nof food they consume [18, 56]. Across the country, the aver-\nage household spends 65% of its income on food expenditure\n[57]. The unaffordability of nutritious food can be a signifi-\ncant barrier to consuming a healthy diet. The cost of a nutri-\ntionally adequate diet (CoNA) measures the minimum daily\ncost of meeting nutrient and energy requirements for a refer-\nence healthy adult woman, as a percent of daily per capita\nhousehold spending on food and non-alcoholic beverages. In\nNigeria, the CoNA is 92%, whereas in South Africa it is 42%\nand in the USA it is 32% [53]. Climate Vulnerability for Nigeria and Nigeria’s\nFood System In Nigeria, an increase in the number of days\nwith extreme temperature will reduce the output of cassava,\ncocoyam, sweet potatoes, cowpeas, and maize [63] By 2050,\nhotter temperatures are expected to reduce output of rice, sor-\nghum, millet, maize, yam, and cassava [68]. Increased rainfall\nwill also compromise sweet potato and rice production [64]. In the short term, warmer temperatures are associated with\nincreased production of millet, onions, tomatoes, and melon\nin northern Nigeria [63]; however, erratic rainfall patterns may\nreduce any positive effects of temperature. Pricing feedback loops highlight the economic effects of\nclimate change. Lowered or shifted yields will impact liveli-\nhoods for 60% of Nigeria’s labor force involved in agricultur-\nal production, 90% of which are smallholder farmers [27]. The\nprice effects of lowered yields will also affect non-farming\nand urban populations reducing their access to high quality\nand nutritious diets. Additional challenges affecting the food\nsystem include certain push factors such as increasing move-\nment from rural to urban areas, putting more stress on food\nsupply chains to urban areas and encroachment of urbaniza-\ntion into adjacent rural land (hinterlands) that are near major\nurban centers [26]. Figure 3 summarizes some effects of cli-\nmate change on food system elements (focusing on food sup-\nply chains and food environments), intermediary diet out-\ncomes, and nutrition and health outcomes in Nigeria using\nthe food systems framework in Fig. 1. Nutrient content will also shift in major food crops and\nconsumers’ access to nutrients will be negatively affected by\nchanges to food storage. Increased concentrations of atmo-\nspheric carbon dioxide will reduce zinc, iron, and protein in\nrice [69] and cassava will have increased concentrations of\ncyanide [60]. Heat will also make storage of vegetables more\ndifficult [29]. Consumers will also have elevated exposure to\nmycotoxins, such as aflatoxin, and mold increases because\nonly 10% of maize traders across the country fumigate and\ndry their wares [64]. Increased surface water temperature, sea level rise, and\nocean acidification will negatively affect aquaculture and fish-\neries [70, 71]. This will affect nutrient access for much of\nNigeria since fish make up about ½ of protein intake [70]. Human control may make aquaculture more resilient to cli-\nmate change [70]; however, farmed fish may hold less nutri-\ntional value than wild caught fish [72]. Nigeria is one of the\nseveral countries identified by Golden et al. Climate Vulnerability for Nigeria and Nigeria’s\nFood System The Nigerian government predicts that the major impacts for\nagricultural production will include lower yields and poor\nlivestock performance in the Sahel and savanna agro-\necological zones, rapid farm wastage, decreased soil fertility,\nand leaching. Seawater incursion and desertification will re-\nduce arable land [29]. Flooding will also affect coastal and\nfreshwater fisheries production negatively through siltation\nand contamination [61]. Some climate models suggest that\nrainfed agriculture may decline up to 50% by 2080, while\noverall agricultural productivity could decline by 10–25%\n[65]. irrigation. Only 1% of agricultural land currently uses modern\nirrigation [26, 28, 73]. The north central region may have\nlower sensitivity to rainfall changes in the short run, if lake\nwater in the region is harnessed to provide irrigation [26]. However, irrigation is not a panacea given uncertain changes\nin rainfall. Extreme weather may also cause flooding and a\nwidespread lack of flood control will make erratic rain partic-\nularly damaging [29, 62]. The agricultural impact of severe flooding caused by heavy\nrains in 2012 provides an example of the multiple pathways in\nwhich climate change may affect household nutrition. In\n2012, heavy rainfall strained dam infrastructure and flooded\nsettlements along the Niger, Benue, and Gongola rivers, caus-\ning $16.9 billion dollars worth of damage [62]. The disaster\ndestroyed 30% of the year’s rice production. The majority of\nfamilies affected by floods that relied on agricultural produc-\ntion to meet their family’s nutritional needs were forced to turn\nto markets to buy the majority of their food [62]. Increased\ndemand created a subsequent negative feedback loop elevat-\ning the price index for food and making market consumption\nmore inaccessible. Scarcity coping strategies used by families\nincluded borrowing food, reducing or skipping meals, and\nrelying on less preferred foods [62]. Countrywide surveys\nshow that even absent crisis level shocks, many families resort\nto food coping strategies. In 2010 and 2011, countrywide\nsurveys showed that between 7 and 9% of those in the lowest\nincome quintile used severe coping strategies such as going to\nbed hungry or borrowing food from the community or local\nNGOs as food assistance in the past 7 days [56]. Increasing temperature [63], shifts in rainfall, [64, 66] and\natmospheric carbon dioxide concentration [60] will drive\nshifts in agricultural yield. Across West Africa, a hotter dry\nseason will cause rice yields to decline, even with increased\nirrigation [67]. Climate Vulnerability for Nigeria and Nigeria’s\nFood System Temperatures in Nigeria are estimated to rise between 1.1 and\n2.6 °C by 2060 [60] leading to losses of up to 30% of GDP\n[61]. Sea level rise is expected to reach 1 m by 2050 [27]\naffecting coastal regions and river systems. Temperature in-\ncreases will affect agriculture mainly through changes in rain-\nfall. Climate change is projected to make the northern zones\ndrier and the southern zones more wet [27, 28, 60] Increasing\ndesertification will harm the northern part of the country,\nwhile sea level rise, saltwater intrusion, and flooding will af-\nfect the southern part of the country [62–64]. While GON analysis focuses mainly on staple crops, low\nconsumption of fruits and vegetables are also associated with\nnegative health outcomes, and the combination of both do-\nmestically produced and imported fruits and vegetables in\nNigeria do not meet the dietary requirements for daily vege-\ntable consumption [58]. Climate change will impact Nigeria’s agro-ecological\nzones in different ways. Nigeria has two main types of\nvegetation—tropical rainforest in the south and savannah in\nthe north. Erratic rainfall and unpredictable severe weather\nevents may affect agricultural production across both areas. While specific climate risks will differ depending on the agro-\necological zone, most key challenges will be related to water Figure 2 shows how diets have changed in Nigeria com-\nparing data from 1990 to 2010 (the most recent available)\nfrom the Global Dietary Database. There are interesting\ntrends. Whole grains and legumes have decreased from 1990 27.8\n98.1\n1.8\n100.2\n37.9\n15.3\n5.8\n25.6\n25.4\n53.2\n113.9\n58.9\n28.1\n103.3\n2.8\n82.3\n41.3\n17.5\n5.7\n25.3\n26.2\n72.3\n117.6\n44.8\n0.0\n20.0\n40.0\n60.0\n80.0\n100.0\n120.0\n140.0\nfiber\nfruits\nfruit juices\nlegumes\nmilk\nnuts and seeds\nprocessed meats\nred meats\nseafood\nsugar-sweetened beverages\nvegetables\nwhole grains\ngrams/day\n2010\n1990\nFig. 2 Changes in the Nigerian diets, 1990–2010. Source: [59] 2010\n1990 Fig. 2 Changes in the Nigerian diets, 1990–2010. Source: [59] Curr Envir Health Rpt (2020) 7:392–403 396 resources. In the north, dryness and drought will affect both\nagricultural production and livestock, while in the south, soil\nporosity issues will heighten the effects of sudden increased\nrain and flooding [26]. Climate change will impact each zone\ndifferently but will also shift the boundaries between the zones\n[27], challenging traditional agricultural cultivation patterns. Climate Vulnerability for Nigeria and Nigeria’s\nFood System as most reliant on\nfish, which also has estimated marine catch reductions of over\n20% by 2050 [72]. Climate Change-Driven Food System Impacts Nutrition and Health Outcomes Intermediary Dietary Outcomes Intermediary Dietary Outcomes Climate Change-Driven Food System Impacts\nIntermediary Dietary Outcomes\nNutrition and Health Outcomes\nReduced caloric intake\nReduced dietary quality and diversity\nIncreased risk of foodborne illness\nIncreased consumption of less \nperishable staple grains and tubers\nWasting \nStunting \nMicronutrient Deficiencies \nOverweight and Obesity\nNCDs\nFood supply chains\nReduced fruit and vegetable production\nIncreased loss of perishable foods\nIncreased contamination of foods with aflatoxin and molds\nIncreased concentration of cyanide in cocoyams\nDisrupted transportation and distribution\nLowered livestock productivity\nReduced fisheries and aquaculture production\nLowered production of rice, cocoyam, sweet potatoes\nReduced micronutrient content in rice and cassava\nFood environments\nIncreased vulnerability to infrastructure of markets\nReduced ability to get to markets Less foods available \nin markets\nIncrease waste of perishable foods\nIncreased cost of food\nFig. 3 Impacts of climate change on food systems for Nigeria. Sources: [26, 27, 29, 60, 61, 63, 64, 67] Food supply chains Increased risk of foodborne illness Wasting \nStunting \nMicronutrient Deficiencies \nOverweight and Obesity\nNCDs Reduced caloric intake\nReduced dietary quality and diversity Increased consumption of less \nperishable staple grains and tubers Fig. 3 Impacts of climate change on food systems for Nigeria. Sources: [26, 27, 29, 60, 61, 63, 64, 67] CCN), the National Policy on Food and Nutrition (NPFN), the\nAgriculture Sector Food Security and Nutrition Policy\n(ASFS), and the National Strategic Plan of Action on\nNutrition (NSPAN), to determine whether they used a food\nsystems framing to develop interventions targeting the\nsyndemic—malnutrition, nutrition-sensitive agriculture, and\nclimate change. Nigeria committed to the creation of a health\nand environment strategic alliance in the Libreville declara-\ntion, acknowledging the important link between environmen-\ntal risks and health outcomes [74]. Overall, Nigeria has devel-\noped strong policy guiding documents and national strategies\non climate change, nutrition, and agriculture. However, policy\nimplementation has been limited due to budgetary constraints\nand stakeholder fragmentation [23, 75] with state budgets and\npolicies not matching federal planning [23, 76]. affordability of diets or the choice architecture of these built\nplaces to facilitate healthier eating. More significantly, only 3\nof the policies contain a cost analysis of implementing the\ngoals and actions set out in those policies. All of the policies\ncontained a monitoring and evaluation (M&E) plan. Assessment of Relevant Nigerian Food-\nand Climate-Related Policies Using the food systems framework as a guide, we assessed\nfive national policies in Nigeria, the Agriculture Promotion\nPolicy (APP), the National Adaptation Strategy (NASPA- Rainfall change will have particularly negative effects on\nagriculture because of Nigeria’s dependence on rainfed Curr Envir Health Rpt (2020) 7:392–403 397 Climate Change-Driven Food System Impacts The most\ncomprehensive set of interventions along the supply chain\ncomes from the Agriculture Sector Food Security and\nNutrition Policy; however, this plan does not explicitly inte-\ngrate the effects of climate change on the supply chain. Table 1 below summarizes the results of the analysis. Challenges to Implementation While Nigeria’s policy documents highlight actionable items\nto create nutrition-sensitive and climate-smart interventions,\nsignificant challenges remain. The NASPA-CCN was devel-\noped in 2011, but fragmented implementation of the strategy\nhas made farm-level adaptation to climate change difficult to\nachieve [78]. The agricultural section of the National Climate\nPolicy focuses on the diversification of livestock, increased\naccess to drought resistant crops, better soil management prac-\ntices, national early warning systems, and increased use of\nirrigation and crop cover [61]. The government also suggests\nthe increased provision of crop insurance at subsidized rates\nfor smallholder farmers, provided by non-profits, or through\npublic-private partnerships. However, adaptation results have\nbeen limited. While the climate policy has ostensibly been in\neffect for 9 years, a 2019 study in two northern communities\nfound that while farmers were using adaptation strategies, they\nwere not utilizing the strategies best suited for their dryland The Nigerian government has made inroads to combine\nagriculture and nutrition policies using a systems perspective. However, the explicit integration of climate change adaptation\nand mitigation actions into nutrition policy has been limited. Three of 5 policies recognize the double burden of undernu-\ntrition and overweight/obesity, while two policies focus on\nundernutrition exclusively. The three nutrition polices have a\nweak focus on climate change, while the climate change pol-\nicy has little focus on nutrition. All five policies make target\ninterventions at various points along the supply chain, but\nagriculture production focused interventions are the most\nprevalent in the climate change and agriculture policies, while\ndemand focused interventions to improve diets are stronger in\nthe nutrition policies. Food environment interventions overall\nwere limited with very little focus on how to change the Curr Envir Health Rpt (2020) 7:392–403 Table 1\nNational policy environment in Nigeria\nNational strategy document\nYears active\nDoes the policy address\nmultiple burdens of\nmalnutrition? Does the policy address the ag-environment-nutrition\nnexus? Does the policy include\ninterventions along food systems? Challenges to Implementation Cost\nanalysis\nM&E plan\nNational Adaptation Strategy\nand Plan of Action\n(NASPA-CCN)\n2011–current No\nUndernutrition only\nNo\n•Focus on climate impacts on\nagricultural production\n•Focus on food access\n•No mention of nutritional\nquality of food\nNo\nFood supply chains\n•Improved agriculture systems for\ncrops and livestock\n•Increase irrigation\nFood environment/markets\n•Facilitate credit to smallholder\nfarmers\nNo\nYes*\n*Focused on building\nout framework, not\ninterventions\nAgriculture Promotion\nPolicy\n(APP)\n2016–2020\nYes\nRecognizes double burden of\nunderweight, overweight,\nand obesity\nNo\n•Focus on agribusiness supply chains\n•Integrating climate-smart agriculture*\n•Integrating nutrition-sensitive programming*\n*Mentioned as a goal to integrate throughout all\nobjectives, but many objectives lack integration of\nnutrition and climate\nYes\nFood supply chains\n•Fertilizer, increased soil nutrients\n•Irrigation\n•Pest and disease control services\n•National grain storage centers\nFood environment/markets\n•Infrastructure improvement\nDiets\n•Import substitution to reduce\neffects of exchange rate policy\non consumers\n•School feeding programs\n•Strategic reserves of food\nNo\nYes\nNational Policy on Food and\nNutrition\n(NPFN)\n2016–Current Yes\nRecognizes burden of\nmalnutrition in all its forms\nNo\n•Links agriculture and food security\n•Does not mention climate change\nYes\nFood supply chains\n•Promote priority value chain\ncrops\n•Improved on-farm storage\n•Myco-toxin prevention\n•Improve processing and\npreservation\nFood environments/markets\n•Effective food distribution\nsystems\n•Improved infrastructure\nDiets\n•Biofortification of staple foods,\nvitamin provision\n•Food quality standards\n•National buffer stock to reduce\nprice volatility\n•School feeding programs\nNo\nYes\n2016–2025\nYes\nNo\nYes\nYes\nYes\n8\nCurr Envir Health Rpt (2020) 7:392–403 398 p\ny\ng\nNational strategy document\nYears active\nDoes the policy address\nmultiple burdens of\nmalnutrition? Does the policy address the ag-environment-nutrition\nnexus? Does the policy include\ninterventions along food systems? Challenges to Implementation Cost\nanalysis\nM&E plan\nNational Adaptation Strategy\nand Plan of Action\n(NASPA-CCN)\n2011–current No\nUndernutrition only\nNo\n•Focus on climate impacts on\nagricultural production\n•Focus on food access\n•No mention of nutritional\nquality of food\nNo\nFood supply chains\n•Improved agriculture systems for\ncrops and livestock\n•Increase irrigation\nFood environment/markets\n•Facilitate credit to smallholder\nfarmers\nNo\nYes*\n*Focused on building\nout framework, not\ninterventions\nAgriculture Promotion\nPolicy\n(APP)\n2016–2020\nYes\nRecognizes double burden of\nunderweight, overweight,\nand obesity\nNo\n•Focus on agribusiness supply chains\n•Integrating climate-smart agriculture*\n•Integrating nutrition-sensitive programming*\n*Mentioned as a goal to integrate throughout all\nobjectives, but many objectives lack integration of\nnutrition and climate\nYes\nFood supply chains\n•Fertilizer, increased soil nutrients\n•Irrigation\n•Pest and disease control services\n•National grain storage centers\nFood environment/markets\n•Infrastructure improvement\nDiets\n•Import substitution to reduce\neffects of exchange rate policy\non consumers\n•School feeding programs\n•Strategic reserves of food\nNo\nYes\nNational Policy on Food and\nNutrition\n(NPFN)\n2016–Current Yes\nRecognizes burden of\nmalnutrition in all its forms\nNo\n•Links agriculture and food security\n•Does not mention climate change\nYes\nFood supply chains\n•Promote priority value chain\ncrops\n•Improved on-farm storage\n•Myco-toxin prevention\n•Improve processing and\npreservation\nFood environments/markets\n•Effective food distribution\nsystems\n•Improved infrastructure\nDiets\n•Biofortification of staple foods,\nvitamin provision\n•Food quality standards\n•National buffer stock to reduce\nprice volatility\n•School feeding programs\nNo\nYes\n2016–2025\nYes\nNo\nYes\nYes\nYes\n8\nCurr Envir Health Rpt (2020) 7:392–403 No No Curr Envir Health Rpt (2020) 7:392–403 399 (continued)\nl strategy document\nYears active\nDoes the policy address\nmultiple burdens of\nmalnutrition? Does the policy address the ag-environment-nutrition\nnexus? Does the policy include\ninterventions along food systems? Challenges to Implementation Cost\nanalysis\nM&E plan\nure Sector Food\nity and Nutrition\ny\nRecognizes burden of\nmalnutrition in all its forms\n•Strong focus on agriculture and nutrition\n•Climate change mentioned once\nFood supply chains\n•Expand production of\nbio-fortified foods\n•Scale up fruit and vegetable\nproduction\n•Reduce post-harvest loss through\nincreased cold storage and\nprocessing\n•National aflatoxin control\ninitiative\nFood environments/markets\n•Facilitate access to credit for\nsmallholder farmers\n•Improved nutrition labeling\nDiets\n•Develop improved food-based\ndietary guidelines\nSector Component of\nnal Food and\ntion Policy\nN)\n2014–2019\nNo\nFocus on undernutrition,\nspecifically for mothers and\nchildren\nDoes not address overweight\nand obesity\nNo\n•No mention of agriculture or climate change\nNo\nDiets\n•Vitamin supplementation\n•Feeding centers for severe acute\nmalnutrition\n•Breastfeeding promotion\nYes\nYes\n[30, 31, 52, 61, 77] National strategy document\nYears active\nDoes the policy address\nmultiple burdens of\nmalnutrition? Does the policy address the ag-environment-nutrition\nnexus? Does the policy include\ninterventions along food systems? Cost\nanalysis\nM&E plan\nAgriculture Sector Food\nSecurity and Nutrition\nPolicy\n(ASFS)\nRecognizes burden of\nmalnutrition in all its forms\n•Strong focus on agriculture and nutrition\n•Climate change mentioned once\nFood supply chains\n•Expand production of\nbio-fortified foods\n•Scale up fruit and vegetable\nproduction\n•Reduce post-harvest loss through\nincreased cold storage and\nprocessing\n•National aflatoxin control\ninitiative\nFood environments/markets\n•Facilitate access to credit for\nsmallholder farmers\n•Improved nutrition labeling\nDiets\n•Develop improved food-based\ndietary guidelines\nHealth Sector Component of\nNational Food and\nNutrition Policy\n(NSPAN)\n2014–2019\nNo\nFocus on undernutrition,\nspecifically for mothers and\nchildren\nDoes not address overweight\nand obesity\nNo\n•No mention of agriculture or climate change\nNo\nDiets\n•Vitamin supplementation\n•Feeding centers for severe acute\nmalnutrition\n•Breastfeeding promotion\nYes\nYes 400 Curr Envir Health Rpt (2020) 7:392–403 environment [79]. Irrigation policies are also critical for cli-\nmate adaptation, but efforts to scale up irrigation have been\nfragmented and duplicative [73, 80]. large strides to improve its policy environment and coherence. Its 2017 agricultural and nutrition policy highlights a systemic\nunderstanding of the drivers of malnutrition. Integrating cli-\nmate change into a more coherent food systems framework\nwould provide nuance to future assessments of malnutrition\nand will allow Nigeria to address and mitigate climate risks for\nnutrition. Doing so would require some major revisions to the\npolicy. Ecker et al. Challenges to Implementation (2020) also recommended that Nigeria’s\npolicymakers consider adopting a food systems framework to\nreformulate national food and nutrition policies in order to\nimprove household diets and reduce the multiple burdens of\nmalnutrition [89]. Numerous surveys of Nigerian farmers have highlighted\nthe continued challenges to increase the use of adaptation\nstrategies. Studies of farmers’ perceptions of climate change\nhave shown that farmers accurately perceive changes in tem-\nperature and rainfall [81], but climate adaptation strategies\ndepend on farmer income, perception of risk, and environment\n[82–84]. Other surveys have found that adaptation strategies\nfor farmers differ between older and younger farmers [82] and\nthat in some communities, challenges remain regarding\nfarmers’ perception of the causes of climate change. Farmers\nwith larger farms, higher incomes, and more schooling are\nmore likely to practice adaptation strategies [82] while those\nthat link climate change to religious beliefs are less likely to\nproactively use adaptive strategies [81]. A significant infor-\nmation gap exists between farmers [83]. Without increasing\nexpenditure on agriculture and other related industries such as\naquaculture and agroforestry, smallholder farmers are likely to\nbe left out of mitigation and adaptation strategies. One revision would be to fulfill the Comprehensive Africa\nAgriculture Development Program (CAADP) pledge to invest\nin agriculture, and, within agriculture, nutrition-climate-\nsensitive agriculture. Nigeria is a signatory to CAADP, which\nhas set a goal of 10% of government budget going towards\nagriculture. However, Nigeria has consistently\nunderperformed on this metric. Average yearly agricultural\nspending between 2008 and 2012 only totaled 4.6% of the\nnational budget. [90]. The most recent CAADP performance\nassessment gave Nigeria 0% progress on its three goals related\nto modernized agriculture, well-nourished citizens, and envi-\nronmentally sustainable and climate resilient economies [91]\nNigeria must increase domestic funding for climate, agricul-\nture, and nutrition in order to address malnutrition. Nutrition policies also lack budget allocations. The Scaling\nup Nutrition (SUN) Movement reports that Nigeria has met\n70% of the criteria for an enabling policy environment for\nnutrition as of 2019 [85]. However, budget allocations have\nnot matched the goals outlined in the costed strategies. Surveys of government and NGO stakeholders in 2015 and\n2016 found concerns about appropriate resource allocation at\nthe federal and state level [86]. In 2019, Nigeria only spent\n0.2% of its budget on nutrition specific interventions [85]. Budget delays lead to limited implementation. Challenges to Implementation A review of\nfederal stakeholders with responsibility for the NSPAN\n(2014–2019) found that only 1/3 had begun implementation\nof the plan by 2018 [87]. Limited domestic resource mobili-\nzation means that nutrition programs rely heavily on interna-\ntional donors for funding [85] and those resources can vary\nwidely depending on donor motivations. A second revision would be to consider double- or triple-\nduty actions. Double- and triple-duty actions simultaneously\nact on two or three of the epidemics (undernutrition, over-\nweight, and/or climate). One example could be reducing red\nmeat consumption to prevent cancer/obesity (obesity/NCDs),\nincrease land efficiency to grow food for human consumption\n(undernutrition), and lower greenhouse gas emissions (climate\nchange). These actions are carefully calibrated so that a focus\non undernutrition does not lead to an increase in overweight\nand obesity for certain populations [92••]. Swinburn and col-\nleagues articulated areas in which double- or even triple-duty\nactions could mitigate the sydemic, many with direct actions\nwith food environments, the place where consumers engage\nwith food systems [15••]. However, if Nigeria’s national pol-\nicies that touch on food systems do not address the impacts of\nclimate change, today’s investments may have little impact in\nthe next three decades. An integrated, holistic food systems\nframing linking climate-smart and nutrition-sensitive agricul-\nture on both land and water could identify priority areas for\ntriple-duty actions to make a more significant impact on the\nsyndemic in Nigeria [9••]. Some examples include scaling up\nnutrition-sensitive agriculture programs with climate adapted\nseeds and irrigation technologies, adapting food-based dietary\nguidelines to promote nutrient-dense and climate-conscious\ndiets, taxing highly processed foods and devoting revenue\nstreams to climate-smart agricultural policies, and developing\nmore urban agriculture. While some of these elements are Strong policy guidance with a lack of implementation\nfunding has been a continued problem for the agriculture,\nclimate, and nutrition sectors in Nigeria. Increased govern-\nment spending on agriculture has been linked to economic\ngrowth [88]. However, Nigerian budgets are heavily depen-\ndent on world oil prices [23, 26], and current price slumps\njeopardize programming for agriculture and nutrition\n(Table 1). • Of importance • Of importance •• Of major importance 1.• Popkin BM, Corvalan C, Grummer-Strawn LM. Dynamics of the\ndouble burden of malnutrition and the changing nutrition reality. Lancet. 2020;395(10217):65–74 This study describes the chang-\ning nutrition dynamics across the world as malnutrition stag-\nnates and overweight/obesity rises. They argue the double bur-\nden of malnutrition is rising in low- and middle-income\ncountries. 16.•• Fanzo J, Mclaren R, Davis C, Choufani J. Climate change and\nvariability: What are the risks for nutrition, diets and food systems? IFPRI Discussion Paper 01645. 2017. This report provides a\nthorough analysis of the multiple ways climate change will im-\npact nutrition along the supply chain. The report stresses the\nneed to create solutions for vulnerable populations that will be\nparticularly affected by climate-related food insecurity. 2. Dietz WH. Double-duty solutions for the double burden of malnu-\ntrition. Lancet. 2017;390(10113):2607–8. 2. Dietz WH. Double-duty solutions for the double burden of malnu-\ntrition. Lancet. 2017;390(10113):2607–8. 17. IPCC. Global Warming of 1.5°C. An IPCC Special Report on the\nimpacts of global warming of 1.5°C above pre-industrial levels and\nrelated global greenhouse gas emission pathways, in the context of\nstrengthening the global response to the threat of climate change,\nsustainable development, and efforts to eradicate poverty [Masson-\nDelmotte V, Zhai P, Pörtner HO, Roberts D, Skea J, Shukla JR\net al]. In Press. 2018. 3. FAO, IFAD, UNICEF, WFP and WHO. The State of Food Security\nand Nutrition in the World 2019. In: Safeguarding against econom-\nic slowdowns and downturns. Rome: FAO; 2019. 3. FAO, IFAD, UNICEF, WFP and WHO. The State of Food Security\nand Nutrition in the World 2019. In: Safeguarding against econom-\nic slowdowns and downturns. Rome: FAO; 2019. 4. Drewnowski A, Popkin BM. The nutrition transition: new trends in\nthe global diet. Nutr Rev. 1997;55(2):31–43. 4. Drewnowski A, Popkin BM. The nutrition transition: new trends in\nthe global diet. Nutr Rev. 1997;55(2):31–43. 5. Popkin BM. Relationship between shifts in food system dynamics\nand acceleration of the global nutrition transition. Nutr Rev. 2017;75(2):73–82. 18. FAO. Small family farms country factsheet: Nigeria. Food and\nAgriculture Organization. 2018. http://www.fao.org/3/i9930en/\nI9930EN.pdf. Accessed 15 Mar 2020. 6. Steyn NP, Mchiza ZJ. Obesity and the nutrition transition in sub-\nSaharan Africa. Ann N Y Acad Sci. 2014;1311(1):88–101. 19. World Bank Group. Population total- Nigeria. https://data. worldbank.org/indicator/SP.POP.TOTL?locations=NG. Accessed\n15 Mar 2020. 7. Conclusion and Recommendations While budgetary dependence on oil revenues and the frag-\nmentation of the federal system will challenge Nigeria’s abil-\nity to implement policies that focus on the common systemic\ndrivers of malnutrition and climate change, Nigeria has made Curr Envir Health Rpt (2020) 7:392–403 401 environment, and reduce climate change for a growing\npopulation. already present in Nigeria’s national policies, budgetary\nguidelines do not explicitly promote double- or triple-duty\nactions. Given Nigeria’s budget constraints and oil revenue\nvolatility, double- or triple-duty actions should become prior-\nity strategies. p p\n10.•• IPCC. Climate Change and Land: an IPCC special report on climate\nchange, desertification, land degradation, sustainable land manage-\nment, food security, and greenhouse gas fluxes in terrestrial ecosys-\ntems [Shukla PR, Skea J, Calvo E, Buendia V, Masson-Delmotte\nHO, Pörtner DC et al (eds.)]. In Press. 2019. 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Metabolomics for understanding stomatal movements
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Theoretical and Experimental Plant Physiology
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Metabolomics for understanding stomatal movements David B. Medeiros
. Luana M. da Luz . Hellen O. de Oliveira . Wagner L. Arau´jo . Danilo M. Daloso . Alisdair R. Fernie Received: 15 October 2018 / Accepted: 5 January 2019 / Published online: 22 January 2019
The Author(s) 2019 Received: 15 October 2018 / Accepted: 5 January 2019 / Published online: 22 January 2019
The Author(s) 2019 Abstract
Stomata control the exchange of CO2 and
water in land plants. For this reason, plants evolved to
quickly respond the surrounding environment and
endogenous cues in order to maintain their photosyn-
thetic rates, but avoiding excessive water loss. Although guard cell has been used as model for
characterization of signaling pathways, mainly regard-
ing abscisic acid (ABA) response, several important
questions
about
its
functioning
remain
elusive. Recently, transcriptomics, proteomics, and metabolo-
mics studies carried out using guard cells as model
have contributed substantially for our understanding
on how guard cells sense and respond to relative air
humidity, CO2, ABA, and sucrose. Comparatively,
proteomics and metabolomics platforms need sub-
stantial improvement to increase the number of
analytes detected. However, with the introduction of
metabolomics-based technologies, several
studies have been published increasing our knowledge on
guard cell function. Here, we review these new
exciting findings as well as discuss the importance of
using these new data to improve prediction when
modelling stomatal behavior. Keywords
Stomata Guard cells Metabolites
Metabolite profiling Mass spectrometry Theor. Exp. Plant Physiol. (2019) 31:91–102
https://doi.org/10.1007/s40626-019-00139-9 (0123456789().,-volV)(
01234567
89().,-volV) D. M. Daloso
Departamento de Bioquı´mica e Biologia Molecular,
Universidade Federal do Ceara´, Fortaleza,
Ceara´ 60440-970, Brazil D. B. Medeiros (&) A. R. Fernie
Max-Planck-Institut fu¨r Molekulare Pflanzenphysiologie,
Am Mu¨hlenberg 1, 14476 Potsdam-Golm, Germany
e-mail: medeiros@mpimp-golm.mpg.de 1 Introduction Stomata, microscopic pores surrounded by two spe-
cialized cells—the guard cells -in the leaf epidermis,
control the exchange of carbon dioxide (CO2) and
water in land plants. Therefore, they are the main gate
for two essential process in plants; photosynthesis and
transpiration. For this reason, plants need to adapt
quickly and efficiently the environment and endoge-
nous cues in order to gain sufficient CO2 to maintain
their photosynthetic rates and avoid excessive water
loss and thus prevent dehydration (Lawson et al. 2014;
Schroeder et al. 2001). D. B. Medeiros (&) A. R. Fernie
Max-Planck-Institut fu¨r Molekulare Pflanzenphysiologie,
Am Mu¨hlenberg 1, 14476 Potsdam-Golm, Germany
e-mail: medeiros@mpimp-golm.mpg.de L. M. da Luz H. O. de Oliveira W. L. Arau´jo
Max-Planck Partner Group at the Departamento de
Biologia Vegetal, Universidade Federal de Vic¸osa,
Vic¸osa, Minas Gerais 36570-900, Brazil Guard cells have been extensively studied for over a
century, and considerable efforts have been made to
better understand their structure, development, phys-
iology, and metabolism (Bergmann and Sack 2007;
Daloso et al. 2017; Dow and Bergmann 2014; Kim
et al. 2010; Mott 2009; Santelia and Lawson 2016). These advances have rendered the guard cell as one of D. M. Daloso
Departamento de Bioquı´mica e Biologia Molecular,
Universidade Federal do Ceara´, Fortaleza,
Ceara´ 60440-970, Brazil 12 3 3 Theor. Exp. Plant Physiol. (2019) 31:91–102 92 Covering the guard cell metabolome is still not
technically viable using a single analytical system but
recent developments have considerably extended our
ability to analyze this cell type. In fact, recent efforts to
comprehend the guard cell metabolome have shed
considerable light on metabolic regulation of stomatal
movement in response to environmental stimuli. In
this review we cover the most recent studies applying
guard cell metabolomics approaches in order to
understand the roles of important players in stomatal
movement such as abscisic acid (ABA), CO2, sucrose,
organic acids as well as plant cell–microbe interaction. We further discuss the importance of integrating
metabolomics data into mathematical modelling in
order to optimize predictions of stomatal behavior
under challenging conditions. the best studied plant cell models for membrane
transport, signaling, and homeostasis (Blatt 2000;
Engineer et al. 2016; Hetherington and Brownlee
2004; Hills et al. 2012; Israelsson et al. 2006). Stomatal pores are structurally very simple, but the
surrounding guard cells are one of the most specialized
cell in land plants. 1 Introduction They are morphologically distin-
guished from other epidermal cells and possess a
complex signal transduction network, tightly regu-
lated membrane ion system, and specialized metabolic
pathways which modulate guard cell turgor and
subsequently promote opening and closure of stomata
(Santelia and Lawson 2016). Generally, the stomatal
pores open in response to increases in guard cell
volume driven by decreases in the water potential of
the guard cell as result of the osmolytes accumulation
and subsequently water influx. Conversely, during
stomatal closure, efflux of osmolytes from guard cells
is required with associated increase in guard cell water
potential and in turn efflux of water (Assmann and
Wang 2001). Potassium (K?) and chloride (Cl-) act as
the main inorganic ions, while malate2- and sucrose
(Suc) are considered as the main organic osmolytes
during stomatal movements (Kollist et al. 2014;
Roelfsema
and
Hedrich
2005;
Vavasseur
and
Raghavendra 2005). 2 Metabolomics studies in guard cells While earlier studies on guard cells focused on
changes in accumulation of sugars, organic acids,
and K? in guard cell (Amoedo et al. 1996; Lu et al. 1995; Talbott and Zeiger 1993, 1996), the recent
introduction of new high throughput technologies has
reignited guard cell metabolism research. Metabolo-
mics approaches have been applied to analyze changes
in guard cell metabolism, being highly useful in
providing a broader understanding of guard cell
function. However, despite the improvements in guard
cell separation methods, all the studies involving
guard cell metabolome still face the bottleneck of high
quality and rapid cell isolation, in order to disturb as
few as possible guard cell primary metabolism. In the
last 5 years several studies were published, in which
metabolomics approaches were used for answering
several unsolved questions on guard cell metabolism,
below we review these exciting new findings. The
platforms used in each study are summarized in
Table 1. Most of the knowledge we have regarding stomatal
movements came from the discovery and characteri-
zation of several components of the protein transport-
ing system in guard cells (Eisenach and De Angeli
2017; Jezek and Blatt 2017). However, lately guard
cell metabolism has been revisited and we have
recently witnessed great efforts in assessing the
importance of a range of metabolites in the regulation
of stomatal function in elegant biochemical and
physiological studies. The knowledge from these
studies has increased our understanding on how guard
cells regulate their primary metabolism through
organic acid, sucrose, starch, and lipid biosynthetic
and degradative pathways in order to control stomatal
movements. Although we discuss some of these
studies in the following sections the focus of this
review is to give an update on how metabolomics
approaches have been applied in guard cells studies
rather than providing an overview of the regulation of
guard cell metabolism for which several recent
reviews document the most important recent findings
(Daloso et al. 2017; Lima et al. 2018; Santelia and
Lawson 2016). 2.1 Metabolic signatures for ABA response napus in response to ABA
Zhu and Assmann
(2017)
GC–TOF–MS
Validation of modeling flux-based predictions using mesophyll and guard
cell protoplasts fed with NaH13CO3
Robaina-Estevez
et al. (2017)
GC–TOF–MS
Feeding with [U-13C]-sucrose in EF from Arabidopsis
Medeiros et al. (2018b)
LC–MRM–MS
EF incubated with bacteria (Pst DC3000) and labeled with 13C and 15N
isotopes
Pang et al. (2018)
LC liquid chromatography, MS mass spectrometry, GC gas chromatography, MRM multiple reaction monitoring, TOF time of flight (Zhu and Assmann 2017). Non-targeted metabolomics
was used as a complementary approach to targeted
metabolomics—providing not only mass/charge ratios
but also the mass spectra of all detectable precursor
molecules. The platform combination resulted in a
metabolite profile of 390 non-redundant metabolites,
of which 77 were ABA responsive. On performing an
enrichment analysis of the metabolic changes flavone
and flavonol biosynthesis and metabolism of amino
sugar, nucleotide, sugar, sucrose, and starch were
highly affected by ABA (Zhu and Assmann 2017). a time-course experiment utilizing liquid chromatog-
raphy–multiple reaction monitoring–mass spectrome-
try (LC–MRM–MS) in order to perform targeted
analysis of 85 signaling-related metabolites in Ara-
bidopsis guard cell protoplasts (Jin et al. 2013). Arabidopsis wild-type plants and gpa1 mutants dis-
playing ABA-hyposensitive stomata were evaluated. This targeted metabolomics study revealed, as would
perhaps be anticipated, coordinated regulation of
signaling metabolites even in unrelated pathways. For instance, hormone related metabolites were more
responsive to ABA treatment in wild type than in gpa1
guard cells. Additionally, the majority of hormones
also displayed different ABA responses in guard cell
metabolite profiles when compared with those in the
mesophyll cell metabolome, suggesting that ABA is
most likely acts upstream of the other hormones (Jin
et al. 2013). Recently, primary metabolism in guard cells has
received increased attention mainly regarding the
roles of organic acids, sucrose (discussed below) and
starch metabolism (we suggest the read of Santelia and
Lawson (2016), Santelia and Lunn (2017), and Daloso
et al. (2017)). On the other hand, secondary metabo-
lism of guard cells is poorly understood. New insights
have, however, been published showing the connec-
tion between flavonol biosynthesis and ABA signaling
(Watkins et al. 2017, 2014). Flavonol accumulation
was observed in guard cells of Arabidopsis, but not in
surrounding pavement cells (Watkins et al. 2014). 2.1 Metabolic signatures for ABA response Guard cells are well established as a model cell type
for deciphering ABA signaling in plants. The regula-
tory role of ABA signaling pathway on stomatal
regulation has being recently improved by using
metabolomics tools. For instance, the metabolic
response to ABA treatment was recently followed in 12 3 Theor. Exp. Plant Physiol. (2019) 31:91–102 93 Table 1 Technologies applied for metabolomics studies in guard cell
Platform used
Remarks
References
LC–MS
Guard cell protoplasts from Columbia-0 and gpa1 Arabidopsis plants
subjected in response to ABA
Jin et al. (2013)
GC–MS/MS and LC–MRM–MS
Mesophyll and guard cells from B. napus subjected in response to NaHCO3
Misra et al. (2015b)
GC–TOF–MS
Feeding with NaH13CO3 in guard cell-enriched epidermal fragments (EF)
from tobacco
Daloso et al. (2015)
GC–TOF–MS
Feeding with NaH13CO3 in EF from transgenic tobacco plants
Daloso et al. (2016b)
GC–MS/MS, LC–MRM–MS,
and LC–TOF–MS
EF from Arabidopsis under 800 ppm CO2
Geng et al. (2016)
GC–TOF–MS
EF from tdt Arabidopsis mutant, apoplastic leaflet fluid, and non-aqueous
fractionation samples
Medeiros et al. (2017)
GC–MS/MS and LC–TOF–MS
Guard cell protoplasts from B. napus in response to ABA
Zhu and Assmann
(2017)
GC–TOF–MS
Validation of modeling flux-based predictions using mesophyll and guard
cell protoplasts fed with NaH13CO3
Robaina-Estevez
et al. (2017)
GC–TOF–MS
Feeding with [U-13C]-sucrose in EF from Arabidopsis
Medeiros et al. (2018b)
LC–MRM–MS
EF incubated with bacteria (Pst DC3000) and labeled with 13C and 15N
isotopes
Pang et al. (2018)
LC liquid chromatography, MS mass spectrometry, GC gas chromatography, MRM multiple reaction monitoring, TOF time of flight Table 1 Technologies applied for metabolomics studies in guard cell
Platform used
Remarks
References
LC–MS
Guard cell protoplasts from Columbia-0 and gpa1 Arabidopsis plants
subjected in response to ABA
Jin et al. (2013)
GC–MS/MS and LC–MRM–MS
Mesophyll and guard cells from B. napus subjected in response to NaHCO3
Misra et al. (2015b)
GC–TOF–MS
Feeding with NaH13CO3 in guard cell-enriched epidermal fragments (EF)
from tobacco
Daloso et al. (2015)
GC–TOF–MS
Feeding with NaH13CO3 in EF from transgenic tobacco plants
Daloso et al. (2016b)
GC–MS/MS, LC–MRM–MS,
and LC–TOF–MS
EF from Arabidopsis under 800 ppm CO2
Geng et al. (2016)
GC–TOF–MS
EF from tdt Arabidopsis mutant, apoplastic leaflet fluid, and non-aqueous
fractionation samples
Medeiros et al. (2017)
GC–MS/MS and LC–TOF–MS
Guard cell protoplasts from B. 2.2 Response to high CO2 concentration biosynthetic
enzyme,
was
demonstrated
to
be
expressed in guard cells, but not pavement cells. Interestingly, null mutation for the gene encoding
CHALCONE SYNTHASE increased the levels of
reactive oxygen species (ROS) in the guard cells. Further, guard cells of mutant plants showed faster
ABA-induced closure compared to wild type, suggest-
ing that flavonols may reduce the ABA-dependent
ROS burst that drives stomatal closing (Watkins et al. 2014). Curiously, flavonols levels were positively
regulated in guard cells by ethylene treatment in the
wild type, but not in an ethylene-insensitive mutant,
suggesting an ethylene-regulated mechanism of flavo-
nols controlling ROS burst under ABA signaling
(Watkins et al. 2014). Similarly, it was also demon-
strated that in tomato (Solanum lycopersicum), ABA-
induced ROS accumulation is followed by stomatal
closure (Watkins et al. 2017). Ethylene treatment of
wild-type tomato plants increased flavonol accumula-
tion in guard cells; however, no flavonol increases were
observed in Neverripe (Nr) plants, an ethylene receptor
deficient mutant. Taken together, these results strongly
suggest the roles of flavonols as important players
during the ABA-dependent ROS production for the
regulation of stomatal opening. Intriguingly, the link
between flavonols and stomatal functioning was also
observed in an independent recent study investigating
metabolic components of water stress in a Meditter-
anean vineyard (Gago et al. 2017). High ambient CO2 concentrations mediate closure of
stomatal pores in plants and conversely low ambient
CO2 concentration triggers the opening of stomatal
pores. Within the context of the raising atmospheric
CO2 concentration to understand how guard cells
adjust their physiology and metabolism in response to
CO2 is a key step for the development of plants better
adapted to the shifting climate condition (Geng et al. 2017). Indeed, although the whole network of CO2
response is not fully clear, recent studies have
addressed the molecular and cellular mechanisms
mediating CO2 regulation of stomatal movements
(Engineer et al. 2016; Zhang et al. 2018). For instance,
changes in the metabolite profile in response to
increases in CO2 concentration was investigated over
time. For this propose mesophyll and guard cells
protoplasts from B. napus were fed with sodium
bicarbonate (NaHCO3) (Misra et al. 2015b). In this
study, targeted metabolite profiling in untreated and
NaHCO3 treated mesophyll and guard cell was
performed using LC–MRM–MS and GC–MS/MS
based technologies, in order to identify 268 metabo-
lites related to both, primary and secondary metabo-
lism. 2.1 Metabolic signatures for ABA response Accordingly, CHALCONE SYNTHASE, a flavonol More recently, a non-targeted metabolomics study
utilizing a combination of gas chromatography–mass
spectrometry (GC–MS/MS) and liquid chromatogra-
phy–time of flight–mass spectrometry (LC–TOF–MS)
was applied to identify the metabolic signatures in
response to ABA in B. napus guard cell protoplasts 12 12 3 3 Theor. Exp. Plant Physiol. (2019) 31:91–102 94 2.2 Response to high CO2 concentration 2.3 Understanding the roles of Suc in guard cells Despite the above-discussed evidence, the direct
proof that sucrose acts as an energetic substrate during
the dark-to-light transition was still missing until
earlier this year when the work using a combination of
stomatal aperture assays and kinetic [U-13C]-sucrose
isotope
labelling
experiments
was
published
(Medeiros et al. 2018b). This work aimed to directly
follow the fate of the carbons released from sucrose
breakdown during light-induced stomatal opening. In
this study GC–TOF–MS was used revealing rapid and
high 13C enrichment in fructose and glucose following
dark-to-light transition. Clear 13C enrichment in gly-
colysis and TCA cycle intermediates as well as
glutamate and glutamine was also observed indicating
activation of these pathways during light-induced
stomatal opening. These new findings allowed a
reevaluation of the current models concerning the
role of sucrose in stomatal opening. It is now clear that
during stomatal opening sucrose is degraded providing
carbon skeletons for glutamine biosynthesis, however,
the physiological reason behind this is, as yet, far from
being understood (Lima et al. 2018; Medeiros et al. 2018b). For a long time sucrose was believed to exclusively
play an osmolytic role in stomatal opening (Daloso
et al. 2016a). However, by using metabolomics tools
recent findings have not only challenged this claim but
also proposed an energetic role of sucrose during light-
induced stomatal opening (for a complete overview
about sucrose roles during stomatal movements please
read Daloso et al. (2016a) and Lima et al. (2018)). The
section below compiles interesting reports focused on
the application of metabolomics tools to solve the
open questions about guard cell metabolism. The metabolic changes in guard cell-enriched
epidermal fragments from tobacco (Nicotiana taba-
cum L.) during light-induced stomatal opening were
evaluated alongside with isotope kinetic labelling
experiment by feeding the guard cell-enriched epider-
mal fragments with NaH13CO3 under light conditions
in order to understand the distribution of photosyn-
thetic fluxes in guard cells (Daloso et al. 2015). By
using a GC–TOF–MS platform it was observed that
concomitant with the decrease in sucrose, there were
increases in the level and 13C enrichment of the TCA
cycle-related metabolites. This report was the first
evidence supporting the hypothesis that sucrose is
catabolized within guard cells in order to provide
carbon skeletons for organic acid production. 2.2 Response to high CO2 concentration Interestingly, opposing metabolic responses
were observed between the two cell types, in that
while mesophyll cells presented increases in amino
acid, phenylpropanoid, redox metabolite, auxin and
cytokinin contents all of which were decreased in
treated guard cells. The outcome from both cell type
metabolite profile revealed that 26 and 27 metabolites
in mesophyll and guard cells were exclusively iden-
tified, respectively. Regarding primary metabolism,
constant increase of malate was observed across the
experiment in NaHCO3 treated guard cells. Addition-
ally, in these cells increases in fructose, glucose, and
mannose were observed at the early time-points
(Misra et al. 2015b). In
another
study
by
using
three
different y
g
Returning to the hormone response it is clear that
hormones are good targets to investigate guard cell
metabolomics. Indeed, considerable attention has
been paid to ABA, but the crosstalk between ABA
and other hormones can potentially also affect stom-
atal movements. For instance, brassinosteroid and
jasmonate can act concurrently with ABA promoting
senescence and programmed cell death (Hossain et al. 2011; Zhang et al. 2009). Other hormones such as
cytokinin, auxin, salicylic acid and ethylene have been
showed to act antagonistically to ABA under water
deficit conditions (Chen et al. 2013; Ha et al. 2012;
Meguro and Sato 2014; Tanaka et al. 2005). Therefore,
we believe that metabolomics tools for specific cells
can be useful in order to evaluate the metabolic
changes provoked by the hormone signaling network
and improving methods for single cell hormone
measurements will likely greatly increase our ability
to understand and model hormonal aspects of the
control of stomatal movements. In
another
study
by
using
three
different
approaches; GC–MS/MS, LC–MRM–MS, and LC–
TOF–MS it was able to successfully annotate 358
metabolites (Geng et al. 2016). In this study the
metabolic responses in guard cell-enriched epidermal
fragments of B. napus were evaluated in response to
elevated CO2 (800 ppm) in a time-course experiment
(over 1 h). Interestingly, these authors reported
changes in primary metabolites such as sugars, fatty
acids, amino acids and nucleotides under elevated CO2 123 123 Theor. Exp. Plant Physiol. (2019) 31:91–102 95 treatment. Additionally, ROS production increased
and stomatal aperture decreased over the time. 2.3 Understanding the roles of Suc in guard cells Another
important finding from this work was the qualitative
demonstration that CO2 fixation in tobacco guard cells
can be catalyzed both by ribulose-1,5-biphosphate
carboxylase/oxygenase
(Rubisco)
and
phos-
phoenolpyruvate carboxylase (PEPc) (Daloso et al. 2015). 2.2 Response to high CO2 concentration Curi-
ously, following the previous findings in metabolic
guard cell response to ABA (Zhu and Assmann 2017),
alterations in flavonoid, organic acid, sugar, fatty acid,
phenylpropanoid and amino acid metabolic pathways
indicated changes in both primary and specialized
metabolic pathways in guard cells. However, the
novelty presented by this work was that after 10 min
under elevated CO2 treatment the jasmonic acid
biosynthesis pathway was significantly induced (Geng
et al. 2016). SYNTHASE 3 (SUS3) in guard cells (Daloso et al. 2016b). A kinetic isotope labeling experiment follow-
ing the metabolic fate of NaH13CO3 during the dark-
to-light transition was performed using guard cell-
enriched epidermal fragments which were analyzed
via GC–TOF–MS. Minor changes were observed in
the metabolite profile of whole leaves, whereas
increased fructose and decreased organic acids levels
were observed in the transgenic plants. In good
agreement with the results from the previous study,
increased labeling of the TCA cycle-related metabo-
lites was observed, suggesting that breakdown of
sucrose is a mechanism providing carbon skeletons
during stomatal opening (Daloso et al. 2016b). SYNTHASE 3 (SUS3) in guard cells (Daloso et al. 2016b). A kinetic isotope labeling experiment follow-
ing the metabolic fate of NaH13CO3 during the dark-
to-light transition was performed using guard cell-
enriched epidermal fragments which were analyzed
via GC–TOF–MS. Minor changes were observed in
the metabolite profile of whole leaves, whereas
increased fructose and decreased organic acids levels
were observed in the transgenic plants. In good
agreement with the results from the previous study,
increased labeling of the TCA cycle-related metabo-
lites was observed, suggesting that breakdown of
sucrose is a mechanism providing carbon skeletons
during stomatal opening (Daloso et al. 2016b). 2.4 Metabolic changes due to altered organic
acids transport in guard cell Malate and fumarate have been highlighted as impor-
tant players during stomatal movements. Given their
importance in controlling stomatal movement, chan-
nels and transporters were recently identified and
functionally characterized to be involved with organic
acid transport at both plasma membrane and tonoplast
of guard cells (Eisenach and De Angeli 2017; Marti-
noia 2018). In this context metabolomics approaches A follow-up study was performed using transgenic
tobacco plants specifically overexpressing SUCROSE 12 3 3 Theor. Exp. Plant Physiol. (2019) 31:91–102 96 fragments, intermediates of the tricarboxylic acid
cycle were altered differently in the two tissues. For
instance, lower malate and fumarate accumulation was
observed in leaves but not in epidermal fragments
(Medeiros et al. 2017). These findings confirmed the
previous hypothesis of functional redundancy for
organic acids transport at the tonoplast in guard cells
(Meyer et al. 2011). have been used in order to evaluate changes in the
metabolism of both leaves and guard cells (Medeiros
et al. 2016, 2017). QUAC1 (Quick activating Anion Channel 1), also
known as ALMT12, is a member of ALMT (Alu-
minum activated malate transporter) family, was
identified as an ABA-dependent anion-selective chan-
nel at the guard cell plasma membrane responsible for
malate efflux during stomatal closure in a voltage-
dependent manner (Meyer et al. 2010). Loss-of-
function of QUAC1 resulted in an impaired kinetic
of stomatal closure in response to dark and high CO2
levels as well as reduced ABA-induced stomatal
closure coupled with significant changes in organic
acid accumulation as well as increases in both stomatal
and mesophyll conductance (Medeiros et al. 2016). By
using the GC–TOF–MS system 40 metabolites related
to primary metabolism were successfully annotated,
showing relatively few changes in the metabolite
profile of mutant leaves. Interestingly, looking at the
organic acids levels, it was observed that maleic acid,
malate, fumarate, succinate increased in the knockout
lines. These results indicate that changes in organic
acids homeostasis in guard cell can affect the whole
leaf
metabolism
highlighting
the
coordination
between mesophyll and guard cell metabolism. Recently the report of a new ALMT member
(ALMT4) demonstrated it to be a phosphorylation-
dependent anion channel, releasing malate from the
vacuole during stomatal closure in response to ABA
(Eisenach et al. 2017). 2.4 Metabolic changes due to altered organic
acids transport in guard cell almt4 knockout mutants
displayed normal stomatal opening, but by contrast
to other organic acids transporters at the tonoplast,
almt4 plants presented impaired stomatal closure in
response to ABA, but not to darkness (Eisenach et al. 2017). Altogether these exciting findings suggest that
independent mechanisms may be activated under
specific conditions and individual organic acid trans-
porters are regulated independently in a stimulus-
dependent manner at the tonoplast (Medeiros et al. 2018a). 2.5 The guard cell metabolome during plant cell–
microorganism interaction A vacuolar malate transporter (tonoplast Dicar-
boxylate Transporter—tDT) was also recently char-
acterized in Arabidopsis. tDT was considered the main
malate transport system at the tonoplast and required
for the proper accumulation of malate in Arabidopsis
leaves (Emmerlich et al. 2003; Hurth et al. 2005). tDT
was only recently functionally characterized (Frei
et al. 2018). Highly purified tDT protein showed
transport activity as malate and citrate 1:1 in an
antiport mode, also accepting fumarate and succinate
as substrates (Frei et al. 2018). However, the func-
tional role of tDT in guard cells remained unclear until
recently when the metabolic impact of the tDT
absence was studied (Medeiros et al. 2017). This
study also used GC–TOF–MS technology to assess
metabolic changes in leaves and guard cell-enriched
epidermal fragments of wild type and tdt knockout
lines. This study showed that manipulating vacuolar
organic acid transport by suppressing tDT greatly
impacts mitochondrial metabolism but has minor
effects on stomatal and photosynthetic capacity. Interestingly, by comparing the metabolite profiles
of
leaves
and
guard
cell-enriched
epidermal The interaction between plants and microorganisms is
known to triggers signaling cascade and metabolic
responses. However, the main issue of analyzing
changes in the plant metabolic profile during the
interaction plant cell–microorganism is avoiding con-
tamination with metabolites from the coexisting
microorganism (Pang et al. 2018). A very recent work
addressed this issue by implementing an effective
stable isotope labeling approach coupled to LC–
MRM–MS measurements (Pang et al. 2018). The
plant bacterial pathogen Pseudomonas syringae pv. tomato DC3000 was incubated in isotope-labeled
medium (Celtone complete medium with [ 98% 13C
and [ 98% 15N). This procedure was used in order to
differentiate plant metabolites from bacterial metabo-
lites. This study suggested that bacterial infection may
affect stomatal movement by reprograming the guard
cell signaling network and primary metabolism. It was
observed that at 30 min after bacterial infection the
most affected pathways were related to amino acid
metabolism. Whereas at 180 min, aminoacyl-tRNA
biosynthesis, the tricarboxylic acid (TCA) cycle, and 123 123 Theor. Exp. Plant Physiol. (2019) 31:91–102 97 Despite the fact that mass spectrometry-based
metabolomics platforms have been widely used in
plant biology, these platforms have only been recently
used to investigate guard cell metabolism (Medeiros
et al. 2015; Misra et al. 2015a). 2.5 The guard cell metabolome during plant cell–
microorganism interaction The establishment of a
reproducible and reliable analytical platform is crucial
for the performance of -omics studies, which can
subsequently aid in the generation of mathematical
models. Some models for leaf metabolism of C3, C4,
and CAM plants have been published (Arnold and
Nikoloski 2014; Cheung et al. 2014, 2015; Chew et al. 2014; Masakapalli et al. 2010, 2013; Pfau et al. 2018;
Seaver et al. 2015; Shameer et al. 2018; Sweetlove
et al. 2013; Wang et al. 2012). Recently the AraCORE
model created for leaf primary metabolism (Arnold
and Nikoloski 2014) has been adapted to model guard
cell metabolism (Robaina-Estevez et al. 2017). Large-
scale omics data have been used to constrain different
models and thus more faithfully predict metabolic
fluxes through metabolic networks (Nikoloski et al. 2015; Orth et al. 2010). This is part of a systemic
characterization of plants, in which the data generated
is used either to create or to improve mathematical
models, following a ‘‘design–build–test–learn’’ cycle
(Sweetlove et al. 2017). carbon fixation as well as the biosynthesis of pheny-
lalanine, tyrosine, and tryptophan were dramatically
affected. In summary, metabolite profiling has aided the
identification of several novel interactions between the
TCA cycle, photosynthesis and stomatal function as
well as guard cell responses to both biotic and abiotic
stresses. Due to the vast complexity of the metabolic
and molecular regulatory pathways underlying guard
cell function they remain not fully understood. How-
ever, the improvements of new technologies toward
metabolomics approaches will help us to better
understand stomatal responses. Furthermore, the
usage of metabolic data into mathematical models
can optimize predictions of stomatal behavior in a
changing environment. Studies in this direction have
been performed and are discussed in the following
section. 3 Metabolomics data for improving stomatal
behavior predictions Transcriptomics, proteomics, and metabolomics stud-
ies carried out using guard cells as model have
contributed substantially for our understanding on
how plant cells sense and respond to environmental
and endogenous signals such as relative air humidity,
CO2, ABA, and sucrose (Medeiros et al. 2015). All of
the knowledge gained from these studies are useful for
the establishment of systems biology approaches. Transcriptomic data has advantage over other omics
platforms for the use in mathematical models given the
higher
coverage
of
transcriptomic
techniques
(Alseekh and Fernie 2018). Comparatively, pro-
teomics and metabolomics platforms need substantial
improvement to increase the number of analytes
detected. This is an important issue for the creation
of genomic scale metabolic models, in which several
reactions of the metabolic network are included in the
model (Hyduke et al. 2013). However, it is noteworthy
that gene expression does not always correlate to
protein expression/activity and subsequently to meta-
bolic changes (Fernie and Stitt 2012; Lehmann et al. 2009). In this scenario, metabolomics approaches
provide vital information by which to understand the
structure and the regulation of metabolic networks as
well as to enhance the predictive power of metabolic
models. Existing models and their combinations have
clearly contributed to the predictive modelling and
refinement of the relationship between stomatal con-
ductance and the players regulating stomatal function. Unfortunately, limited success in clarifying how
changes in stomatal movements, by consequence
water loss and CO2 gain, are affected by the interplay
of environment, cellular levels, as well as the coordi-
nation between mesophyll and guard cells. The
OnGuard platform is one of the first modelling
approaches created specifically for guard cells (Hills
et al. 2012). Although the possibility to analyze guard
cell signaling pathways using the theory of biological
networks was postulated much earlier (Hetherington
and Woodward 2003). OnGuard is a software built
based on the knowledge of transport and accumulation
of important molecules for guard cell osmoregulation
such as K?, Cl-, Ca?2, malate, and sucrose. Simula-
tions of the transport of these molecules through both
plasma and tonoplast membranes of guard cells can be
now performed by OnGuard (Chen et al. 2012; Hills
et al. 2012; Wang et al. 2014). Despite its great
contribution to understand important process that
regulates stomatal movements, OnGuard software is 12 3 3 Theor. Exp. Plant Physiol. 3 Metabolomics data for improving stomatal
behavior predictions (2019) 31:91–102 98 output of the model can be manipulated according to
the aim of the study (Medeiros et al. 2015; Orth et al. 2010). The AraCOREred model indicates that tran-
scriptome data can be used to predict cell-specific
metabolic changes. This model can then be used in the
future to predict guard cell metabolic responses to
ABA and sucrose, given the availability of transcrip-
tome data under ABA and sucrose treatment (Bates
et al. 2012; Bauer et al. 2013; Leonhardt et al. 2004;
Pandey et al. 2010). However, it is important to
highlight that AraCOREred model was created assum-
ing the guard cell metabolism at steady states. Due to
the dynamic nature of the guard cell metabolism, the
current challenge is to model guard cells under non-
steady state condition, which would seem to be more
physiologically relevant given the high plasticity of
guard cell metabolism under varying conditions. Modelling strategies such as dynamic flux balance
analysis seems to be great option to fulfil this gap. The
establishment of such non-steady state modelling,
while challenging, may increase the reliability of
predictions by the current available models for guard
cell functioning. not able to predict metabolic alterations in guard cells. This is partially due to the relative scarcity of guard
cell metabolomics data. However, given that recent
metabolomics approaches (reviewed above) being
adapted to study guard cells, this hurdle is no longer
valid. LC–MS and GC–MS-based metabolomics studies
have provided important information regarding the
guard cell response to ABA, bicarbonate, and sucrose
(Daloso et al. 2015; Jin et al. 2013; Medeiros et al. 2018b; Misra et al. 2015b; Zhu and Assmann 2017). Furthermore, the establishment of a methodology to
perform
13C-feeding experiments with the use of
labelled substrates resolve a long debate regarding the
role of sucrose for guard cell regulation and the mode
of CO2 fixation found in guard cells (Daloso et al. 2015; Medeiros et al. 2018b). RubisCO-mediated CO2
fixation has long been thought to be absent in guard
cells (Outlaw and Manchester 1979). However, the
13C distribution found in guard cells subjected to light
indicate a clear CO2 fixation mediated by both
RubisCO and PEPc (Daloso et al. 2015). The results
of this study were substantially different from those
observed in the Arabidopsis rosette which is largely
composed of mesophyll cells (Szecowka et al. 2013). 3 Metabolomics data for improving stomatal
behavior predictions Taking this into account, a systems biology study
compared the NaH13CO3 distribution in mesophyll
and guard cell protoplasts by combining flux predic-
tions using a genome scale metabolic model and the
13C distribution measured in both cells via GC–TOF–
MS (Robaina-Estevez et al. 2017). The modelling
strategy of this work was based in previous published
transcriptome data (Bates et al. 2012; Pandey et al. 2010) that were integrated into the AraCORE model
(Arnold and Nikoloski 2014). As output, the new
model, namely AraCOREred, provides cell-specific
metabolic predictions of 455 reactions which com-
prise 374 primary metabolites of mesophyll and guard
cells (Robaina-Estevez et al. 2017). Interestingly, the
results of this work confirmed that guard cells have
higher anaplerotic CO2 fixation by PEPc than meso-
phyll cells as well as regarding the source of sucrose
and malate within guard cells. 123 4 Concluding remarks Over the past 5 years the application of metabolomics
approaches in order to better understand guard cell
metabolism and the mechanisms underlying the
stomatal responses to environmental and endogenous
cues has increased. Metabolite profiling of isolated or
enriched-guard cells extracts has aided the identifica-
tion of several novel interactions between the TCA
cycle, photosynthesis, and stomatal function. Further-
more, guard cell metabolomics studies have fulfilled
some important unsolved questions regarding the roles
of sucrose and organic acids transport and metabolism
during stomatal movements as well as the guard cell
responses to water deficit, high CO2 concentrations,
and plant defense against pathogens. Alongside other omics technologies, metabolomics
studies of the highly specialized guard cell have
proven to be a powerful tool for understanding the
hormonal and metabolic regulation of stomatal move-
ments. This is particularly important since plant
growth, development, and yield depend greatly of
the fine control of plant–environment gas exchanges. Given the complexity of the interconnected networks The use of such genome scale, constraint-based
metabolic models is important to understand the
regulation of plant metabolism (Sweetlove et al. 2017). Mainly because the model can predict meta-
bolic changes at subcellular level under different
conditions, given that the input of signals and the 123 Theor. Exp. Plant Physiol. (2019) 31:91–102 99 governing stomatal movements under varying condi-
tions, we argue that metabolomics approaches are
highly valuable. An emerging technology that may
greatly aid in this vein is metabolite imaging (Bjarn-
holt et al. 2014), which may soon be able to aid our
understanding of metabolite movements between the
mesophyll and guard cells. Ongoing systems biology
approaches, combining data from different omics
technologies will aid in elucidating the mechanisms
underlying stomatal control and furthermore could
ultimately unravel targets for modulation of stomatal
responses to environment and towards the design of
new strategies for engineering better plant perfor-
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doi.org/10.1104/pp.112.197350 Acknowledgements
Open access funding provided by Max
Planck Society. This work was supported by funding from Max
Plank
Society,
Conselho
Nacional
de
Desenvolvimento
Cientı´fico e Tecnolo´gico (CNPq-Brazil, Grant 402511/2016-6,
W.L.A), Fundac¸a˜o de Amparo a` Pesquisa do Estado de Minas
Gerais (W.L.A., FAPEMIG, Grants APQ-01078-15 and RED-
00053-16, W.L.A.). Research fellowships from CNPq-Brazil to
W.L.A. and D.M.D and scholarships from Coordenac¸a˜o de
Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES-Brazil)
to L.M.L and H.O.O. are gratefully acknowledged. Acknowledgements
Open access funding provided by Max
Planck Society. This work was supported by funding from Max
Plank
Society,
Conselho
Nacional
de
Desenvolvimento
Cientı´fico e Tecnolo´gico (CNPq-Brazil, Grant 402511/2016-6,
W.L.A), Fundac¸a˜o de Amparo a` Pesquisa do Estado de Minas
Gerais (W.L.A., FAPEMIG, Grants APQ-01078-15 and RED-
00053-16, W.L.A.). Research fellowships from CNPq-Brazil to
W.L.A. and D.M.D and scholarships from Coordenac¸a˜o de
Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES-Brazil)
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induced reactive oxygen species are modulated by 123 12
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The sustainability of human resource planning for construction projects
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cc-by
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The sustainability of human resource planning for construction
projects Adegbenga Raphael Ashiru1 and Adebimpe Lucia Ashiru2 1 Lecturer- Department Of Building Technology, Federal Polytechnic, Kaura - Namoda. Zamfara - State. Nigeria
2 Lecturer- Department Of Business Administration and Management, Federal Polytechnic, Kaura - Namoda. Zamfara -
State. Nigeria Abstract. Human Resource planning for construction project is described as a way of managing human
resources within a growing nature of construction industry. Nonetheless, management of human resource
is an extremely stimulating task. However, in spite of the substantial resources allocated to construction
planning, there are rising concerns over its failure to achieve its goals and different application of planning
strategies. It is against this backdrop that this research was carried out to examine the sustainability of
Human resource planning for construction projects to justify the aforesaid statement. Furthermore, the
researchers adopted a quantitative approach that provided a broader understanding of the research. SPSS
Version 20 was used to obtain the descriptive and inference statistical analysis. Interestingly, majority of
the literature findings were justified by the empirical findings noting that there is correlation between the
two findings except where the respondent chose combination of the causes of neglect of HR planning on
construction projects stressing that one cause cannot significantly neglect the process on construction
projects. Conclusively, the research results indicated that through improving the practicality of Project HR
planning by construction organisations, it would contribute significantly towards improving performance
both at organisational and project level. The findings recommended guidelines for improving the visibility
and adaptability of sustainable HR planning for construction projects and further provided justification for
implementing its policies and procedures. © 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/). * Corresponding Author: ashiruadegbenga@fedponam.ed.ng , 0
(2019)
MATEC Web of Conferences 258
SCESCM 2018
2018 , 0
(2019)
MATEC Web of Conferences 258
SCESCM 2018
2018 https://doi.org/10.1051/matecconf/201925802018 P Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
es/by/4.0/). ces. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
/) 2.1.1 Evaluation of Current Resources Evaluation current human resource is another stage in
HR planning through assessing employee’s interest,
abilities, skills and experiences. After comparing these
with SHRM plans, resource shortage that required filling
of skills gaps will be outlined [17]. Through this,
efficient database must be created to accommodate
information about employees’ qualification, interests,
training and experience during employment lifespan. 2.1.3 Internal Availability Analysis Before
carrying
out
external
recruitment,
the
organisation may check the availability of existing
employee through employee future movement analysis
in relation to transfers, promotions, resignations,
terminations and retirements. However, organisations
can predict these trends to prevent temporary shortage of
employee [18]. An organisation could compare internal
resources availability with the future needs through HR
approach. Through this, internal employee-development
and external recruitment could be determined. 1 Introduction of sustainable HR planning for construction
projects. of sustainable HR planning for construction
projects. •
To draw conclusions on the relevance of
sustainable HR planning for construction projects
and makes recommendations to practitioners and
for future research. As human resource (HR) is significant to all
industries,
including
the
construction
industry,
maintaining and attracting skilled employees is a
priority. Hence, complexities in human contacts within
construction industry will bring about construction-based
issues of HR planning in such organisations [7]. However, it is against this backdrop that this research
was carried out to answer the following fundamental
research questions: Nonetheless, construction firms must ensure that
current and future market analysis are carried out to meet
functions of HR planning thereby ensuring that right
percentage of individuals with right skill are considered
[15]. By analysing the current human resources as well
as linking these to potential opportunities in future
market
may
allow
recruitment
and
employee-
development plans decisions to be made [16]. Subsequently, Leng suggested HR planning approaches
that could use to ease HR problems as follows [15]; •
What is the relationship between HR planning and
construction project productivity; •
How can sustainable Human Resource planning be
used to improve individual and team performance
at both construction organisation and project levels To ease the problem which underperformance have
placed on construction industry, therefore, researcher
will discuss basic ways in which HR planning could be
used to manage construction projects to justify
Loosemore’s opinion that team or individual behavior
have posed a rising concern on risks in construction
projects and recommended that effective human resource
planning is the possible way to alleviate those
construction risks [8]. 2 General Overview of Construction
Industry Construction industry offers opportunity to mankind
through job creation and the industry offers substantial
responsibilities for those who work in it [9]. Nonetheless, the industry creates built environment as it
designs, produce, conserve and demolishes structures
[10]. A main contractor (Builder) tenders for
construction contracts and when contract is awarded, the
Builder further award subcontract to specialised
contractors. Furthermore, micro organisations dominates
construction sector with various organisation merging
together to form teams to carry-out a project [11]. Those
involved in project delivery include professionals such
as clients/owner and general contractor, suppliers and
subcontractors forms a team of contractor. Also,
Architects, Civil Mechanical, Structural, Electrical
Engineers,
Land
and
Quantity
Surveyors
forms
Consultant’s team [12]. However, construction industry
remains a poorly understood sector despite its
contribution to economic and social growth of nations
[11]. Finally, the agenda of performance improvement
has not been discharging by management of workforce
within the sector. 2.1.2 Job Analysis and Design Job analysis is a process of analysing organisational
restructuring methods to meet current and future
business needs through collection of information of
existing human resource and this further includes the
following: •
Assigning individual responsibilities to future
needs
by
examining
the
organisational
structure; •
Identifying shortages or misfits; Redesigning misfit positions and individuals •
Redesigning misfit positions and individuals
responsibilities to suit the firm’s future needs. responsibilities to suit the firm’s future needs. 1 Introduction of each employee performance measurement through
effective training
[4]. A system that
measures
performance is needed in order to monitor improvements
within construction teams [5]. Also, Langford et al
argued that construction industry has a habit to shed
personnel as part of a survival plan, retraining those
whose skills are replaceable [6]. To justify the aforesaid
statement, the research aimed to examine and promote
the sustainability of HR planning for construction
projects and to achieve this, the under listed objectives
are set to achieve the aim of the research: The word planning is defined as a detailed system for
achieving an objective meaning. However, a detailed
system includes the method, the activities to carryout,
time of those activities and resources including finance
[1]. Moreover, an organisational planning creates
efficiency and effectiveness in an organisation. Its
provides clearer focus which bonds managers and
subordinates in creating strong teams and produces
limitless fulfillment among planners with common
vision. Nonetheless,
effective
planning
before
implementation is significant for successful delivery of
every project but there are indications of inadequate
effort and time in planning construction projects
especially at tender and pre-contract stages [2]. •
To critically review literature relating to challenges
of HR planning for construction projects and
develop proper understanding of current situation
in Construction industry; •
To collect data from contracting organisations
including
Wolverhampton
city
council
on
challenges of HR planning for construction
projects; Furthermore, in spite of the substantial resources
allocated to construction planning, there are rising
concerns over its failure to achieve its goals and the
different
application
of
planning
strategies
[3]. Nevertheless, the significant factor in implementing
organisation’s planning policy in construction is the need •
To analyse data collected and validate a framework
that could help stakeholders focuses their priorities , 0
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between HRM and the overall organisational strategic
plan. Nevertheless, Bulla and Scott argued that HR
planning is the process of acknowledging organisational
HR requirements and plans to satisfy those requirements
are made [13]. Furthermore, Reilly maintained that HR
planning is a way that an organization estimates the
demand for workforce and evaluates its nature, size and
sources of supply to meet the demand [14]. HR planning
further includes creation of strategies in recruitment and
selection,
retention,
talent
management,
absence
management, flexibility and employer brand. 2.1.4 Employee Turnover Analysis 2.1.4 Employee Turnover Analysis 2.1.4 Employee Turnover Analysis
Employee turnover analysis can be carried out for two
purposes, namely; to ascertain possible future employee
replacement and identify purpose for employee transient
and best action to retain them. In fact, an assessment
could be carried out on the effect of promoting and
transferring of individuals in an organisation through HR
planning [19]. •
Interpersonal interfaces – this could be formal
or informal relationships between various
individuals on a site. (II) Labour requirement- This required individual or
team competencies at different time frame. General
resource requirements may involve labour requirement
which form part of resource planning. (III) Organisational Constraints For efficiency, effectiveness and upward growth in an
organisation, there must be agreed objectives to be able
to manage risks with establishment of goals and targets
through organisational strategic level [6]. Apparently,
the effective use of individuals involved in the project is
the essence of project HRM. This revolves round
identifying,
assigning
project
responsibilities,
documenting and reporting relationship. Although,
necessity for planning is very vital in construction due
to; •
Collective bargaining– prescribed agreements
with labour
union
may require assured
responsibilities or relationships; •
Project Team Preferences– Success of a team
in
previous
project
can
probably
be
recommended for similar project in future. B) Techniques
And
Tools
For
Project
Organisational Human Resource Planning B) •
project complexity which requires a huge
number of human input; •
The overall competitive approach in an unstable
market and aggressive environment. •
Project Templates- Since every project is
distinctive, definitions of responsibility of a
comparable project can help hasten the process
of organisational planning in a new project. Therefore, project HR manager must possessed wide
knowledge about market, product and method the
organisation is operating. This will allow project work
analysis and labour requirement be carried out. However, Leng identified in Table 1, a synopsis of
project HR planning as follows [15]: •
Human resource procedures and practices –
Many
organisations
operates
different
guidelines, policies and procedure with series of
facets of organisational planning that can
support project team. Example is where
organisations allow a manager to perform his
duties like a coach. •
Human resource procedures and practices –
Many
organisations
operates
different
guidelines, policies and procedure with series of
facets of organisational planning that can
support project team. Example is where
organisations allow a manager to perform his
duties like a coach. Table 1- Overview of Project HR Planning
INPUT
TOOLS &
TECHNIQUES
OUTPUT
Interfaces in the
project
Project
Templates
Roles and
responsibility
Labour
Requirement
Human resource
procedures
&
practices
Labour
management plan
Organisational
Constraints
Analysis
of
stakeholder
Organisational
chart and
Supporting
Documents
) Inputs to Project Human Resource Planning Table 1- Overview of Project HR Planning •
Analysis of Stakeholder– The needs of
stakeholders should be identified, analysed and
met. C) Outputs from the project organisational human
resource planning C) Outputs from the project organisational human
resource planning (I) Roles and Responsibility – This is about who does
what and who decides what. This should be allocated to
suitable
project
professional. Most
roles
and
responsibilities may vary over time and allocated to
active professionals involved in project activities. (I) Roles and Responsibility – This is about who does
what and who decides what. This should be allocated to
suitable
project
professional. Most
roles
and
responsibilities may vary over time and allocated to
active professionals involved in project activities. A) Inputs to Project Human Resource Planning A) 2.1 Human Resource Planning in Construction A
process
that
ascertains
current
and
future
organisational human resources needs to achieve
organisational goals is described as Human Resource 2 , 0
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Technical interfaces- Technical interfaces
could be formal or informal relationship
between professionals on a project and this
happen within project phases where civil
engineer work could be compatible with design
of structural engineer. 3 Methodology •
Training needs- an employee skill is very
important on a project and those skills could be
developed as part of the project. However, the nature of work, organisation strategy,
economic and social changes are vital HR planning
factors
influencing
for
labour
force
supply
in
construction industry and it is very vital for the
management of the organisation to ensure the effective
use of HR planning and avoid inconsistency so that it
can bring coherence and compatibility between project
team members as well as the different group within the
structure of the firm [20]. The researcher distributed twenty (20) well-
structured questionnaires to selected respondents who
are managers involved in decision making at strategic
level for their respective organisations [6]. The
researcher recovered twelve (12) responses achieving
60% of the feedback. The data collected was analysed by
descriptive and inference statistics. The research
instrument also measures a reliability and validity test of
0.766. Furthermore, data relating to challenges of HR
planning were extracted from four questions which were
adopted from [15] to develop varying challenges of HR
planning for the research work. Indeed, the paper
presented various improvement methods that can be
adopted to encourage the practicality of the subject
matter as SPSS levene test was used to ascertain the
equality of variance for the research work. 3 Methodology This research was carried out in Wolverhampton West
Midlands, United Kingdom to identify the various
impacts of sustainability of Human Resource planning
on construction projects in Wolverhampton city. Nonetheless, non-probability sampling was adopted as
this enables us to select participants based on the
research area in Wolverhampton city. This further
assumed
a
purposive
sampling
strategy
where
respondents were selected at the discretion of the
researcher but with the sole purpose to be typical of the
whole population and the research question will be best
examined to evaluate respondent engagement. The
researcher adopted a quantitative approach that provided
a broader understanding of the research problem [20]
and [21]. SPSS Version 20 was used to obtain and
analyse the descriptive and inference statistical analysis. The researcher distributed twenty (20) well-
structured questionnaires to selected respondents who
are managers involved in decision making at strategic
level for their respective organisations [6]. The
researcher recovered twelve (12) responses achieving
60% of the feedback. The data collected was analysed by
descriptive and inference statistics. The research
instrument also measures a reliability and validity test of
0.766. Furthermore, data relating to challenges of HR
planning were extracted from four questions which were
adopted from [15] to develop varying challenges of HR
planning for the research work. Indeed, the paper
presented various improvement methods that can be
adopted to encourage the practicality of the subject
matter as SPSS levene test was used to ascertain the
equality of variance for the research work. This research was carried out in Wolverhampton West
Midlands, United Kingdom to identify the various
impacts of sustainability of Human Resource planning
on construction projects in Wolverhampton city. Nonetheless, non-probability sampling was adopted as
this enables us to select participants based on the
research area in Wolverhampton city. This further
assumed
a
purposive
sampling
strategy
where
respondents were selected at the discretion of the
researcher but with the sole purpose to be typical of the
whole population and the research question will be best
examined to evaluate respondent engagement. The
researcher adopted a quantitative approach that provided
a broader understanding of the research problem [20]
and [21]. SPSS Version 20 was used to obtain and
analyse the descriptive and inference statistical analysis. •
Job descriptions- an outlines of job title,
responsibilities,
Skills,
authority,
physical
environment, and other requirements to execute
a job. 4 Findings The literature sources agreed to varying challenges of
HR planning on construction projects. The four
identified factors considered are presented in table 2, 3, 4
and 5. •
Increases
Personal
Growth-
Project
team
commitment towards professional development
and challenges; Table 2. Factors HR Planning
Factors to look into when performing
HR planning operation for construction
projects
Mean
Std. Deviation
Does your organisation appraise labour
resources such as interest, abilities, skills
and experience? 3.99
1.187
Does
your organisation designs job
specifications
to
make
sure
right
individuals are in right positions
3.76
1.404
Source: Field Survey, 2017 •
Boost Business Performance- Ability of business
activities to perform through achieving low
operational
control,
employee
turnover
and
repetitive business. (I) Interfaces in the project •
Organisational
interfaces-
Organisational
interfaces
can
be
formal
or
informal
relationships between departments and could be
extremely complex or simple. Example could
be
an
emergence
compound
telecommunications
system
to
coordinate
different subcontractors on a project. (II) Labour Management Plan – It described project
team take off time and defines when and how labour will
be utilised. The labour plan could be very detailed and
be formal or informal which is subjected to project
requirements. 3 , 0
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Poor management and leadership commitment
•
Poor organisation’s management support;
•
Poor commitment of stakeholders concerning HR
practices
•
Resistance to change in new HR techniques and
tools
•
Neglect of significance of HR involvement in a
project (III) Organisational chart- This is a project reporting
relationships
in
graphical
representation. An
organisational
chart
is
a
typical
example
of
organisational breakdown structure (OBS) which could
be formal or informal and detailed framed that shows
departments in an organisation and their responsibilities
depending on project necessities. (III) Organisational chart- This is a project reporting
relationships
in
graphical
representation. An
organisational
chart
is
a
typical
example
of
organisational breakdown structure (OBS) which could
be formal or informal and detailed framed that shows
departments in an organisation and their responsibilities
depending on project necessities. (IV) Supporting document – Organisational planning
supporting detail is subjected to project size and area. Supporting detail information may include the following, 2.3 Impacts of Sustainable HR Planning on
Performance for Construction Projects •
Enhance Technical Performance- To achieve
technical performance of a project, technical
requirements
must
be
specified
at
the
commencement of the project in order to eliminate
human error, possible delay and low skill level. •
Increases Technical Innovations- Effective ways
of identifying technical problems during the project
with ability of solving them. Such innovation
includes ways of enhancing critical thinking and
problem solving abilities. •
Improves Effectiveness In execution Of Project-
This is an effective way of meeting time and cost
targets. This involves achieving quality standard,
time and cost targets. 2.4 Causes of Failure in HR Planning for
Construction Projects Causes of failure in Project HR Planning are highlighted
by [15] as follows: 4 , 0
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and widely practice by construction organisations. The empirical result indicated that most of the
respondents Strongly Agree that evaluation the current
and existing resources is paramount important for their
organisation where individual interest, abilities, skills, or
experience in HR Planning could be carried out before
beginning of any construction projects with table 2
reflecting opinion with the mean 3.99. Also, job
specification as a major factor is considered at an
Agreeable rate. This indicated that efficient design of job
specifications could make responsibilities be allocated
right individuals reflecting a mean of 3.76. Therefore,
the literatures justified empirical result. Similarly, [23] opined that through effective HR
technical innovation and performance could be improved
to reduce human error, delays and enhance team problem
solving skills. It is strongly agree that effective Project
HR planning in practice will improve project deliveries
to cost, time and standard quality through selection of
right individuals for the right job reflecting mean 3.69. Continuous training of teams to enhance competencies
will reduce chance of delay in project team task
performance, human error and further improve problem
solving abilities. Table 3. Techniques and Tools in HR Planning
Application of techniques and tools in HR
planning
Mean
Std. Deviation
Does your organisation practices HR
procedures and guidelines in retaining
project teams
3.53
1.605
Does your organisation enhance labour
management plan to reduce cost and
increase motivation
3.71
1.456
Source: Field Survey, 2017 Table 3. Techniques and Tools in HR Planning Table 5. Failure in HR Planning
Causes of failure
in HR planning for
construction
Mean
Std. Deviation
A, B, C, D, E
3.90
0.508
B,D
3.09
1.023
B,E
3.06
0.799
A,D
3.07
1.995
A,C,D,E
3.79
1.478
A,B,D
3.57
1.282
B,D,E
3.48
1.285
A,D,E
3.18
1.240
A,E
3.89
0.891
Source: Field Survey, 2017 Analysis of table 3 shows that respondents strongly
agreed that organisation set standard practices for HR
procedures in allocating team responsibilities and
producing guidelines for job activities reflecting mean of
3.53. 2.4 Causes of Failure in HR Planning for
Construction Projects This shows that job guidelines ought to obviously
outline the work scope, job function, what to be carried
out with essential skills and capabilities required from
project team to successfully carryout the construction
project. Eventually, the scale with mean 3.71 shows a
strong positive approach practices when an organisation
utilise standard HR procedures and guidelines and the
feedback from labour management plan shows that
respondents justified the literature that organisation
should look into a suitable rewarding plan that would
enhance level of employee’s motivation and cost
reduction to improve effectiveness. Source: Field Survey, 2017 Identified failures are represented in table 5 with
alphabet A-E and define as - A- Poor of effective
management and leadership commitment; B- Poor
organization’s
management
support;
C-
Poor
stakeholders commitment in regards to HR practices; D-
Resistance to change in new HR techniques and tools; E-
Neglect of important of HR involvement in a project). g
j
Nevertheless, the scales with mean 3.90; 3.09; 3.06;
3.07; 3.79; 3.57; 3.48; 3.18 and 3.89 shows that the
entire occurrence have greater contribution to the neglect
of important construction Project HR planning. Table 4. HR Planning on Performance
Effect of HR planning on performance
for construction projects
Mean
Std. Deviation
Does your organisation considers improving
team performance to improve efficiency on
the
construction
project
through
in
individual skills, competency and team
behaviour. 3.08
1.569
Does your organisation retrain project team
to enhance efficiency and performance
3.18
1.368
Does effective Project HR planning aids
technical
innovation
improvement
and
efficient performance by eliminating delays,
labour error and upsurge manner of problem
solving
3.69
1.625
Source: Field Survey, 2017 Table 6- Independent Sample t Test for Perceptions Factors
in HR Planning
Variables
M
SD
T
df
P-
VALUE
Does
your
organisation
undertakes labour
resources
appraisals such as
interest, abilities,
skills
or
experience. Project managers
1.80
0.76
-3.21 24
0.004*
Non Project
managers
3.10
1.37
Does
your
organisation
design
job
specifications
to
make
sure
that
responsibilities are
given
to
right
individuals. Project managers
1.70
0.66
-2.096
5.765
0.087
Non Project
managers
2.69
1.26
*significant at 0.05 Level of P-Value Table 6- Independent Sample t Test for Perceptions Factors
in HR Planning
Variables
M
SD
T
df
P-
VALUE
Does
your
organisation
undertakes labour
resources
appraisals such as
interest, abilities,
skills
or
experience. 2.4 Causes of Failure in HR Planning for
Construction Projects Project managers
1.80
0.76
-3.21 24
0.004*
Non Project
managers
3.10
1.37
Does
your
organisation
design
job
specifications
to
make
sure
that
responsibilities are
given
to
right
individuals. Project managers
1.70
0.66
-2.096
5.765
0.087
Non Project
managers
2.69
1.26
*significant at 0.05 Level of P-Value Table 6- Independent Sample t Test for Perceptions Factors Source: Field Survey, 2017 The observation of [23] argued that to improve
efficiency and performance on construction projects,
organisations need to place project team on formal
training either on-job or off-job training. Nevertheless,
the scenario as seen in table 4 having mean 3.08 and
3.18 specifies that formal developmental training of
project team may improve efficiency and effectiveness
of Human Resource. Nonetheless, on-job training offers 5 , 0
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2018 https://doi.org/10.1051/matecconf/201925802018 Table 6 is the result of perception factors on Project
Managers and Non-Project Managers. At 0.05 P- value,
both Project managers and non- Project managers seems
to strongly agreed on factors to look into when
performing HR planning operation for construction
projects such that their respective organisations create
job specifications to ensure that right responsibilities are
given to right individuals (t= -2.096, df = 5.765). On the
other hand, non-Project managers hold stronger
perception than Project managers for their respective
organisations
to
undertake
employee
resources
appraisals such as skills, abilities, experience and
interests (t = -3.21, df = 24). The implication of this is
that non-Project managers agree more than Project
managers that their respective organisations always
undertake employee resources appraisals such as skills,
abilities, experience and interests. management and avoid inconsistency so that HR
planning for construction project can bring sustainable
coherence and compatibility between project team
members as well as the different group within the
structure of the organisation. Lastly, transformational
leadership from the organisation top management is
necessary to help in creating a positive organisational-
project culture. 5 Conclusion Spearman’s Rho Correlations Between Five Principal
Causes of Neglect of HR Planning
HR Tools,
Techniques
And Output
Poor
effective
management
and
leadership
commitment
Poor
organisation’
s
management
Support
Poor
stakeholde
rs
commitme
nt in
regards to
HR
practices
Resistance
to change in
new
HR
techniques
and tools
Neglect
of
important of
HR
involvement
in a project
Project
Templates
Correlation
0 .006
0.269
0.044
-0.098
-0.105
Sig (p<0.05)
0.976
0.169
0.848
0.634
0.616
Analysis of
stakeholder
Correlation
-0.066
-0.136
0.087
0.014
0.284
Sig (p<0.05)
0.742
0.500
0.682
0.938
0.157
Human
resource
procedures
and practices
Correlation
0.064
0.005
0.146
0.371(*)
0.273
Sig (p<0.05)
0.762
0.982
0.485
0.048
0.181
Roles and
responsibility
Correlation
-0.312
0.026
0.334
-0.124
0.032
Sig (p<0.05)
0.125
0.904
0.096
0.555
0.888
Employee
management
plan
Correlation
-0.113
0.065
0.111
0.188
0.234
Sig (p<0.05)
0.579
0.757
0.584
0.379
0.247
*Correlation significant at 0.05 Level of P-Value Table 7. Spearman’s Rho Correlations Between Five Principal
Causes of Neglect of HR Planning *Correlation significant at 0.05 Level of P-Value Spearman’s Rho correlation findings in Table 7 for
five principal causes of HR planning neglect indicated
that at 0.05 level of P-value the research shows that there
is a strong correlation between resistance to change in
new HR techniques and tools and Human resource
procedures and practices (correlation = 0.371 and p-
value= 0.048) and was found to have better effect on site
productivity since correlation significant is less than
0.05. The implication of this correlation is that the more
a project manager resists changing to adopt new HR
techniques and tools, the more the neglect of human
resource procedures and practices exist on construction
project. Therefore, to achieve optimal productivity on
construction project, it is advisable that an organisation
allows the project manager to perform his responsibility
as a coach to create a positive approach to people based 5 Conclusion The strategic finding established factors to look into
when
performing
HR
planning
operations
for
construction projects. Initially, effective utilisation of
labour appraisals to assess interest, abilities, skills and
experience and design job specifications to ensure the
responsibilities are given to right individuals were
identified in the literature review by different scholars as
part of causes of desertion of HR planning on
construction projects. However, the key consideration
factors
for
Sustainable
Project
HR
Planning
(Organisation’s structure with responsibilities given to
right individuals and assessment of current resources)
will bring about putting into practice of new HR
Planning technique and tools for construction project. Essentially, the main reoccurring theme identified was
that management of the construction organisations needs
to look into the essential factors needed to be considered
at the strategic level and the leaders leading a
construction project team to consider those essential
factors needed at operational level to clarify the
numerous functions of HRM in the construction
organisations and avoid inconsistencies among several
practices on construction projects. Nevertheless, the
impact of sustainable HR planning towards performance
should include its efficient practices as advocated and
strongly considered by both literature and empirical. Lastly, objectives of the paper were met as the
sustainability of HR planning for construction projects
were
examined
through
literature
review
and
substantiated with the responses from participant to
obtain empirical findings which were justified in the
research work. Table 7. Spearman’s Rho Correlations Between Five Principal
Causes of Neglect of HR Planning
HR Tools,
Techniques
And Output
Poor
effective
management
and
leadership
commitment
Poor
organisation’
s
management
Support
Poor
stakeholde
rs
commitme
nt in
regards to
HR
practices
Resistance
to change in
new
HR
techniques
and tools
Neglect
of
important of
HR
involvement
in a project
Project
Templates
Correlation
0 .006
0.269
0.044
-0.098
-0.105
Sig (p<0.05)
0.976
0.169
0.848
0.634
0.616
Analysis of
stakeholder
Correlation
-0.066
-0.136
0.087
0.014
0.284
Sig (p<0.05)
0.742
0.500
0.682
0.938
0.157
Human
resource
procedures
and practices
Correlation
0.064
0.005
0.146
0.371(*)
0.273
Sig (p<0.05)
0.762
0.982
0.485
0.048
0.181
Roles and
responsibility
Correlation
-0.312
0.026
0.334
-0.124
0.032
Sig (p<0.05)
0.125
0.904
0.096
0.555
0.888
Employee
management
plan
Correlation
-0.113
0.065
0.111
0.188
0.234
Sig (p<0.05)
0.579
0.757
0.584
0.379
0.247
*Correlation significant at 0.05 Level of P-Value Table 7. 5.2 Recommendations for Future Research The results from the research showed that it is preferable
to explore all HR planning for construction project best
practices and be adopted by construction organisations
and leaders leading construction projects. However,
further research can be carried out on implementation of
Project HR Planning by stakeholders for its success. 11. A. Dainty,
I. Grugulis
and
D. Langford,
Understanding Construction Employment: The Need
for a Fresh Research Agenda Personnel Review. 36,
501-508 (2007) 12. G. Dessler, “Human Resource Management”, 8th Ed. Upper Saddle River, New Jersey: Prentice Hall
Incorporated. (2000) 5.1 Recommendations Obviously, the paper findings suggested that the
inconsistency in the various practices of HRM have led
desertion of planning for construction project. Critically
empirical findings results highlighted that construction
industry recognised the use HR planning, therefore,
adaptability
and
visibility
of
implementation
of
guidelines, policies and procedures of sustainable HR
planning on construction project should be justified and
improved. The following are recommended: •
Strategic plan should be incorporated into
construction organisation system; •
HR planning objective should be incorporated
into the construction organisational strategic
plans; 6 , 0
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2018 https://doi.org/10.1051/matecconf/201925802018 7. A. Dainty and M. Loosemore, Human Resource
Management in Construction: Critical perspectives”,
London: Spon Press, (2012) •
Concerted efforts should be maintained by
strategic decision makers in construction
organisations
to
encourage
contemplative
learning and incessant improvements through
training for individuals and teams; 8. M. Loosemore, Construction Crisis Management,
American Society of Civil Engineers, New York. (2000) •
Sustainable application of HR technique and
tools application should be encouraged to assess
the need organisation and projects. 9. |J. Schervayder and R.S. Mayo, Construction
Fundamentals, 2nd Ed. New Yoon: McGrew Hill. (2004) 10. L. J. Mullins, “Management and Organizational
Behaviour”, 5th Edition, Essex: Pearson Education. (1999) References 13. D N. Bulla and P M. Scott, Manpower requirements
forecasting: a case example, in Human Resource
Forecasting and Modelling, 1st Ed, Human Resource
Planning Society: New York (1994) 1. A.R.J. Dainty, B.M. Bagilhole and R.H. Neale ‘The
Compatibility of Construction Companies’ Human
Resource Development Policies with Employee
Career Expectations,’ Engineering, Construction and
Architectural Management 7, 169-78, (2000) 14. P. Reilly, Guide to Workforce Planning in Local
Authorities, Employers Organization for Local
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Information Systems (HRIS) in HR planning and
development in mid to large sized organizations”
Proceedia of Social and Behavioural Science, 133,
61-67 (2014) 15. T. H. Leng, Human resource planning and human
resource development in construction. Master’s
Thesis, University Technology of Malaysia (2005) 16. P. Boxall and J. Purcell, Strategy and Human
Resource Management, London: Palgrave Macmillan
(2016) 3. C. Fogarassy, K. Szaboand, and J. Poor, Critical
issues of human resource planning, performance
evaluation and long-term development on the central
region and non-central areas: Hungarian case study
for investors. International Journal of Engineering
Business Management 9, 1–9 (2017) 17. K. Akingbola, "A Model Of Strategic Non-profit
Human
Resource
Management."
Voluntas:
International Journal of Voluntary & Non-profit
Organizations 24, 214-240 (2013) 18. M. Loosemore, A. Dainty and H, Lingard, “Human
Resource Management in Construction Projects-
Strategic and Operational Approaches”, London:
Spon Press (2003) 4. A. Laufer and R. Tucker, “Is Construction Planning
Really Doing its Job? A Critical Examination of
Focus, Role and Process” Construction Management
and Economics, London, E. & F. N. Spon, 5, 243-66
(1987) 19. S.G. Naoum,
Peoples
and
organisational
management in construction: 1st ed. London: Thomas
Telford Publishing (2001) 5. L.Nesan, H. Jawahar and D. Gary. Empowerment in
Construction: The way Forward for Performance
Improvement, Baldock, Hertfordshine, England:
Research Studies Ltd, (1999). 20. P. Farrel, Writing a built environment dissertation. West Sussex, UK: Blackwell. 79. (2011). 21. S.G Naoum, Dissertation Research and Writing for
Construction Students, 3rd ed Abingdon, Oxon:
Routledge. 67-97 (2013). 6. D. A. Langford R. Fellows, M. Hancock and A. Gale,
Human Resource Management in Construction, 2nd
ed. London: Longman, (1995) 7 7
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Hindawi
Complexity
Volume 2024, Article ID 9873842, 1 page
https://doi.org/10.1155/2024/9873842
Retraction
Retracted: Audit Analysis of Abnormal Behavior of Social Security
Fund Based on Adaptive Spectral Clustering Algorithm
Complexity
Received 23 January 2024; Accepted 23 January 2024; Published 24 January 2024
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References
[1] Y. Wu, Y. Chen, and W. Ling, “Audit Analysis of Abnormal
Behavior of Social Security Fund Based on Adaptive Spectral
Clustering Algorithm,” Complexity, vol. 2021, Article ID
4969233, 11 pages, 2021.
Hindawi
Complexity
Volume 2021, Article ID 4969233, 11 pages
https://doi.org/10.1155/2021/4969233
D
E
Research Article
Audit Analysis of Abnormal Behavior of Social Security Fund
Based on Adaptive Spectral Clustering Algorithm
Yan Wu,1 Yonghong Chen,2 and Wenhao Ling
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1
School of Business, Xi’an University of Finance and Economics, Shaanxi, Xi’an 710100, China
School of Public Administration, Xi’an University of Finance and Economics, Shaanxi, Xi’an 710061, China
3
Institute of Local Government and Societal Governance, Henan University, Kaifeng, Henan 475004, China
2
Correspondence should be addressed to Wenhao Ling; lwh@henu.edu.cn
Received 7 April 2021; Revised 27 April 2021; Accepted 10 May 2021; Published 19 May 2021
A
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Academic Editor: Zhihan Lv
Copyright © 2021 Yan Wu et al. 'is is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abnormal behavior detection of social security funds is a method to analyze large-scale data and find abnormal behavior.
Although many methods based on spectral clustering have achieved many good results in the practical application of clustering,
the research on the spectral clustering algorithm is still in the early stage of development. Many existing algorithms are very
sensitive to clustering parameters, especially scale parameters, and need to manually input the number of clustering. 'erefore, a
density-sensitive similarity measure is introduced in this paper, which is obtained by introducing new parameters to transform the
Gaussian function. Under this metric, the distance between data points belonging to different classes will be effectively amplified,
while the distance between data points belonging to the same class will be reduced, and finally, the distribution of data will be
effectively clustered. At the same time, the idea of Eigen gap is introduced into the spectral clustering algorithm, and the verified
gap sequence is constructed on the basis of Laplace matrix, so as to solve the problem of the number of initial clustering. 'e strong
global search ability of artificial bee colony algorithm is used to make up for the shortcoming of spectral clustering algorithm that
is easy to fall into local optimal. 'e experimental results show that the adaptive spectral clustering algorithm can better identify
the initial clustering center, perform more effective clustering, and detect abnormal behavior more accurately.
R
1. Introduction
'e development history of the social security fund market is
less than 20 years, but it has developed rapidly and has
become an indispensable part of the national economic life
[1, 2]. 'eoretical and empirical studies have shown that, in a
market dominated by individual investors, investment decisions lack sufficient rationality due to limited information
acquisition capabilities. From the perspective of the domestic securities market, individual investors are obviously
speculative, often fast in and out for short-term combat, are
susceptible to market sentiment, and often blindly follow the
trend, causing disorderly volatility in my country’s securities
market, leading to significant market price fluctuations.
Divorced from the actual situation of the operation of the
national economy, it has failed to effectively guide resource
allocation and industrial structure optimization, and it is
also difficult to use market forces to evaluate the benefits of
listed companies [3, 4]. 'erefore, the detection of abnormal
behaviors of social security funds is beneficial to the development of the fund market [1, 5].
Cluster analysis is a statistical method for studying
sample classification, as well as a method of data mining,
which can effectively realize the exploration of the internal
connections between things [6, 7]. 'e fundamental purpose
of the clustering algorithm is to automatically classify the
given samples into corresponding classes under certain
standards. Manual classification of data has great limitations
in practical applications [8]. Clustering belongs to the category of unsupervised classification. In the process of
classification, the degree of similarity between the given
classification objects is distinguished according to some
characteristics of the objects themselves. Clustering algorithms have received more and more attention from
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Complexity
researchers and are currently widely used in many fields.
With various abnormal behaviors in the fund market in the
past two years, many researchers have used clustering algorithms to detect such abnormal behaviors [9, 10]. 'e
spectral clustering algorithm is a relatively new research
direction in clustering algorithms, which uses the properties
of the feature vector in the similarity matrix of the data set to
cluster the data set. 'e idea of the spectral clustering algorithm is to use the similarity between data points to cluster
the data set. 'is method can also be applied to the cluster
analysis problem in the nonmeasure space [11].
Many methods based on spectral clustering have
achieved many good results in the practical application of
clustering, but the study of spectral clustering algorithms is
still in the early stage of development. Many existing algorithms are very important for clustering parameters, especially scale parameters. It is sensitive and requires manual
input of the number of clusters. 'erefore, this paper
proposes an adaptive spectral clustering algorithm. 'e main
contributions of the algorithm are as follows:
(1) 'is paper introduces a density-sensitive similarity
measure, which is obtained by introducing new
parameters to deform the Gaussian function. Under
this metric, the distance between data points belonging to different classes will be effectively enlarged, while the distance between data points
belonging to the same class will be reduced, and
finally the distribution of data will be effectively
clustered.
(2) 'e idea of the intrinsic gap is introduced into the
spectral clustering algorithm, and the intrinsic gap
sequence is constructed on the basis of the Laplacian
matrix to solve the problem of the number of initial
clusters.
(3) 'e advantage of the artificial bee colony algorithm’s
strong global search ability is used to make up for the
shortcoming of the spectral clustering algorithm that
it is easy to fall into the local optimum. At the same
time, in order to prevent the premature phenomenon of artificial bee colony algorithm, its location
search formula is improved.
and accurately identify abnormal and nonabnormal behaviors, which is also one of the current research hotspots
[14–17].
Spectral clustering algorithms are derived from the
theory of spectrograms. Based on different segmentation
methods, researchers have proposed some more classic
spectral clustering algorithms. PF algorithm [18], as the
original prototype of spectral clustering, has been extensively
studied in the field of machine learning. Wang et al. [19]
proposed a canonical cut set to divide the graph and proposed the famous SM algorithm. 'e clustering result of the
SM algorithm is also significantly higher than that of the PF
algorithm. Xu et al. [20] proposed the famous SLH algorithm. After the algorithm constructs the similarity matrix
according to the multipath canonical cut set criterion, the
data of eigenvalues and eigenvectors is determined by the
number of cluster inputs. If the number of clusters is 3, then
the number of feature vectors is 3. Although its consideration is comprehensive, its high complexity affects the
calculation speed and the efficiency is poor, but it still
achieves a good clustering effect. Ding et al. [21] proposed
the NJW algorithm. 'e algorithm is the same as the SLH
algorithm. 'e number of eigenvalues is determined by the
number of clustering groups after obtaining the eigenvectors. Shiotsuka et al. [22] proposed the Mcut algorithm. 'is
algorithm deeply studies the vector and uses this vector as a
criterion as a feature vector. 'e clustering results obtained
tend to be in a balanced state, and it takes a long time for
larger data sets. Zhu et al. [23] proposed the MS algorithm.
Similar to the SLH algorithm and the NJW algorithm, the
selection method of the feature vector is similar. Contrary to
Mcut, it has a poor clustering effect when the data set is small
or when the segmented image is required to be small.
At present, many scholars have carried out extensive
research on spectral clustering, mainly focusing on the
determination of the number of clustering groups, the
construction of feature vectors, and the construction of
similarity matrices.
Aiming at the adaptive problem of the number of
clustering groups, literature [24] converts large data sets into
small data sets, uses correlation to merge the grouped data,
and after fully studying its principle, proposes a new spectral
clustering algorithm; the algorithm can automatically obtain
the number of clustering groups. Literature [25] calculates
the difference between adjacent eigenvalues after arranging
the eigenvalues by calculating the eigenvectors and eigenvalues of the data similarity matrix. If the previous feature
value minus the current feature value is the largest, the
number of current feature values counted is the number of
cluster groups. Literature [26] first studied the distribution
of data, estimated the proportional parameter of each data
point based on this, and proposed the STSC algorithm, but
the calculation consumes too many resources and runs
slowly.
Aiming at the problem of feature vector selection and
similarity matrix construction, literature [27] sets a
threshold based on the feature requirements of the data and
runs the NJW algorithm multiple times. 'e number of runs
is manually set, and the adaptation of the threshold
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'e organizational structure of this article is as follows.
'e second part shows the relevant research content of this
article. 'e third part shows the research algorithm of this
article. 'e fourth part is the experimental results. 'e fifth
part is the conclusion.
2. Related Knowledge
At this stage, the computer network has basically realized
globalization, and the huge network system facilitates the
exchange and transmission of fund information. Facing the
increasingly complex network environment, it is of great
significance to accurately detect the abnormal behavior of
fund investment [12, 13]. In this regard, experts and scholars
apply clustering algorithms to detection technology, relying
on unsupervised and semisupervised methods to quickly
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3
parameter a is obtained, which eliminates the interference of
manual input on the construction of the similarity matrix,
and improves the clustering effect. Literature [28] uses the
rough clustering method to cluster the constructed left and
right singular vectors and proposes rough spectral clustering, which is successfully applied in text data mining. Literatures [29–31] proposed that the existing feature vectors
do not fully reflect the characteristics of the data itself, and
these features themselves are also difficult to extract.
'erefore, some control information is added to the feature
vector selection of the spectral clustering algorithm, and a
semisupervised spectral clustering feature vector selection
algorithm is proposed. Some supervision information is
added to the algorithm, and the feature vector is selected on
this basis.
control parameters, simple and easy to implement algorithm, and strong robustness. In the artificial bee colony
algorithm, the members of the algorithm are nectar, lead bee,
follow bee, and scout bee, among which scout bee is also
called hire bee. Among the four members, the nectar source
and the leading bee have a one-to-one correspondence in
number. 'e three types of bees search in their respective
search neighborhoods and compare their search results
continuously to obtain the optimal solution. “Swing dance”
is an important way for these three bees to transmit information. Figure 1 is a schematic diagram of the artificial
bee colony algorithm.
'e realization process of the artificial bee colony algorithm is as follows.
3. Adaptive Spectral Clustering Algorithm
3.2.1. Initialize Parameters. Suppose that the total number
of nectar sources is n, the maximum number of loops of the
algorithm is max_m, the maximum number of iterations is
max_it, the maximum number of searches is max_s, the
number of bees is collected m, and the number of honey bees
staying in a nectar source is recorded, and m � 0. By using
formula (5), we can randomly generate n honey source
information.
3.1. Spectral Clustering Algorithm. 'e spectral clustering
algorithm is based on the division of spectral graphs, and its
essence is to transform the clustering problem into the
optimal segmentation problem of graphs [32–35]. 'e
spectral clustering algorithm regards the data samples as the
vertices in the graph, which is represented by the set J, the
vertices are connected by edges, and the edges are represented by the set B. Assign a weight value w according to the
similarity between samples, and an undirected weighted
graph T � (J, B) based on the similarity of the samples can be
obtained. In this way, in the graph T, the clustering problem
is transformed into the optimal graph segmentation problem
on the graph T, so that the internal similarity of each
subgraph after division is very high, and the similarity between different subgraphs is very low.
Let Di denote the i-th data sample point, and let dis (di,
dj) denote the distance between di and dj. 'is distance is
usually expressed by the European distance ‖di-dj‖, and σ is
expressed as a scale parameter. We can get the similarity
matrix:
T
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2
− dis di ,dj /2σ 2
Sij � e
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.
(1)
'e distribution of surrounding data can be effectively
reflected by the degree of the data point. 'e degree matrix is
a diagonal matrix composed of diagonal elements with all
degree values, expressed as
Duiij � Sij .
(2)
'e noncanonical Lagrangian matrix is expressed as
Feiij � Duiij − Sij .
(3)
Iij � min i × rand(max j − min j) + min j.
(5)
In the formula, j ∈ {1, 2, . . ., m}, rand is a random
number between (0, 1), and max_j and min_j are the upper
and lower limits of the j-th dimension.
3.2.2. Lead the Bee Stage. Lead the bee to search for the local
nectar source location based on the greedy selection strategy
in the nectar source neighborhood. In the process of
searching for a nectar source, if a new and better nectar
source is found, the lead bee will compare the nectar source
with the best nectar source searched in history. If F (Jij) > F
(Iij), select the nectar source and pass the formula (6) to
update the position of the nectar source, change the position
of the nectar source from Iij to Jij, and lead the bee position to
update the position of the food source by the following
formula:
Jij � fij + λfij − fkj .
(6)
In the formula, k is generated randomly. Jij represents the
new nectar source position generated near fij, λ ∈ (− 1, 1) is a
random number, and the new nectar source position is
constrained by λ to be generated near the original nectar
source fij.
'e canonical Laplace matrix is expressed as
Feiij � Dui−ij(1/2) Sij Dui−ij(1/2) .
(4)
3.2. Artificial Bee Colony Algorithm. 'e artificial bee colony
algorithm is an artificial intelligence bionic algorithm with
strong searchability, which has the advantages of fewer
3.2.3. Follow the Bee Phase. 'e leading bee transmits the
nectar source information it carries to the follower bee in the
form of “swing dance,” and the follower bee selects a higherquality nectar source to follow according to the principle of
roulette. Calculate the probability pi selected by the bee by
the following formula. Fi represents the fitness value of the
nectar source Fi. 'e larger the fitness value of Fi, the higher
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Complexity
Hiring bee stage: multiple search mechanisms
Start
Cg
Initialization
Bg
Group initialization
Rg
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Individual selection; search method
Train data set
Test data set
Constructor group
Train data
Cg
Bg
Rg
Val data
Test data
Feature
extraction
Nectar or
nonnectar
UF
Bee
S
R
S
Nectar or
nonnectar
R
Unloading
UF
honey
Recruit a dancing
room A
area for A
EF1
Recruit a dancing
area for B
UF
S
T
C
Nectar A
EF1
S
EF1
UF
Unloading
honey
room B EF1
Nectar
EF1
Calculate
population fitness
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Multiple bee colony optimization model
Record the
optimal value
Nectar B
EF1
Yes
Whether there is a
scout bee
Yes
End of
iteration?
Scout bee
search
No
End
No
Figure 1: Artificial bee colony algorithm.
the probability of being selected by the bee. Fi is generated by
the formula (8).
pi �
fi
,
fi
1
⎪
⎧
⎪
,
⎪
⎪
⎪
1
+
f
Fi
⎨
Fi � ⎪
⎪
1
⎪
⎪
⎪
⎩ 1 + ����f F ����.
i
R
(7)
(8)
3.2.4. Reconnaissance Bee Stage. If the nectar source Fi has
not changed after mining, the nectar source is abandoned, a
new nectar source is generated from equation (6), and a new
search is continued. At this time, the lead bee attached to the
nectar source Fi becomes a scout bee.
3.2.5. Record the Optimal Solution. Compare the fitness
values of the current n feasible solutions, select the optimal
solution, and judge whether max_it is greater than max_m at
this time, and if it is greater than max_m, the algorithm ends.
Because the artificial ant colony algorithm is a parallel algorithm in nature, the search process of each ant is independent
of each other and communicates only through pheromone.
'erefore, the artificial ant colony algorithm can be regarded as
a distributed multiagent system. It starts independent solution
search at multiple points in the problem space at the same time,
which not only increases the reliability of the algorithm but also
makes the algorithm have a strong global search capability.
3.3. Overview of Density-Sensitive Similarity Measures. In
general, clustering is an unsupervised machine learning
process. Using some prior knowledge of the data set can
improve the effectiveness of clustering. 'e most important
thing is the consistency assumption of the data set, that is,
local consistency and global consistency.
(1) Local consistency: adjacent data points have higher
similarity in spatial position
(2) Global consistency: data points located on the same
structure have a higher similarity
For example, in Figure 2, there are two types of points, point
a belongs to one of them, and points b, c, d, and e belong to the
other. Local consistency is reflected in that the similarity between point d and points b and e is higher than the similarity
between point d and points f and c. Global consistency is reflected in that the similarity between point c and point d is
higher than the similarity between point c and point a. However,
in this example, the traditional Euclidean distance can only
reflect the local consistency of the data, not the global
Complexity
5
10
8
c
6
4
b
a
2
0
–10
–8
–6
–4
–2
–2
0
2
4
6
8
10
d
–4
e
–6
f
–8
–10
Figure 2: Data points on different trends.
consistency of the data. Assuming that in Figure 2, points c and f
belong to the same class, and point a belongs to another class.
'en, we expect the similarity between c and f to be greater than
the similarity between c and a, but the point that is under the
Euclidean distance measure c is closer to point a.
Based on the above problems, we design a similarity
measure that can satisfy both local consistency and global
consistency density-sensitive similarity measures. 'is metric
can shorten the distance between data points in the same class
and, at the same time, enlarge the distance between data points
in different classes and effectively describe the actual distribution of data points, so as to achieve a good clustering effect.
Define the following similarity measures with adjustable
density:
Sij � e−
number of clusters before clustering. However, in practical
applications, the value of the number of clusters k cannot be
determined in advance under normal circumstances. At the
same time, the calculation of similarity in traditional spectral
clustering algorithms is also greatly affected by parameter
values. Aiming at these two problems, this paper proposes an
adaptive spectral clustering algorithm by introducing density-sensitive similarity measures and an artificial ant colony
algorithm.
'e algorithm uses the characteristics of the size of the
intrinsic gaps in the cluster to automatically determine the
number of initial clusters and then uses the orthogonal
feature vector to classify the data. Suppose that, in an ideal
state, there are k separable classes for a given data set S. For
the normalized similarity matrix, there is a conclusion: the
first k largest eigenvalues of the matrix are 1. At the same
time, the k + 1 eigenvalue is less than 1, and the actual
distribution of the k clusters determines the size of the
difference between the two eigenvalues. 'e more obvious
the distribution, the greater the difference in eigenvalues,
and conversely, the smaller the difference. At the same time,
this difference is defined as the intrinsic gap. According to
the perturbation theory of the matrix, the larger the value of
the intrinsic gap, the more stable the nature of the subspace
formed by the selected k eigenvectors.
'e idea of the Eigen gap is developed based on the
matrix disturbance theory. Based on the obtained Laplace
matrix, the idea of eigenvalues λ is arranged in descending
order, that is, λ1 > λ2 > . . .> λn. 'e gap sequence G represents the difference between the k and k + 1 eigenvalues, that
is, T � λk − λk+1. Among them, the larger the intrinsic gap,
the more stable the subspace constructed by the selected k
feature vectors. Usually, the number of clusters in the
original data set is determined according to the first maximum value of the gap sequence in this card. 'e initial
number of clusters k is generated as shown in
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αdis di ,dj /2σ 2
− 1.
(9)
In equation (9), dis (di, dj) represents the Euclidean distance
between data points di and dj. 'e variable α is the density
parameter. For simple data sets, α generally takes a natural
number greater than 1. When the data set is complex and its
probability distribution function is not convex, the value of α
can be smaller, generally set to 0.2.
'e given data point corresponds to a vertex set J of an
undirected weighted graph T � (J, B). Since the density-sensitive
similarity measure does not satisfy the triangular inequality, it
cannot be directly used to construct the similarity matrix.
'erefore, we redefine a distance measure based on this
measure.
Use L � {L1, L2, . . ., Ln} to represent the path between the
connecting points L1 and Ln with n vertices on the graph, where
Lk ∈ J, (Lk, Lk+1) ∈ J. Use Lij to represent the set of all paths
between the data point pair di and dj, and define the distance
sensitive to the density between di and dj:
R
D Sij � min Lk , Lk+1 .
(10)
3.4. Adaptive Spectral Clustering Algorithm. 'e traditional
spectral clustering method requires manual input of the
k � max T1 , . . . , Tn .
(11)
In the follow-up phase, the follower bee generates a new
nectar source in its vicinity according to equation (6) and
makes comparison choices. But in formula (6), Fij represents
the nectar source near fij that is superior to fij in the previous
nectar source. Since the parameter λ constrains Fij near fij,
there is a lack of overall search and comparison. 'erefore,
when the nectar source location is updated by formula (6),
the algorithm is easy to fall into the local optimum during
operation. 'is article will introduce the concept of a
globally optimal solution into this formula. Experiments
show that the improved position update formula (12) has a
strong purpose and directionality in the search process, and
the algorithm has a fast convergence speed, which makes it
easy to jump out of the local optimal phenomenon.
Fij � fij + λfij − fkj + αfbest,j − fij ,
(12)
where Fij is the location of the new nectar source near fij.
Both k and j are random numbers generated by random
formulas. 'e algorithm implementation process is shown in
Figure 3.
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Complexity
Train data set
Introduce improved algorithm for
clustering
Test data set
D
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Initialize parameters
set related parameters
Enter the number of
clusters k
Nectar
Global search factor introduces update
formula of the follower bee position
Construct
similarity matrix
Find the laplace
matrix and
normalize it
Find the eigenvalues
and eigenvectors of
the matrix
Construct an
eigenvector matrix
and arrange the
eigenvectors in
descending order
Use feature vector space as
search domain
T
C
Improve ABC algorithm to search
Construct the
intrinsic gap
sequence T and
determine the
value of k
Whether to reach the maximum
number of iterations
If the i-th row in the matrix
is divided into the j-th
category, then the i-th row is
marked as the j-th category.
Treat each row in the
eigenvector matrix
as a point in the
clustering space
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Output clustering
results
Figure 3: Algorithm flow.
700
4.1. Clustering Criterion Function. In order to verify the
rationality of the initial clustering center selected by the
algorithm, the experiment uses the first clustering criterion
function value after the initial clustering center is selected to
judge.
If the function value is smaller, it means that the selection of the initial cluster center is more reasonable and
closer to the true cluster center. In addition, due to the
decrease of the function value of the clustering criterion, the
quality of the clustering is improved, and the algorithm is
more efficient. 'e experimental results are shown in
Figure 4.
It can be seen from Figure 4 that when k � 2, the clustering criterion function value of the improved algorithm is
significantly higher than that of the contrast algorithm.
However, starting from k � 3, the clustering criterion
function value of the improved algorithm has dropped
significantly. As the number of clusters k increases, the
clustering criterion function values of the comparison algorithm and the improved algorithm tend to be parallel to
each other, and there is always a certain gap. 'is is because
the improved algorithm pays more attention to the distribution of the initial cluster centers. When k � 2, the improved algorithm selects the least tight data point as the
second initial cluster center. Due to the small number of
clusters, the selection of the second initial cluster center is far
from the true cluster center, resulting in an increase in the
value of the clustering criterion function. However, the
improved algorithm takes into account the distribution of
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Clustering criterion function value
4. Results and Discussion
600
500
400
300
200
100
0
1
2
PF
Mcut
SM
3
4
5
6
7
8
Number of cluster centers
MS
SLH
STSC
9
10
NJW
Our
Figure 4: Clustering criterion function.
real cluster centers and pays more attention to the uniform
distribution. When the number of clusters k increases, the
uniformly distributed data points are more in line with the
distribution of the optimal clustering center, which effectively reduces the clustering criterion function value of the
improved algorithm, thereby improving the clustering
quality. It can be seen from Figure 4 that when k � 8, the
algorithm in this paper can achieve the best performance.
'erefore, the initial number of clusters in this paper is
selected as 8.
Complexity
7
4.2. Analysis of Convergence Time. In order to verify the
execution efficiency of the algorithm, the experiment uses
the convergence time to judge. If the convergence time is
shorter, the algorithm runs faster and the execution efficiency is higher. In addition, because the convergence time is
reduced, the processing efficiency of the algorithm is increased, and the effect of clustering is improved. 'e experimental results are shown in Figure 5.
It can be seen from Figure 5 that as the number of
clusters k increases, the convergence time of the improved
algorithm is the fastest. 'is is because the improved algorithm takes into account the distribution of the true
clustering centers and selects the initial clustering centers
from the data points with a low degree of compactness,
which greatly reduces the amount of data for distance
calculations, and greatly improves the algorithm’s performance while ensuring the clustering effectiveness. Although,
when the number of clusters k increases, the initial cluster
center selection process of the two algorithms becomes
similar, and the convergence time gradually approaches, the
convergence time of the improved algorithm is still lower
than that of the comparison algorithm.
the selection result is more reasonable. In addition, due to
the reduction in the number of iterations, the accuracy of
clustering increases, and the algorithm is more efficient. 'e
experimental results are shown in Figure 7.
It can be seen from Figure 7 that the improved algorithm
takes into account the distribution of real cluster centers and
pays more attention to data points with low tightness. When
the number of clusters k increases, the data points with low
tightness are closer to the clusters of the new cluster, which
effectively reduces the number of iterations of the improved
algorithm, resulting in an increase in the number of iterations. 'e convergence time is shown in Figure 8.
It can be seen from Figure 8 that as the number of
clusters k increases, the convergence time of the improved
algorithm is significantly less than that of the contrast algorithm. 'is is because the improved algorithm proposed
in this paper guarantees that there is a significant distance
between the selected data points, so that the selected data
points belong to different clusters to the greatest extent while
ensuring the efficiency of the algorithm execution, which
improves the execution efficiency of the algorithm, without
losing a good initial clustering center; this also significantly
reduces the convergence time of the algorithm, accelerates
the convergence of the algorithm, and improves the execution efficiency of the algorithm.
D
E
A
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T
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T
C
4.3. Performance Analysis of Clustering Algorithm. 'e
clustering results of the improved new algorithm in this
paper are given below and compared with the clustering
results of the traditional spectral clustering method (STSC).
'e experimental results are shown in Figure 6, where
Figures 6(a) and 6(c) are the clustering results of the STSC
algorithm. Figures 6(b) and 6(d) are the results of the algorithm in this paper. Figure 6(a) contains three irregularly
shaped data sets. It can be seen from the classification results
that, for a simple data set, both the STSC algorithm and the
algorithm in this paper have achieved ideal clustering results.
Figures 6(c) and 6(d) show two concentric circular data sets.
'e traditional spectral clustering algorithm got the wrong
classification result for the intersection of the parabola, while
the algorithm in this paper got the correct classification
result.
From the above experimental analysis, we can see that
for some simple data sets with obvious classification, both
the STSC algorithm and the algorithm in this paper can get
the correct classification results. However, for some complex
data sets, such as concentric circles, the clustering results of
the STSC algorithm have large errors. Because the algorithm
in this paper introduces a density-adjustable similarity
measure, the similarity between different types of data points
is reduced, and the ideal clustering result is obtained, and the
number of clusters is accurately calculated. 'e STSC algorithm must manually input the number of clusters and use
Euclidean distance as the similarity measure, which does not
accurately and effectively reflect the actual clustering distribution of the data, so the clustering effect is relatively
poor.
To verify the clustering quality of the algorithm, the
experiment uses the number of iterations to judge. If the
number of iterations is less, it proves that the initially selected cluster center is closer to the real cluster center, and
R
4.4. Abnormal Behavior Analysis. We injected outliers into
the data set to generate a synthetic data set to evaluate the
effect of anomaly detection. 'e specific detection effect is
shown in Figure 9.
As shown in Figure 9, as the number of clusters k
increases, the detection rate of the algorithm in this paper
gradually increases. 'e improved algorithm proposed in
this paper first selects the points with the largest and
smallest compactness, so as to ensure that the two initial
cluster centers belong to different clusters. In addition,
the initial cluster centers are randomly selected to ensure
that the selected data points are evenly distributed to the
greatest extent. When the number of clusters k increases,
the uniform distribution of data points is more consistent
with the distribution of the optimal clustering center,
thereby improving the clustering performance and the
effect of anomaly detection. Finally, despite the slight
increase in execution time, the detection effect of the
improved algorithm is still better than the comparison
algorithm.
DE algorithm has strong global convergence ability
and robustness and does not need to rely on the characteristic information of the problem. It is suitable for
solving some optimization problems in complex environments that cannot be solved by conventional mathematical programming methods. 'erefore, this article
will compare the use of DE optimization, no DE optimization, no artificial ant colony algorithm, and artificial
ant colony algorithm. 'e experimental results are shown
in Figure 10. It can be seen from Figure 10 that the artificial ant colony algorithm used in this paper has the
best performance.
8
Complexity
30
Convergence time
25
D
E
20
15
10
5
0
1
2
3
4
5
6
7
8
Number of cluster centers
PF
Mcut
SM
T
C
9
10
NJW
Our
MS
SLH
STSC
Figure 5: Convergence time.
10
A
R
T
E
8
6
4
2
10
8
6
4
2
0
0
–10
–8
–6
–4
–2
–2
0
2
4
6
8
10
–10
–8
–6
–4
–2
–2
–4
–4
–6
–6
–8
–8
–10
–8
R
–6
–4
–2
10
8
8
6
6
4
4
2
2
0
–2
2
4
6
8
10
–10
–8
–6
–4
6
8
10
2
4
6
8
10
0
–2
–2
–4
–4
–6
–6
–8
–8
–10
–10
(c)
4
(b)
10
0
2
–10
(a)
–10
0
0
(d)
Figure 6: Comparison of clustering results between SC algorithm and this algorithm.
Complexity
9
1
0.9
Accuracy of detection
0.8
D
E
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
2
3
4
5
6
7
Number of cluster centers
PF
Mcut
SM
T
C
8
9
MS
SLH
STSC
10
NJW
Our
Figure 7: Analysis of the number of iterations and clustering performance.
30
A
R
T
E
Convergence time
25
20
15
10
5
0
1
2
3
PF
Mcut
SM
4
5
6
7
8
Number of cluster centers
9
MS
SLH
STSC
10
NJW
Our
Figure 8: Convergence time.
0.8
Accuracy of detection
R
1
0.9
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
2
PF
Mcut
SM
3
4
5
6
7
8
Number of cluster centers
9
MS
SLH
STSC
Figure 9: Detection effect of abnormal behavior.
10
NJW
Our
10
Complexity
References
1
Accuracy of detection
0.98
0.96
0.963
0.949
0.936
0.94
0.92
0.9
0.892
0.88
0.86
0.84
0.82
With-DE
Without-DE With-ABC
Different techniques
Without-ABC
Figure 10: Detection effect of different optimization techniques.
5. Conclusion
'is paper first introduces a density-sensitive similarity
measure, which is obtained by introducing new parameters to deform the Gaussian function. Secondly, in view
of the defect that the number of clusters cannot be automatically determined, this paper quotes the idea of the
Eigen gap, constructs an Eigen gap sequence, calculates
the first maximum value, and determines the number of
clusters k, which solves the problem that the spectral
clustering algorithm has the problem of insufficient
sensitivity of cluster centers. Finally, in order to improve
the global search ability of the algorithm, it is combined
with the artificial bee colony algorithm with strong global
searchability. In order to further enhance the algorithm’s
global searchability, the global search factor is introduced
into the artificial bee colony algorithm, which effectively
improves the artificial bee colony algorithm’s tendency to
fall into local optimality, appear premature, and slow the
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achieve a good clustering effect. However, the improved
algorithm in this paper also has limitations, such as the
problem that the algorithm takes a long time in the
clustering process. 'erefore, how to combine the advantages of artificial bee colony algorithm and spectral
clustering algorithm and reduce the time complexity of
the algorithm will be the problem to be studied and solved
in the next step.
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Data Availability
'e data used to support the findings of this study are
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Conflicts of Interest
'e authors declare that they have no known competing
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Indonesian
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Analisis Kadar Zat Besi (Fe) Pada Tepung Kulit Kentang
|
Jurnal Ners
| 2,023
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cc-by-sa
| 2,615
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Jurnal Ners Volume 7 Nomor 2 Tahun 2023 Halaman 1005 - 1008
JURNAL NERS
Research & Learning in Nursing Science
http://journal.universitaspahlawan.ac.id/index.php/ners Jurnal Ners Volume 7 Nomor 2 Tahun 2023 Halaman 1005 - 1008
JURNAL NERS
Research & Learning in Nursing Science
http://journal.universitaspahlawan.ac.id/index.php/ners Jurnal Ners Volume 7 Nomor 2 Tahun 2023 Halaman 1005 - 1008
JURNAL NERS
Research & Learning in Nursing Science
http://journal.universitaspahlawan.ac.id/index.php/ners ANALISIS KADAR ZAT BESI (FE) PADA TEPUNG KULIT
KENTANG Ifa Nurhasanah
Program Studi Sarjana Kebidanan Fakultas Ilmu Kesehatan Universitas Ibrahimy
nurhasanah_ifa@yahoo.com Ifa Nurhasanah Program Studi Sarjana Kebidanan Fakultas Ilmu Kesehatan Universitas Ibrahimy
nurhasanah_ifa@yahoo.com Abstrak Kentang adalah salah satu buah yang tumbuh di negara Indonesia. Buah ini memiliki manfaat yang baik bagi
tubuh bila mengkonsumsinya. Beberapa bagian dari buah ini juga dapat dimanfaatkan dengan biak, salah
satunya adalah pada kulitnya. Kulit kentang yang biasanya sering dibuang dan tidak digunakan, memiliki
kandungan zat besi yang diperlukan oleh tubuh. Tujuan penelitian ini adalah untuk mengetahui dan
menganalisis kandungan kadar zat besi pada tepung kulit kentang. Metode yang digunakan pada penelitian
yaitu dilakukan pada uji analisis kuantitatif dan kualitatif. Alat yang digunakan untuk menganalisis kadar zat
besi dalam tepung kulit kentang adalah Spektrofotometer Serapan Atom (SSA) yang dilakukan di
Laboratorium Sucofindo Surabaya pada bulan Maret-juli 2023. Hasil penelitian ini menunjukkan bahwa
terdapat kandungan zat besi pada tepung kulit kentang sebanyak 111, 8 dalam 100 gr. Kesimpulannya bahwa
kulit kentang dapat dimanfaatkan sebagai bahan makanan karena memiliki kandungan zat besi yang tinggi
bila dibandingkan dengan daging kentang, sehingga dapat meningkatkan kadar hemoglobin dalam tubuh
seseorang agar dapat mencegah atau sebagai penanganan dari penyakit anemia. Kata Kunci: Analisis, tepung kulit kentang, zat besi Jurnal Ners Volume 7 Nomor 2 Tahun 2023 Halaman 1005 - 1008
JURNAL NERS
Research & Learning in Nursing Science
http://journal.universitaspahlawan.ac.id/index.php/ners METODE Metode penelitian ini adalah penelitian
kualitatif dan kuantitatif dengan pendekatan
eksperimen laboratorium. Penelitian dengan jenis
kualitatif bertujuan untuk mengetahui adanya
kandungan zat besi (Fe) pada tepung kulit kentang. Sedangkan jenis penelitian kuantitatif memiliki
tujuan untuk menganalisis dari nilai kadar zat besi
atau Fe yang terdapat pada tepung kulit kentang. Penelitian ini dilakukan di Laboratorium Sucofindo
Surabaya pada bulan Maret- Juli 2023. Alat yang
digunakan pada penelitian ini diantaranya corong
gelas, gelas kimia, pipet tes, labu ukur, kertas
saring, kaca arloji, dan spektrofotometer Serapan
Atom (SSA). Sedangkan bahan yang dipakai
adalah aquadest, sampel buah pepaya, tablet zat
besi
(multivitamin). Selanjutnya
peneliti
melakukan preparasi sampel, analisis kualitatif dan
analisis kuantitatif. Kulit kentang bila tidak diolah maka akan
menjadi sampah dan tidak bisa dikonsumsi. Akan
tetapi, bila kulit kentang diolah dan dibuat menjadi
bahan makanan, hal ini dapat dikonsumsi. Salah
satunya dapat dibuat menjadi tepung kulit kentang. Tepung ini dapat dijadikan bahan membuat
makanan
yang
dapat
divariasikan
menjadi
makanan yang lagi tren saat ini (Fajiarningsih,
2013). Kentang adalah sejenis buah yang dapat
dengan mudah diperoleh dan tidak mahal. Buah ini
juga dapat ditanam sendiri dipekarangan rumah. Sehingga setiap hari dapat mengkonsumsi dari
buah ini. Meskipun kandungan zat besi yang
terdapat pada buah ini tidak terlalu besar bila
dibandingkan dengan buah lainnya, akan tetapi
kulit kentang dapat memenuhi kebutuhan zat besi
yang dibutuhkan oleh tubuh manusia. Abstract Potato is a fruit that grows in Indonesia. This fruit has good benefits for the body when consumed. Some
parts of this fruit can also be used multiplied, one of which is the skin. Potato skins, which are usually thrown
away and not used, contain iron which is needed by the body. The purpose of this study was to determine and
analyze the iron content in potato skin flour. The method used in this research is carried out on quantitative
and qualitative analysis tests. The tool used to analyze the levels of iron in potato skin flour is the Atomic
Absorption Spectrophotometer (AAS) which was carried out at the Sucofindo Laboratory in Surabaya in
March-July 2023. The results of this study indicate that there is an iron content in potato skin flour as much
as 111.8 in 100 gr. The conclusion is that potato skin can be used as a food ingredient because it has a high
iron content when compared to potato flesh, so it can increase hemoglobin levels in a person's body in order
to prevent or as a treatment for anemia. Keywords: Analysis, potato skin flour, iron @Jurnal Ners Prodi Sarjana Keperawatan & Profesi Ners FIK UP 2023 @Jurnal Ners Prodi Sarjana Keperawatan & Profesi Ners FIK UP 2023 Corresponding author :
Address : Situbondo
Email
: nurhasanah_ifa@yahoo.com
Phone
: 085335724774 Corresponding author :
Address : Situbondo
Email
: nurhasanah_ifa@yahoo.com
Phone
: 085335724774 ISSN 2580-2194 (Media Online) Jurnal Ners Universitas Pahlawan PENDAHULUAN dilakukan
oleh
Soelarso
tahun
1997
yang
menyebutkan bahwa kulit kentang memiliki
kandungan gizi lima kali lebih besar bila
dibandingkan dengan dagingnya. Menurut peneliti
yang sama yang mengatakan bahwa kandungan
gizi dari kulit kentang diantaranya adalah kalori
115 kal, serat 5 gr, vitamin C 7,8 gr, asam folat 5,5
mg, kalsium 19,8 mg, zat besi 4,1 mg, potassium
322 mg, dan sodium 3,1 mg (Soelarso, 2012). Negara Indonesia merupakan salah satu
negara penghasil kentang terbesar di Asia
Tenggara. Hal ini membuat masyarakat Indonesia
sering mengkonsumsi kentang setiap harinya
(Soelarso, 2012). Kentang adalah tanaman yang
dapat dimanfaatkan untuk dijadikan sayur yang
dapat dikonsumsi oleh banyak orang (Rahma,
2017). Selain rasanya yang enak, kentang ini juga
memiliki
kandungan
gizi
yang
baik
dan
dibutuhkan oleh tubuh. Kandungan gizi yang terdapat pada kulit
kentang dapat menjadi daya tarik bagi konsumen
untuk mengkonsumsi tidak hanya pada daging
kentang, namun juga dapat memanfaatkan kulit
kentang. Kulit kentang dapat dikonsumsi dengan
dibersihkan terlenih dahulu dan dikeringkan
sehingga dapat dihaluskan menjadi tepung. Tepung
kulit kentang dapat menjadi bahan dasar seseorang
membuat olahan makanan seperti kue, atau jajanan
lain yang dapat dikonsumsi dan sesuai selera
konsumen. Selain itu, tepung kulit kentang ini tidak
memiliki efek samping setelah mengkonsumsinya
(Fajiarningsih, 2013). Sehingga aman dikonsumsi
bagi penderita anemia atau untuk mencegah dari
penyakit ini, utamanya bagi remaja perempuan dan
ibu hamil yang rentan terkena anemia (Rahma,
2017). Salah satu kandungan gizi dari kentang
adalah zat besi (Fe). Zat besi merupakan salah satu
zat gizi yang diperlukan tubuh untuk memproduksi
hemoglobin dalam sel darah merah yang dapat
mencegah dari penyakit anemia yang rentan terjadi
pada remaja perempuan dan ibu hamil. Dengan
adanya kentang, dapat membantu seseorang yang
mengalami anemia untuk meningkatkan kadar
hemoglobin dalam darah (Rahma, 2017). Kentang terdiri dari daging, biji dan kulit. Daging
dan
biji
pada
kentang
seringkali
dimanfaatkan bahkan dapat dikonsumsi setiap hari. Namun, pada kulit kentang sering dijadikan
sampah atau dibuang dan tidak dimanfaatkan. Padahal beberapa penelitian meneybutkan bahwa
kulit kentang memiliki kandungan gizi yang tidak
kalah dengan buah kentang itu sendiri. Salah satu
kandungan gizi yang terdapat pada kulit kentang
adalah zat besi (Fe). Hal ini diperkuat dengan
penelitian yang dilakukan oleh Rahma Widyastuti
tahun 2017 yang mengatakan bahwa penelitian
dengan in vivo menyebutkan kulit kentang dapat
meningkatkan kadar hemoglobin pada mencit
(Rahma, 2017). Tujuan
penelitian
ini
adalah
untuk
mengetahui dan menganalisis kadar zat besi (Fe)
pada tepung kulit kentang. Jurnal Ners Universitas Pahlawan Analisis kuantitaif Uji analisis kuantitatif pada tepung kulit
kentang bertujuan untuk menganalisis nilai kadar
zat besi atau fe pada sampel dengan menggunakan
alat Spektrofotometer Serapan Atom (SSA). Adapun tahap-tahap dalam melakukan uji analisis
ini sebagai berikut. Pengukuran kadar besi (Fe) Sampel tepung kulit kentang yang telah
dipreparasi sebelumnya dimasukkan kedalam SSA
dengan cara diinjeksi. Lalu, ukur serapannya
dengan gelombang yang sama yaitu 283,3 nm dan
catat hasil pengukuran untuk selanjutnya dilakukan
analisis. 1007| ANALISIS KADAR ZAT BESI (FE) PADA TEPUNG KULIT KENTANG tepung kulit kentang yang telah dilakukan dengan
menggunakan alat spektrofotometer serapan atom
ditunjukkan pada tabel dibawah ini. Tabel 1. Hasil analisis kadar zat (Fe) pada tepung
kulit kentang
Sampel
Unit
Hasil
Tepung kulit kentang
PPM
111,8 tepung kulit kentang yang telah dilakukan dengan
menggunakan alat spektrofotometer serapan atom
ditunjukkan pada tabel dibawah ini. kentang dan mudah saat dibersihkan. Selanjutnya
kentang dikupas dan dipisah antara kulit dan
dagingnya. Saat melakukan pengkupasan pada
kulit kentang, usahakan daging tidak ikut pada
kulit kentang (dikupas tipis). Kemudian kulit
kentang yang sudah terpisah dengan daging,
dilakukan penjemuran dibawah sinar matahari
dengan suhu 55˚-60˚C selama 6-7 jam. Setelah
kering,
kulit
kentang
di
haluskan
dengan
menggunakan alat penghalus (blender) dan diayak. Tepung kulit kentang dilarutkan dan disaring, lalu
dilakukan penimbangan sebanyak 10 gr. Filtrat
dari sampel tersebut dimasukkan kedalam labu
ukur sebanyak 100 ml. Selanjutnya, sampel
ditambahkan dengan aqudest sebagai pelarut
hingga tanda batas dan dihomogenkan. Tabel 1. Hasil analisis kadar zat (Fe) pada tepung
kulit kentang g
Sampel
Unit
Hasil
Tepung kulit kentang
PPM
111,8 g
Sampel
Unit
Hasil
Tepung kulit kentang
PPM
111,8 Pada tabel 1. Menunjukkan hasil analisis
kadar zat besi pada tepung kulit kentang sebesar
111,8 dalam 100 gr. Hal ini membuktikan bahwa
tepung kulit kentang memiliki kandungan zat besi
yang tinggi bila dibandingkan dengan daging
kentangnya. Analisis kuantitatif Hasil analisis kuantitatif pada tepung kulit
kentang menunjukkan adanya kandungan zat besi
didalamnya. Tepung
kulit
kentang
dapat
dimanfaatkan untuk dikonsumsi karena tidak
memiliki efek samping setelah memakannya dan
justru memiliki manfaat yang baik bagi tubuh yaitu
dapat meningkatkan kadar hemoglobin dalam
darah karena tepung kulit kentang memiliki
kandungan zat besi (Suryowibisono, 2020). Tepung
kulit kentang dapat di jadikan bahan untuk
membuat olahan makanan yang dapat dikonsumsi
oleh masyarakat khususnya bagi yang rentan
terkena anemia yaitu pada remaja putri dan ibu
hamil. Namun, zat besi alami tidak hanya diperoleh
dari kulit kentang, akan tetapi juga dapat
memanfaatkan bahan makanan lainnya, seperti ikan
gabus yang di ekstrak juga dapat meningkatkan
kadar Hb (Azzalina & Galaupa, 2023). Penelitian
lain juga menunjukkan bahwa jus jambu merah dan
jeruk sunkis madu juga berpengaruh terhadap
peningkatan kadar Hb (Safitri, 2019). Bila di telaah
dari banyaknya penelitian yang berkaitan dengan
peningkatan kadar Hb sangatlah banyak dan mudah
untuk didapatkan. Kadar Hb seseorang dapat
meningkat disebabkan karena zat besi dalam darah
terpenuhi
dengan
baik. Banyaknya
sumber
makanan yang dapat diperoleh untuk kebutuhan zat
besi bertujuan untuk menurunkan kejadian anemia
terutama bagi ibu hamil dan remaja. Analisis kualitatif Langkah awal yang dilakukan untuk
mengetahui adanya kandungan Fe pada tepung
kulit kentang adalah sampel uji yang telah
dipreparasi sebelumnya diteteskan kedalam tabung
reaksi sebanyak 5 tetes. Kemudian, teteskan
sebanyak 3 tetes larutan kaliun tiosianida (KSCN)
pada tabung yang sama. Penilaian yang dilakukan
adalah melihat dari perubahan warna yang terjadi
pada tabung reaksi tersebut. Perubahan warna
merah
yang
terjadi
pada
larutan
sampel
menyatakan bahwa terdapat kandungan Fe didalam
sampel. Pengukuran kadar larutan standar logam besi
(Fe) Masing-masing
larutan
baku
Fe
dimasukkan dengan cara menginjeksikan kedalam
alat SSA yang dibuat dengan konsentrasi 0 ppm, 5
ppm, 10 ppm, 20 ppm dan 50 ppm. Kemudian ukur
serapannya pada Panjang gelombang 283,3 nm. Selanjutnya, hasil pengukuran dicatat dan dibuat
kurva kalibrasi untuk mendapatkan persamaan
liniernya. Preparasi sampel Tepung kulit kentang adalah sampel untuk
dilakukan uji analisis kadar Fe. Kentang yang
dipilih menjadi sampel adalalah kentang yang
bagus dan tidak busuk. Langkah awal pembuatan
sampel dimulai dari kentang yang dicuci bersih
sampai tidak ada kotoran yang menempel pada
kulit kentang. Sebelumnya kentang direndam satu
hari agar kotoran tanah tidak menempel pada Dengan adanya penelitian ini, dapat
memberitahukan bahwa kulit pada kentang juga
dapat dimanfaatkan menjadi olahan makanan yang
dapat dikonsumsi. Sehingga kulit kentang tidak
lagi menjadi sampah atau tidak dimanfaatkan
dengan baik oleh para konsumen. Penelitian yang ISSN 2580-2194 (Media Online) Jurnal Ners Universitas Pahlawan Jurnal Ners Universitas Pahlawan Analisis kualitatif Uji analisis kualitatif bertujuan untuk
mengetahui hasil kadar zat besi pada tepung kulit
kentang. Bila
melihat
pada
tabel
1
yang
menunjukkan bahwa kadar zat besi pada tepung
kulit kentang sebnayak 111,8 dalam 100 gr
(Rahma, 2017). Kadar zat besi pada tepung kulit
kentang sangat besar bila dibandingkan dengan
daging kentang itu sendiri. Hal ini dapat menambah
informasi bagi masyarakat khususnya ibu yang
sering memasak buah ini untuk tidak menjadikan
kulit kentang sebagai sampah dan pada akhirnya
dibuang. Namun,
kulit
kentang
ini
dapat
dimanfaatkan dengan baik dan diolah secara benar
sesuai dengan selera makanan yang diinginkan agar DAFTAR PUSTAKA memenuhi zat besi pada tubuh (Anova, W, & S,
2014). memenuhi zat besi pada tubuh (Anova, W, & S,
2014). Anova, T., W, H., & S, S. (2014). Substitusi
Tepung Terigu dengan Tepung Kentang
(Solanum Sp) pada Pembuatan Cookis
Kentang. J Litbang Ind, 123-131 Vol. 4,
No. 2 . Kebutuhan zat besi yang diperlukan oleh
tubuh harus terpenuhi setiap harinya agar terhindar
dari penyakit anemia. Beberapa data menunjukkan
bahwa kelompok remaja putri dan ibu hamil
memiliki jumlah penderita anemia yang lebih
besar bila dibandingkan dengan kelompok umur
lainnya. Pada kelompok remaja putri beresiko
terkena anemia, disebabkan karena mengalami
menstruasi setiap bulan sehingga berdampak pada
pengeluaran darah dan akhirnya mengalami
kekurangan darah. Hal ini juga terjadi pada ibu
hamil khususnya pada trimester akhir yang
memerlukan asupan zat besi lebih banyak
dibandingkan sebelum hamil. Sehingga dengan
adanya kebutuhan zat besi yang banyak, dapat
didapatkan dari kulit kentang yang dibisa di olah
menjadi makanan atau minuman sehat dan dapat
dikonsumsi setiap hari (Winarno, 2004). Azzalina, N., & Galaupa, R. (2023). Perbandingan
Pemberian
Ekstrak
Ikan
Gabus
dan
Smooty Bayam Terhadap Peningkatan
Kadar Hemoglobin pada Ibu Hamil di
Wilayah UPT Puskesmas Ciomas Tahun
2023. Jurnal Ners, 121-126 Vol. 7, No. 1. Broto, W., T, H., & H, H. (1996). Effect of Sodium
Bisulphite Concentration ang Thickness of
Potato Tuber on Inhibition of Enzymatic
and Non-Enzymatic Browning of. 331-346. Cicilia, S., Basuki, E., Prarudiyanto, A., Alamsyah,
&
Handito,
D. (2018). pengaruh
substansiTepung Terigu dengan Tepung
Kentang
Hitam
(Coleus
Tuberosus)
Terhadap Sifat Kimia dan Organoleptik
Cookies . J Ilmu dan Teknol. Pangan ,
304-310 Vol. 4, No. 1 . Kentang dapat diperoleh di beberapa
tempat dan mudah untuk mendapatkan nya. Selain
itu, buah satu ini juga memiliki harga yang cukup
murah, dan bahkan dapat ditanam sendiri di
pekarangan rumah sehingga lebih mudah untuk
mendapatkan dan dikonsumsi setiap hari (Broto, T,
& H, 1996). Kulit kentang yang akan diolah dapat
dijadikan
tepung
terlebih
dahulu,
agara
memudahkan untuk membuat olahan makanan
yang lagi trend saat ini, utamanya bagi kelompok
remaja yang sering mengikuti jajanan viral saat ini
(Fajiarningsih, 2013). Fajiarningsih, H. (2013). Pengaruh Penggunaan
Komposit Tepung
Kentang
(Solanum
Tuberosum L.) Terhadap Kualitas Cookies. Noscov, S. (2013 ). Pottasium, Physical and
Chemical Properties . Rahma, W. (2017). Bioaktivitas Kulit Kentang
(Solanum
Tuberosum
L.)
Terhadap
Peningkatan Kadar Haemoglobin Secara In
Vivo. Namun, saat mengolah makanan dari
bahan tepung kulit kentang harus diperhatikan
bahan bahan campuran lainnya. HASIL DAN PEMBAHASAN Hasil uji analisis kadar zat besi (Fe) pada Jurnal Ners Universitas Pahlawan ISSN 2580-2194 (Media Online) 1008| ANALISIS KADAR ZAT BESI (FE) PADA TEPUNG KULIT KENTANG DAFTAR PUSTAKA Usahakan kulit
kentang yang telah dibuat tepung, dikomposisikan
lebih banyak sehingga zat besi yang diperlukan
tubuh dapat terpenuhi dari tepung kulit kentang
tersebut (Cicilia, Basuki, Prarudiyanto, Alamsyah,
& Handito, 2018). Buah kentang yang dipilih juga
harus segar, karena akan berdampak pada kulit
kentang tersebut. Sehingga dengan memilih buah
kentang yang segar, maka akan mendapatkan kulit
kentang baik dan bagus serta dapat dijadikan
tepung (Noscov, 2013 ). Membuat makanan
dengan olahan tepung kulit kentang ini juga harus
memperhatikan cara memasaknya, sehingga tidak
merusak kandungan zat besi yang ada dalam
tepung kulit kentang. Safitri, Y. (2019). Pengaruh Pemberian Jus bayam
Merah, Jeruk Sunkis, Madu Terhadap
kadar Haemoglobin pada Ibu Hamil yang
Mengalami Anemia di UPT Puskesmas
Kampar. Jurnal Ners, 72-83 Vol. 3, No. 2. Soelarso, B. (2012). Budi Daya Kentang Bebas
Penyakit . Yogyakarta : Kanisius . Suryowibisono. (2020). Pemanfaatan Limbah Kulit
Kentang
Sebagai
Bahan
Tambahan
Pembuatan Puding. Universitas Telkom
Bandung . Winarno, F. (2004). Kimia Pangan dan Gizi . Jakarta : PT Gramedia Pustaka Utama . SIMPULAN Pada penelitian ini dapat disimpulkan
bahwa tepung kulit kentang memiliki kadar zat
besi sebanyak 111,8 dalam 100 gr. Tepung kulit
kentang dapat dimanfaatkan oleh masyakarat
utamanya pada kelompok remaja putri dan ibu
hamil yang rentan beresiko terkena anemia. Sehingga hal ini dapat meningkatkan kadar
hemoglobin dalam tubuh. ISSN 2580-2194 (Media Online) Jurnal Ners Universitas Pahlawan
|
https://openalex.org/W2146634687
|
https://figshare.com/articles/journal_contribution/Supplementary_Table_4_from_Development_of_Gene_Expression_Based_Score_to_Predict_Sensitivity_of_Multiple_Myeloma_Cells_to_DNA_Methylation_Inhibitors/22496613/1/files/39954636.pdf
|
English
| null |
Development of Gene Expression–Based Score to Predict Sensitivity of Multiple Myeloma Cells to DNA Methylation Inhibitors
|
Molecular cancer therapeutics
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TABLE S4: Genes underexpressed in decitabine treated HMCLs. Five HMCLs were cultured with or without 0.5 µM
decitabine for 7 days and gene expression was profiled with Affymetrix U133 plus 2.0. Genes significantly
differentially expressed between control and decitabine treated cells were identified using SAM supervised paired
analysis with a 5% false discovery rate. When a gene was interrogated by several probe sets, we used the probe set
yielding to a maximum variance across control and decitabine treated cells. TABLE S4: Genes underexpressed in decitabine treated HMCLs. Five HMCLs were cultured with or without 0.5 µM
decitabine for 7 days and gene expression was profiled with Affymetrix U133 plus 2.0. Genes significantly
differentially expressed between control and decitabine treated cells were identified using SAM supervised paired
analysis with a 5% false discovery rate. When a gene was interrogated by several probe sets, we used the probe set
yielding to a maximum variance across control and decitabine treated cells. TABLE S4: Genes underexpressed in decitabine treated HMCLs. Five HMCLs were cultured with or without 0.5 µM
decitabine for 7 days and gene expression was profiled with Affymetrix U133 plus 2.0. Genes significantly
differentially expressed between control and decitabine treated cells were identified using SAM supervised paired
analysis with a 5% false discovery rate. When a gene was interrogated by several probe sets, we used the probe set
yielding to a maximum variance across control and decitabine treated cells. Probeset
Gene
Ratio
Banding
Affymetrix description
Intercellular communication and membrane proteins
202304_at
FNDC3A
0.40
13q14.2
fibronectin type III domain containing 3A
209541_at
IGF1
0.43
12q22-q23
insulin-like growth factor 1 (somatomedin C)
221658_s_at IL21R
0.50
16p11
interleukin 21 receptor
210587_at
INHBE
0.55
12q13.3
inhibin; beta E
219702_at
PLAC1
0.42
Xq26
placenta-specific 1
209606_at
PSCDBP
0.48
2q11.2
pleckstrin homology; Sec7 and coiled-coil domains; binding protein
202375_at
SEC24D
0.31
4q26
SEC24 related gene family; member D (S. cerevisiae)
222838_at
SLAMF7
0.51
1q23.1-q24.1 SLAM family member 7
222690_s_at TMEM39A
0.46
3q13.33
transmembrane protein 39A
Signal transduction
224451_x_at ARHGAP9
0.38
12q14
Rho GTPase activating protein 9 /// Rho GTPase activating protein 9
212954_at
DYRK4
0.42
12p13.32
dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 4
228831_s_at GNG7
0.33
19p13.3
guanine nucleotide binding protein (G protein); gamma 7
209314_s_at HBS1L
0.53
6q23-q24
HBS1-like (S. TABLE S4: Genes underexpressed in decitabine treated HMCLs. Five HMCLs were cultured with or without 0.5 µM
decitabine for 7 days and gene expression was profiled with Affymetrix U133 plus 2.0. Genes significantly
differentially expressed between control and decitabine treated cells were identified using SAM supervised paired
analysis with a 5% false discovery rate. When a gene was interrogated by several probe sets, we used the probe set
yielding to a maximum variance across control and decitabine treated cells. cerevisiae)
219188_s_at LRP16
0.36
11q11
LRP16 protein
229549_at
OPN1SW
0.40
7q31.3-q32
Opsin 1 (cone pigments); short-wave-sensitive (color blindness; tritan)
Cytoskeleton
213500_at
COPB2
0.58
3q23
Coatomer protein complex; subunit beta 2 (beta prime)
204411_at
KIF21B
0.53
1pter-q31.3
kinesin family member 21B
226438_at
SNTB1
0.36
8q23-q24
Syntrophin; beta 1 (dystrophin-associated protein A1; 59kDa; basic component 1)
226181_at
TUBE1
0.38
6q21
tubulin; epsilon 1
Cell cycle
214152_at
CCPG1
0.44
15q21.1
cell cycle progression 1
223569_at
PPAPDC1B
0.42
8p12
phosphatidic acid phosphatase type 2 domain containing 1B
223195_s_at SESN2
0.47
1p35.3
sestrin 2
Metabolism
231202_at
ALDH1L2
0.45
12q23.3
aldehyde dehydrogenase 1 family; member L2
219572_at
CADPS2
0.37
---
Ca2+-dependent activator protein for secretion 2
212816_s_at CBS
0.41
21q22.3
cystathionine-beta-synthase
218923_at
CTBS
0.42
1p22
chitobiase; di-N-acetyl-
201791_s_at DHCR7
0.45
11q13.2-q13.57-dehydrocholesterol reductase
204646_at
DPYD
0.48
1p22
dihydropyrimidine dehydrogenase
203397_s_at GALNT3
0.65
2q24-q31
UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase
203157_s_at GLS
0.42
2q32-q34
glutaminase
226183_at
GSK3B
0.49
3q13.3
Glycogen synthase kinase 3 beta 1555037_a_atIDH1
0.53
2q33.3
isocitrate dehydrogenase 1 (NADP+); soluble
201625_s_at INSIG1
0.34
7q36
insulin induced gene 1
221760_at
MAN1A1
0.43
6q22
Mannosidase; alpha; class 1A; member 1
222805_at
MANEA
0.51
6q16.1
mannosidase; endo-alpha
232488_at
MGC15875
0.55
5q35.3
hypothetical protein MGC15875
225520_at
MTHFD1L
0.41
6q25.1
methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like
202847_at
PCK2
0.47
14q11.2
phosphoenolpyruvate carboxykinase 2 (mitochondrial)
221788_at
PGM3
0.49
6q14.1-q15
Phosphoglucomutase 3
201397_at
PHGDH
0.36
1p12
phosphoglycerate dehydrogenase
205194_at
PSPH
0.38
7p15.2-p15.1 phosphoserine phosphatase
200831_s_at SCD
0.39
10q23-q24
stearoyl-CoA desaturase (delta-9-desaturase)
209610_s_at SLC1A4
0.54
2p15-p13
solute carrier family 1 (glutamate/neutral amino acid transporter); member 4
208916_at
SLC1A5
0.40
19q13.3
solute carrier family 1 (neutral amino acid transporter); member 5
209921_at
SLC7A11
0.41
4q28-q32
solute carrier family 7; (cationic amino acid transporter; y+ system) member 1
Protein binding
215930_s_at CTAGE5
0.30
14q13.3
CTAGE family; member 5
1554462_a_atDNAJB9
0.42
7q31|14q24.2-q24.3
DnaJ (Hsp40) homolog; subfamily B; member 9
222294_s_at EIF2C2
0.52
8q24
Eukaryotic translation initiation factor 2C; 2
235745_at
ERN1
0.35
17q24.2
endoplasmic reticulum to nucleus signalling 1
219118_at
FKBP11
0.38
12q13.12
FK506 binding protein 11; 19 kDa
218361_at
GOLPH3L
0.45
1q21.2
golgi phosphoprotein 3-like
224763_at
RPL37
0.35
5p13
ribosomal protein L37
201915_at
SEC63
0.51
6q21
SEC63-like (S. cerevisiae)
202061_s_at SEL1L
0.56
14q24.3-q31
sel-1 suppressor of lin-12-like (C. TABLE S4: Genes underexpressed in decitabine treated HMCLs. Five HMCLs were cultured with or without 0.5 µM
decitabine for 7 days and gene expression was profiled with Affymetrix U133 plus 2.0. Genes significantly
differentially expressed between control and decitabine treated cells were identified using SAM supervised paired
analysis with a 5% false discovery rate. When a gene was interrogated by several probe sets, we used the probe set
yielding to a maximum variance across control and decitabine treated cells. elegans)
217790_s_at SSR3
0.40
3q25.31
signal sequence receptor; gamma (translocon-associated protein gamma)
202558_s_at STCH
0.47
21q11.1|21q11stress 70 protein chaperone; microsome-associated; 60kDa
222116_s_at TBC1D16
0.70
17q25.3
TBC1 domain family; member 16
218145_at
TRIB3
0.41
20p13-p12.2
tribbles homolog 3 (Drosophila)
Nuclear proteins and transcription factors
227558_at
CBX4
0.49
17q25.3
chromobox homolog 4 (Pc class homolog; Drosophila)
219551_at
EAF2
0.46
3q13.33
ELL associated factor 2
226982_at
ELL2
0.45
5q15
elongation factor; RNA polymerase II; 2
202146_at
IFRD1
0.51
7q22-q31
interferon-related developmental regulator 1
214179_s_at NFE2L1
0.59
17q21.3
nuclear factor (erythroid-derived 2)-like 1
213878_at
RECQL
0.44
12p12
RecQ protein-like (DNA helicase Q1-like)
208763_s_at TSC22D3
0.53
Xq22.3
TSC22 domain family; member 3
225382_at
ZNF275
0.35
Xq28
zinc finger protein 275
227132_at
ZNF706
0.49
8q22.3
zinc finger protein 706
Others
227755_at
---
0.53
---
CDNA FLJ42435 fis; clone BLADE2006849
208725_at
---
0.46
---
Full-length cDNA clone CS0DD001YD20 of Neuroblastoma Cot 50-normalized of
244623_at
---
0.39
---
Transcribed locus
226719_at
---
0.53
---
CDNA FLJ34899 fis; clone NT2NE2018594 230570_at
---
0.37
---
Transcribed locus
229969_at
---
0.41
---
Transcribed locus; moderately similar to XP_508230.1 PREDICTED: zinc finger protein 195 [Pan troglodytes]
223136_at
AIG1
0.41
6q24.2
androgen-induced 1
222545_s_at C10orf57
0.56
10q22.3
chromosome 10 open reading frame 57
220755_s_at C6orf48
0.49
6p21.3
chromosome 6 open reading frame 48
219802_at
FLJ22028
0.44
12p12.1
hypothetical protein FLJ22028
212633_at
KIAA0776
0.49
6q16.1
KIAA0776
229090_at
LOC220930
0.45
10p11.23
hypothetical protein LOC220930 230570_at
---
0.37
---
Transcribed locus
229969_at
---
0.41
---
Transcribed locus; moderately similar to XP_508230.1 PREDICTED: zinc finger protein 195 [Pan troglodytes]
223136_at
AIG1
0.41
6q24.2
androgen-induced 1
222545_s_at C10orf57
0.56
10q22.3
chromosome 10 open reading frame 57
220755_s_at C6orf48
0.49
6p21.3
chromosome 6 open reading frame 48
219802_at
FLJ22028
0.44
12p12.1
hypothetical protein FLJ22028
212633_at
KIAA0776
0.49
6q16.1
KIAA0776
229090_at
LOC220930
0.45
10p11.23
hypothetical protein LOC220930
|
https://openalex.org/W4252808335
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https://journal.tarbiyahiainib.ac.id/index.php/attalim/article/download/281/235
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English
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High Touch Approach and Its Relationship with Students’ Learning Outcomes at IAIN Bukittinggi
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Al-Ta'lim/Jurnal Al-Ta'lim
| 2,017
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cc-by-sa
| 4,325
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AL-TA’LIM JOURNAL, 24 (2), 2017, (165-173) (Print ISSN 1410-7546 Online ISSN 2355-7893)
Available online at http://journal.tarbiyahiainib.ac.id/index.php/attalim Darul Ilmi Darul Ilmi
Institut Agama Islam Negeri Bukittinggi,
Indonesia. E-mail: ilmid10@yahoo.com Institut Agama Islam Negeri Bukittinggi,
Indonesia. E-mail: ilmid10@yahoo.com Keywords: High touch, student-centered and learning outcomes Keywords: High touch, student-centered and learning outcomes How to Cite: Ilmi, D. (2017). High touch approach and its relationship with students’ learning outcomes at IAIN
Bukittinggi. Al-Ta Lim Journal, 24 (2). doi:http://dx.doi.org/10.15548/jt.v24i2.281 How to Cite: Ilmi, D. (2017). High touch approach and its relationship with students’ learning outcomes at IAIN
Bukittinggi. Al-Ta Lim Journal, 24 (2). doi:http://dx.doi.org/10.15548/jt.v24i2.281 High Touch Approach and Its Relationship with Students’ Learning
Outcomes at IAIN Bukittinggi Received: 06th July 2017; Revised: 13th July 2017; Accepted: 17th July 2017
Permalink/DOI: http://dx.doi.org/10.15548/jt.v24i2.281 Abstract: Learning without students’ center tends to create less
passionate students in following the lecture. They tend to ignore
their lecturer. They have less attention, and try to create
ineffective conditions, therefore, the learning atmosphere will
become less effective to achieve the intended goals. The purpose
of the research is to determine the process of learning, the
implementation of High Touch approach, and factors that
influence its implementation. Classroom action research
approach was used at department of mathematics which involve
students who enroll at academic year 2016/2017 IAIN
Bukittinggi. The research was done in two cycles in which one
cycle conducted three meetings. The instruments used were
observation, essay questions, and tasks given to students. The
results show that there is an improvement in students’
participation and activeness in learning when their lecturer builds
learning experience through reinforcement, affection, guide,
directive action, and good modeling by student–center andself-
learning activities, and independent learning skill orientations
showed in cycle I and II. It implies that implementing high touch
may lead students to be more active, creative, and fun in learning. arul Ilmi, High Touch Approach and Its Relationship with Students’ Learning Outcomes… arul Ilmi, High Touch Approach and Its Relationship with Students’ Learning Outcomes… © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) INTRODUCTION resources. Educational investment is human
resources used to develop human potentials in
which includes both benefit and social returns
(Collins & Clark, 2003; Davidsson & Honig,
2003; Weinberger, 1998). Education is a strategic investment to
improve rivalry among countries in politics,
laws, cultures, technologies, and defenses all
over the world. In accordance to this condition,
the
developed
countries
simultaneously
improve their education focusing on human
capital and human investment. This is the
reason to improve the capability to compete
among
nations
by
having
high
quality The quality of education output is
affected by learning quality
(Moore &
Kearsley, 2011; Nightingale & O’Neil, 2012;
Sims, Dobbs, & Hand, 2002). The quality is
determined by prerequisite conditions of 165 © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) Volume 24, Number 2, July, 2017, Page 165-173 166 students’ mastery, learning skills, facilities, and
physical and socio-emotional environment. Thus, learning process as operationalization of
educational practice done by lecturer should
contain high-tech and high-touch. The high-
touch
(the
term
used
“authority”)
is
“educational tool” used by educator to touch
students’ selves in educational relationship
which directs to high-touch condition, in the
meaning that educator’s treatment positively,
constructively, and comprehensively touch
students’ humanity aspects (Anderson, 2004;
Gainor, Goins, & Miller, 2004; Kulchitsky,
2008; Noddings, 2013). atmosphere should be built by lecturer’s
authority through engaging students in learning
processes. Light, Calkins, & Cox (2009)
furthermore, said that education phenomenon
occurs when it grows and develops through
authority actualization reflected in the way
lecturer teaches in the classroom. Through this
process,
the
lecturer
can
assure
the
development of learning situation. Based on the preliminary survey in
the learning process of Mathematics education
students in even semester in academic year
2015/2016 at IAIN Bukittinggi, the researcher
found that the lecturer tended to show bad
attitudes such as talkative, anger, and unfair,
while they prefer good, friendly, smart, and
kind attitudes. The results also show
incompatible relation between lecturer and
students because the lecturer tended to make
the students inferior and passive. Moreover,
students were less passionate to learn, reluctant
to participate in discussion, and used to
compare between one lecturer and another,
thus, efficient learning environment is difficult
to achieve. High-touch implementation or authority
used to develop students’ personals includes
acknowledgment,
affection,
reinforcement,
guidance, directive actions, and modeling. Indicators of Success The indicators of success are presented
as follow: The indicators of success are presented
as follow:
90 - 100 = Very good
80 - 89 = Good
65 - 79 = Adequate
55 - 64 = Quite Good
0 - 54 = Not Good The learning process is success when
students: 1. Solve the problems well
2. Ask many questions
3. Share their ideas
4. Done the task well
5. Present the result of task well
6. Pay attention, listen to, and give opinion
to their peers’ work
7
Participate in giving opinion This research describes the improvement
of learning process through high touch
approach in Islamic education science subject
and its relationship with learning outcomes of
Mathematics education department students in
even semester in academic year 2016/2017 at
IAIN Bukittinggi in which the data are
quantitative data. 7. Participate in giving opinion Setting of the Research The research was done at IAIN
Bukittinggi toward Mathematics education
department students in second semester in
academic year 2016/2017. The samples were
35 students consisting of 8 males and 27
females. The steps in this research is named
cycle in which one cycle consisted of three
meetings. The
researcher
observed
the
planning, implementation, observation, and
reflection activities. The reflection in the first
cycle was used to take further action in the
next cycle. Technique of Data Collection The students were coming from
many
different
backgrounds
including
educational, socio-economic, and cultural
backgrounds gathering in Islamic education
science course class. To collect the data, the researcher used
both direct observation and observation sheet
in each cycle. The observation was done to
observe
the
class
atmosphere,
learning
process, students’ activities, and the results
were written in observation sheet. Tests were
also used at the beginning and the end of
meeting. Test is a series of questions used to
measure students’ competence. The tests were INTRODUCTION On
the other hand, high-tech (education) is
“educational tool” used by educator for
realization of learning goals achievement
directed to the use of high quality technology. It
includes curriculum, learning methods, learning
equipment, learning environment, and learning
assessment. Learning process is done not only
by using appropriate materials and methods, but
also in line with students’ affective and social
developments to achieve purposed goals. High
tech and authority should be in harmony to
improve the learning process and outcome. Regarding to the phenomena above, an
analysis of learning process especially the
implementation of authority and its relationship
with learning outcomes is needed. The research
was done towards Mathematics education
department students in even semester in
academic year 2016/2017 at IAIN Bukittinggi. The lecturer is expected to implement authority
and education in learning process based on
educational science principles which are related
to the learning outcomes. Accordingly, the
problems are identified as follow: The study done by Loughran (2013)
showed that the application of authority and
educational in learning process was not done
well as expected as many problems emerged in
relation with mastering difficulty, low of
learning skill, insufficient facilities, students’
selfhood and physical and socio-emotional
environment. Accordingly, efforts to enable
learning process improve through implementing
authority and education in learning process
(Black, Harrison, Lee, Marshall, & Wiliam,
2004; Gould, 2009; Hord, 2009; Vescio, Ross,
& Adams, 2008). 1. The lecturer’s implementation of authority
(high touch) in learning process g
g p
2. Learning activities tended to be passive
3. Learning outcomes 4. The relationship between authority and
learning outcomes Kriz (2003) stated that in order to create
effective learning environment, the classroom 5. Students’ problems include less passionate,
less motivated, and passive. © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) Darul Ilmi: High Touch Approach and Its Relationship with Students’ Learning outcomes… 167 METHOD science course was presented in even semester
in academic year 2016/2017. This research is classified into Action
Research in which describes facts, conditions
of
learning
process,
or
phenomena
systematically,
factually,
and
actually
Arikunto, (2002); Gay & Airasian (2000)
stated that descriptive research is a research
done to collect the information about a
phenomenon as it is. The analysis used was
descriptive analysis. According to Best (1982)
descriptive analysis is an analysis done to
know the relationship between independent
and dependent variables. The independent
variable in this research was high touch, while
dependent variable was learning outcomes. Cycle I In the first meeting, the writer allowed the
students to study and analyze the materials by
using books, accessing internet and map and
identify the material to be explained, the steps
of learning process: 2. Observation sheet was used to measure
students’ activities during the learning
process. The activities to be observed
included 3. The students who asked questions both
from lecturer or peers 1. The students sat in group and were allowed
to learn the materials to be presented in
discussion in the classroom. 4. The students who shared their ideas 5. The students who discussed with their
peers 2. The lecturer controlled the discussion; the
group presenting was commented by three
other groups. Later general comments were
allowed to be given by all students. 6. Understanding and mapping the materials 7. Presenting the results in group 8. Paying attention to peers’ presentation 9. Participating in every discussion 3. The lecturer observed the discussion, the
students who actively participated in giving
comment. RESULT AND DISCUSSION in essay forms in which the answer is in the
form of discussion, reasoning, and exploring
ideas (Arikunto, 2002). The research was done in Mathematics
Education Department in even semester in
academic year 2016/2017 IAIN Bukittinggi in
which the focus is on improving the students’
role in learning. The research, in general
included students’ activities in learning and
learning outcomes. Time of Research The research was done in four months,
one semester, in which the Islamic education © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) Volume 24, Number 2, July, 2017, Page 165-173 168 Instrument of the Research The instruments used in the research: 1. Students’
activities
sheet
containing
questions and structured tasks based on
the sub-chapters of Educational Bases
course. The structured tasks were given
both in group and individual. Procedure of the Research 4. The lecturer and students concluded the
materials discussed. The research was done in two cycles
in which each cycle consisted of three
meetings, the steps in each cycle: 5. The lecturer re-asked the conclusion made
in
order
to
check
the
students’
understanding. 1. Planning 1. Planning 2. Prepare learning equipment such as
syllabus and SAP 6. The lecturer delivered the next materials
and resources to be used. 3. Prepare the task to be done, questions to
be answered, and observation sheet In the second meeting, the class
discussed educational bases materials in which
the procedure was quiet the same as the first
meeting. The students were asked to bring
visual aids in the form of carton related to the
materials, then, it is presented by groups, the
steps as follow: 4. Action 4. Action 4. Action The actions done by students were
studying, understanding, and analyzing the
materials
based
on
the
syllabus,
and
presenting group works through discussion. In this step, the approach was focused on
students’ center learning in order to improve
learning process quality and gain better
outcomes. Thus, the learning scenario was
constructed in the form of SAP. 1. The students sat in group and were allowed
to learn the materials to be presented in
discussion in the classroom. © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) Darul Ilmi: High Touch Approach and Its Relationship with Students’ Learning outcomes… 169 2. The lecturer controlled the discussion; the
group presenting was commented by three
other groups. Later general comments were
allowed to be given by all students. 1. The lecturer controlled the discussion; the
group presenting was commented by three
other groups. Later general comments were
allowed to be given by all students. 2. The lecturer observed the discussion, the
students who actively participated in giving
comment. 3. The lecturer observed the discussion, the
students who actively participated in giving
comment. 3. The lecturer and students concluded the
materials discussed. 4. The lecturer and students concluded the
materials discussed. 4. The lecturer re-asked the conclusion made
in
order
to
check
the
students’
understanding. 5. The lecturer re-asked the conclusion made
in
order
to
check
the
students’
understanding. 6. The lecturer delivered the next materials
and resources to be used 5. The lecturer delivered the next materials
and resources to be used 6. At the end of the third meeting, the lecturer
re-asked the materials learned from the first
until the third meetings and the students
were asked to collect them. In the third meeting, the class discussed
educational bases materials in which the
procedure was quiet the same as the first
meeting. The students were asked to bring
visual aids in the form of carton related to the
materials, then, it is presented by groups, the
steps as follow: 7. Observation
The observation of learning activities was
done during learning process. The observer
completed the observation sheet and the
data collected was analyzed descriptively
to show students’ activities as described in
the table below: 7. Observation
The observation of learning activities was
done during learning process. 4. Action The observer
completed the observation sheet and the
data collected was analyzed descriptively
to show students’ activities as described in
the table below: The students sat in group and were
allowed to learn the materials to be presented in
discussion in the classroom. Table 3
No
Learning Activities
First
Meeting
Second
Meeting
Third
Meeting
Average
1
The students completed the task given
60
70
80
70
2
The students shared their opinion
30
35
40
35
3. The students discussed each other
40
50
60
50
4. The students presented their works
20
30
40
30
5
The students paid attention to their friends’
presentation
50
60
70
60
6
The students who did not participate
80
60
50
65
7
The students who did not care
50
60
70
60
8
The students who were not discipline
30
20
20
25
9
The students who were chatting during
learning
10
15
10
12,5 Table 3 not discipline was 25 %, there was an
improvement, chatting during learning was 12,5
%, there was an improvement. The data above shows that there is an
improvement from the first until the third
meetings in preparing the task given by the
lecturer by average was 70 %, giving opinion
was 35 %, discussing with other students was
50 %, presenting the works was 30 %, paying
attention to others’ presentation was 60%, not
participating was 65 %, not caring was 60 %, In general, there were improvements in
cycle I including doing tasks, giving opinion,
the way of discussion, and the way of
presenting the works, but there were still many © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) Volume 24, Number 2, July, 2017, Page 165-173 170 5. The lecturer re-asked the conclusion made
in
order
to
check
the
students’
understanding. students did not participate during learning such
as not caring and not discipline as much as
60%. g
6. The lecturer delivered the next materials
and resources to be used. In
specific,
various
strategies
implemented during educational bases course
affected students’ ways in learning process. These
results
were
gotten
through
implementing high touch approach including
affection, mildness, guidance, reinforcement,
directive actions, good modeling, and student
center development oriented to students’
activities. In the second meeting, the class
discussed educational bases materials in which
the procedure was quiet the same as the first
meeting. 4. Action The students were asked to bring
visual aids in the form of carton related to the
materials, then, they were presented by groups,
the steps as follow: The results of cycle I describe that there
was an improvement in the students’ learning
process although the results were not satisfying. Only some students were active and wanted to
present their works. These phenomena might be
caused by great number of groups and lack of
preparation. 1. The students sat in group and were allowed
to learn the materials to be presented in
discussion in the classroom. 2. The lecturer controlled the discussion; the
group presenting was commented by three
other groups. Later general comments were
allowed to be given by all students. 3. The lecturer observed the discussion, the
students who actively participated in giving
comment. © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) Cycle II Accordingly, the writer minimized the
number of groups in cycle II in which four or
five students per group. Each student was given
the materials’ summary and each group mapped
the concept in simple visual aid to be presented,
the steps are described as follow: 4. The lecturer and students concluded the
materials discussed. 5. The lecturer re-asked the conclusion made
in
order
to
check
the
students’
understanding. 6. The lecturer delivered the next materials
and resources to be used In the first meeting, the writer allowed
the students to study and analyze the materials
by using books, accessing internet and map and
identify educational bases to be explained, the
steps of learning process: In the third meeting: 1. The students sat in group and were allowed
to learn the materials to be presented in
discussion in the classroom. 1. The students sat in group and were allowed
to learn the materials to be presented in
discussion in the classroom. 2. The lecturer controlled the discussion; the
group presenting was commented by three
other groups. Later general comments were
allowed to be given by all students. 2. The lecturer controlled the discussion, the
group presenting was commented by three
other groups. Later general comments were
allowed to be given by all students. 3. The lecturer observed the discussion, the
students who actively participated in giving
comment. 3. The lecturer observed the discussion, the
students who actively participated in giving
comment. 4. The lecturer and students concluded the
materials discussed. 4. The lecturer and students concluded the
materials discussed. © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) arul Ilmi: High Touch Approach and Its Relationship with Students’ Learning outcomes… Darul Ilmi: High Touch Approach and Its Relationship with Students’ Learning outcomes… 171 5. The lecturer re-asked the conclusion made
in
order
to
check
the
students’
understanding. 6. The lecturer delivered the next materials
and resources to be used
At the end of the third meeting, the
lecturer re-asked the materials learned from the
first until the third meetings and the students
were asked to collect them. Table 4. The results of the observation in cycle II are described in the table below:
No
Learning Activities
First
Meeting
Second
Meeting
Third
Meeting
Average
1
The students completed the task given
70
80
90
80
2
The students shared their opinion
40
45
50
45
3. Cycle II The students discussed each other
50
65
80
70
4. The students presented their works
30
40
50
40
5
The students paid attention to their friends’ presentation
60
70
85
72,5
6
The students who did not participate
60
40
40
45
7
The students who did not care
60
70
80
70
8
The students who were not discipline
20
15
15
17,5
9
The students who were chatting during learning
10
15
10
12,5 At the end of the third meeting, the
lecturer re-asked the materials learned from the
first until the third meetings and the students
were asked to collect them. Table 4. The results of the observation in cycle II are described in the table below:
No
Learning Activities
First
Meeting
Second
Meeting
Third
Meeting
Average
1
The students completed the task given
70
80
90
80
2
The students shared their opinion
40
45
50
45
3. The students discussed each other
50
65
80
70
4. The students presented their works
30
40
50
40
5
The students paid attention to their friends’ presentation
60
70
85
72,5
6
The students who did not participate
60
40
40
45
7
The students who did not care
60
70
80
70
8
The students who were not discipline
20
15
15
17,5
9
The students who were chatting during learning
10
15
10
12,5 6. The results of the first, second, and third
meetings show that less than 50% students
still not participating in discussion even
some other never participate from cycle I
until cycle II. 7. In the first, second, and third meeting there
was an improvement in not caring, less
motivated due to the students’ social and
cultural background. 2. In the results of the first, second, and third
meetings there was an improvement in
giving opinion as much as 10 % inasmuch
as the lecturer stimulated and respected the
students’ opinion. 8. In cycle II, there were still some students
came late to the class and take much time
when the lecturer allowed them to go out of
the class. 3. In presenting their works, still the same
students in cycle I presented the materials
in cycle II. The materials were presented
well because they were directed by course
book provided by the lecturer, but there
were lack of reasoning and concrete
examples. 9. Cycle II There still some students chatted during the
learning process, but later they were able to
follow the process. In
general,
cycle
II
covered
improvement rather in cycle I. In other words,
high touch approach implementation enables
students to learn better, but when it is related to
learning
outcomes
in
form
of
essay
examination, the results are not sufficient. They
still have difficulty to do reasoning or to give
operational and contextual examples. They may
have lack preparation to follow examination
such as not used to have autonomous learning
or lack of reading outside the class. 4. In participating in discussion, still the
students who were active in cycle I
dominated
the
discussion,
but
some
students who did not participate in cycle I
were able to give their opinion in cycle II. 5. In the first, second, and third meeting, the
students respected their classmates who
presented the materials because the lecturer
directed and gave them the reward. © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) Volume 24, Number 2, July, 2017, Page 165-173 172 CONCLUSION AND
RECOMMENDATION Gainor, S. J., Goins, R. T., & Miller, L. A. (2004). Using online modules in a multi-
modality teaching system: A high-touch,
high-tech approach to geriatric education. Gerontology & Geriatrics Education,
24(4), 45–59. Regarding to the findings of the
research, it is concluded that the high touch
approach implemented by teacher through
affection, mildness, guidance, directive actions,
and good modeling influenced students’
attitude in learning. The lecturer allows
students
to
develop
their
creativity
in
independent
learning
skill,
self-learning
activities and student center, learning involved
reward and punishment, respects students, and
forms the class in U letter form, and gives tasks
for
students
although
the
maximum
achievement is not yet gained. Gay, L. R., & Airasian, P. (2000). Educational
research: Competencies for. Analysis and
Application,(6th Edition). Ohio: Prentice
Hall. Gould,
J. M. (2009). Understanding
organizations
as
learning
systems. Strategic Learning in a Knowledge
Economy, 119–140. Hord, S. M. (2009). Professional learning
communities. Journal
of
Staff
Development, 30(1), 40–43. Based on the findings, the writer
expects that the lecturer varies the learning
through high touch approach. Although this
research cannot be generalized, if the case and
phenomenon is quiet the same, the approach
may help lecturer during teaching learning
process. Kriz, W. C. (2003). Creating effective learning
environments and learning organizations
through
gaming
simulation
design. Simulation & Gaming, 34(4), 495–511. For policy makers, they need to
complete learning medium and facilities to
enable lecturers to improve their potentials. For
the next researcher, further research is needed
to improve learning process quality in order to
help students to gain better learning outcomes. Kulchitsky, J. D. (2008). High-tech versus
high-touch education: Perceptions of risk
in
distance
learning. International
Journal of Educational Management,
22(2), 151–167. Light, G., Calkins, S., & Cox, R. (2009). Learning
and
teaching
in
higher
education: The reflective professional. Sage. © 2017 by Al-Ta’lim All right reserved. This work is licensed under (CC-BY-SA) REFERENCES Anderson, T. (2004). Towards a theory of
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online learning, 2, 109–119. Loughran, J. (2013). Developing a pedagogy of
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approach to ethics and moral education. Univ of California Press. Vescio, V., Ross, D., & Adams, A. (2008). A
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https://www.frontiersin.org/articles/10.3389/feduc.2020.00125/pdf
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Perceived Teacher Support and Communication in Strategizing Possible Selves
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Frontiers in education
| 2,020
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cc-by
| 11,299
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Perceived Teacher Support and
Communication in Strategizing
Possible Selves Rebeca Mireles-Rios1* and Shadi Roshandel2 1 University of California, Santa Barbara, Santa Barbara, CA, United States, 2 University of California, East Bay, Hayward, CA,
United States 1 University of California, Santa Barbara, Santa Barbara, CA, United States, 2 University of California, East Bay, Hayward, CA,
United States During high school, adolescents develop future plans for their lives and profession
and teachers are generally believed to play an important role. At present, however,
there is limited evidence about exactly how this support affects students. This study
uses possible-selves theory to explore the link between perceived teacher support and
communication about the future to adolescents’ own hoped-for and feared possible
selves. Surveys and structured interviews were used to gather views of 85 Latina/o and
White students (Mage = 15.5 years). Results indicate that greater perceived support and
better communication from teachers were both associated with adolescents expressing
more concrete strategies for achieving all types of possible selves. This study offers
initial evidence about the important role teachers may play in supporting and shaping
how adolescents construct an action plan that will lead to their desired future selves. Specialty section: Specialty section:
This article was submitted to
Educational Psychology,
a section of the journal
Frontiers in Education Received: 10 January 2020
Accepted: 25 June 2020
Published: 21 July 2020 Edited by:
Michael S. Dempsey,
Boston University, United States Reviewed by:
Patrick J. Carroll,
The Ohio State University,
United States
Doug Hamman,
Texas Tech University, United States Keywords: possible selves, perceived teacher support, motivation, goal intention, action plan, adolescence,
teacher communication INTRODUCTION Throughout adolescence, there is a dramatic increase in cognitive skills, allowing students to think
more concretely about their future (Piaget, 1964), and focus on who they are and who they want
to become (Erikson, 1968). The construct of “possible selves” has been used to help understand
how adolescents think about their future (Markus and Nurius, 1986; Roshandel and Hudley, 2018). Specifically, possible selves are components of the self-concept and are defined as both positive
and negative mental representations of the self in the future which are constructed from their past
and current perceptions of themselves (Markus and Nurius, 1986). They may include hoped-for
selves (what one hopes to be), expected selves (what one expects to be), and feared selves (what
one wants to avoid being) (Markus and Nurius, 1986). Each of these has been shown useful to help
motivate adolescents to achieve a goal or avoid a fear they may have for their future (Markus and
Nurius, 1986; Zhu and Tse, 2015). Possible selves are more proximal in nature and may serve as
stronger behavioral motivators for attaining future selves than more ambiguous, distal goals for
oneself (Oyserman et al., 2011). *Correspondence:
Rebeca Mireles-Rios
rebeca.rios@ucsb.edu ORIGINAL RESEARCH
published: 21 July 2020
doi: 10.3389/feduc.2020.00125 Citation: Gollwitzer (1993, 1999)
discusses the value of understanding implementation intentions
of goals, which they define as the intentions to form a plan to
meet a goal. Research has found that the connections created by
implementation intentions facilitate goal attainment (Gollwitzer
et al., 2004; Duckworth et al., 2011; Gollwitzer and Oettingen,
2011). Thus, by including strategies to the possible selves concept,
we can further understand why some individuals successfully
achieve desired selves and avoid feared selves, whereas others
with similar expectations and fears do not manage the same
outcome (Clinkinbeard and Murray, 2012). However, empirical
research has yet to fully explore the contexts that facilitate strategy
development. As high school represents an essential time in
transitioning into adulthood, it is an optimal environment to
foster the development of these critical strategies. Current research on possible selves, has linked the content of
one’s identities to the behaviors or strategies individuals develop
in order to achieve a desired expected self or avoid a feared
possible self (Zhu and Tse, 2015). Possible selves have been
associated with improved educational attainment (Destin and
Oyserman, 2009), more gainful employment (Lee and Oyserman,
2009), and reduced riskiness in sexual behavior (Clark et al.,
2007). However, there is limited research on how adolescents
develop these strategies in order to influence their current
behaviors to reach a desired outcome. During high school, adolescents have the potential to develop
effective strategies that will help them achieve a goal, change
their behavior, or take action to avoid a fear as they are further
developing future-oriented thoughts (Markus and Nurius, 1986;
Clinkinbeard and Murray, 2012; Zhu and Tse, 2015). High
school is also a critical time as adolescents are making plans
about their future and transitioning into their autonomous selves
(National Research Council, 2013). In order to help adolescents
develop effective strategies for changing their current behavior
to achieve their possible selves, it is important to understand
the contextual factors, such as support and interactions with
teachers, which contribute to such processes. If students perceive
support from supportive social contexts (e.g., teacher) during
uncertain situations (e.g., starting a new internship or planning
for their future after high school), they are more likely to explore
new opportunities and feel less anxious (Gamboa et al., 2013). Citation: Mireles-Rios R and Roshandel S
(2020) Perceived Teacher Support
and Communication in Strategizing
Possible Selves. Front. Educ. 5:125. doi: 10.3389/feduc.2020.00125 Research shows that some patterns of possible selves are universal, while others are sensitive to
context and culture, resulting in individual differences in the processes related to possible selves
(Cross and Markus, 1991; Knox et al., 1998). Oyserman et al. (2011) found that children in more
disadvantaged neighborhoods were more likely to have school-focused possible identities than July 2020 | Volume 5 | Article 125 1 Frontiers in Education | www.frontiersin.org Possible Selves and Perceived Teacher Support Mireles-Rios and Roshandel children in less disadvantaged neighborhoods. More recently,
Roshandel and Hudley (2018), identified gender differences
in high school adolescents’ college-going possible selves, with
females who perceived their teachers as more supportive being
more likely to aspire and expect to attend a 4-year college
compared to males. Turcios-Cotto and Milan (2013) found that
Latin/o youth were less likely to picture themselves attending
college, when compared to Black and White youth. Taken
together, these studies highlight the importance of accounting for
individual differences, such as gender and ethnicity, which may
contribute to one’s possible selves. allowing one to have many possible selves (Oyserman et al., 2015). Being able to distinguish between expectations and fears for the
future is critical in the development of possible selves as there is
a belief that the current self is malleable, facilitating a confidence
that change is possible (Oyserman et al., 2004). For the purpose
of this study, we will use the term possible selves to refer to
any future orientation, but expected selves and feared selves to
distinguish between positive and negative future possible selves. According to research, adolescents with the most developed
possible selves understand the necessary relationship between
future expectations and fears, and have identified strategies in
place to facilitate the achievement–avoidance of those selves
(Oyserman and Markus, 1990; Oyserman et al., 2004). Strategies
that are associated with expected and feared possible selves may
be the most critical piece of future planning and the element that
is most closely linked to actual behavior change (Oyserman et al.,
2006; Clinkinbeard and Murray, 2012). Ajzen (1985) asserts that
the type of goal chosen and the commitment to that goal are
important determinants in whether an individual carries out the
behaviors necessary for goal attainment. Perceived Teacher Support and
Communication Possible selves research indicates that individuals often look to
others as models for the development of their future selves
(Oyserman and Fryberg, 2006; Anders and Olmstead, 2018). Communicating positive feedback from others fosters hope and
has the potential to increase an individual’s self-efficacy and self-
esteem, whereas negative feedback is associated with negative
affect and lowered expectations that may stifle attempts to
change or further develop (Ruvolo and Markus, 1992; Cross
and Markus, 1994). One of the primary sources of this type of
communication during high school are teachers (Clinkinbeard
and Murray, 2012). Given the connections between feedback
and behavior, it is theoretically possible that how teachers
support and communicate with students can serve as a potential
social resource to the development of strategies that guide
students to achieving their expected selves and/or avoiding
their feared selves. Citation: Research highlighting the predictors of effective planning can
ultimately guide teachers in facilitating and designing curriculum
and programs that enhance adolescents’ effective future-oriented
thinking and the production of strategies (Oyserman et al.,
2004; Dunkel and Kerpelman, 2006; Zhu and Tse, 2015). Yet,
thus far, there is limited insight into how perceived teacher
support and communication may be related to the development
of adolescents’ strategies to achieve a hoped for and expected
possible selves while avoiding their feared possible selves. This
study explores the relationship between adolescents’ perceptions
of their teachers support and communication and their possible
selves. We examine not only their types of expectations
and fears, but also the strategies they develop to meet the
desired outcomes. Strategies Findings show that having strategies represents an “action plan”
to any possible selves (Oyserman et al., 2004). Clinkinbeard
and Murray (2012) make a distinction between “concrete” and
“abstract” strategies by defining a “concrete strategy” as one that
could be repeated by another person. One example of a concrete
strategy to deal with an adolescent’s fear of dropping out of
school would be to “take detailed notes in my math class,” as
compared to an abstract strategy of “change my habits.” If this
“action plan” is more concrete, adolescents have specific next
steps in figuring out how to achieve their goal (Clinkinbeard
and Zohra, 2012; Anders and Olmstead, 2018). These strategies
thus represent the motivational resource most closely tied to
changing behavior (Oyserman et al., 2004; Anders and Olmstead,
2018). Gollwitzer et al. (2004) discuss that the first step for
implementation intention is to identify the goal and then plan
actions that make the goal achievable. If we are more cognizant
of adolescents’ possible selves, then teachers can provide them
with the support of developing these concrete strategies that will
increase the likelihood of adapting current behaviors to achieve
the desired future goals. Thus, it is imperative to explore the role
of perceived teacher support and communication in providing
opportunities to develop the skillset of forming specific strategies
related to possible selves. In order to provide an innovative approach to understanding
the complex research agendas, this study used a mixed-methods
approach to explore adolescents’ perceptions of support, by
integrating both numerical data (e.g., survey) and text data
(e.g., interviews) (Creswell, 2014). Specifically, an explanatory
sequential design was chosen, where we initially collected and
analyzed survey data, followed by qualitative data to help us
better understand the quantitative findings (Mertens, 2007;
Creswell, 2014; Creswell and Clark, 2017). The Present Study The present study explores how teachers may serve as a social
resource to support the cognitive development of concrete
strategies to help adolescents address their possible selves. First,
we want to understand the types of possible selves and strategies
high school students report. In terms of the strategies we also
want to explore if these strategies are categorized as concrete
or abstract. Secondly, we want to understand how perceptions
of teacher support may be associated with the aforementioned
strategies. We hypothesize when adolescents perceive more
teacher support, they are more likely to have concrete strategies
to deal with their possible selves. Specifically, we hypothesize that
greater perceived teacher communication about the future will
predict greater use of concrete over abstract strategies for both
expected and feared possible selves. MATERIALS AND METHODS Participants and Procedures
A total of 85 (41 males and 44 females) adolescents between
the ages of 14 to 17 (M = 15.5, SD = 0.885) were recruited
to participate in a study focusing on their thoughts about
their future. Twenty-seven percent of the participants were in
9th grade, 42% were in the 10th grade, and 31% were in the
11th grade. For the purpose of this study, only results from
participants who identified as White and Latin/o students were
analyzed, as only a few participants identified as “other.” This
ethnic breakdown of the students matched the overall school site
demographics (45% White and 55% Latin/o). The school site has
a 40% population of free or reduced lunch. Distinction Between Expected and
Feared Possible Selves Teachers also create learning environments that provide
students with resources and the space to explore how their
current behaviors may impact their future goals that they Unlike general and ambiguous goals and fears, possible selves
exist in any future domain (e.g., academic, social, and health), July 2020 | Volume 5 | Article 125 Frontiers in Education | www.frontiersin.org 2 Possible Selves and Perceived Teacher Support Mireles-Rios and Roshandel may not receive otherwise (Strati et al., 2017; Roshandel and
Hudley, 2018). Studies show that when adolescents perceive
their teachers as supportive, they report higher levels of interest
and engagement in their school work (Watson et al., 2016),
more positive academic attitudes and values (Fredricks et al.,
2019), and more academic engagement (Wentzel et al., 2016). In
addition, from an adolescent perspective, it is not only interacting
with their teachers that is vital but rather what the teachers
are communicating (Elhay and Hershkovitz, 2019). Thus, these
studies together confirm the significant impact perceived teacher
support and communication has on adolescents’ academic
engagement and motivation. experiences. Martinez and Castellanos (2018) found that
students who were more engaged in school activities had greater
opportunity to develop relationships with teachers who shared
their own academic experiences with students, helping the
students to carve out their own college trajectories. This finding
supports prior research which argues that aspirations to attend
college are not solely based on motivation, but can also be driven
by school context and relationships with teachers, which can give
students the confidence they need to make their aspirations a
reality (Gutman and Akerman, 2008; Martinez and Castellanos,
2018). In addition, such future aspirations, can play a significant
role in impacting current academic behavior and achievement. Given that teachers may influence students’ academic
achievement and school engagement (Wentzel et al., 2016), it is
theoretically possible to expect a relationship between perceived
teacher support and adolescents’ strategies for achieving or
avoiding their possible selves. The more we understand how
teachers can influence students’ development of strategies
through communication and support, the better we can prepare
teachers to create the optimal learning environment that focuses
on academics as well as development of the whole adolescent. Frontiers in Education | www.frontiersin.org Types of Strategies for Possible Selves Instructions and questionnaire matrices were adopted from
Oyserman and James (2011). Participants were asked to write in
up to four strategies for each expected possible selves (“What are
doing to be that way in the future?”) and four strategies for each
feared possible selves (“What are you doing to avoid being that
way in the future?”). Academic Achievement Weighted grade point averages (GPA) were collected from
official school records. Survey Measures
Demographics Students were asked the following questions: Do your teachers
talk about your future? What are the ways that teachers support
you in the classroom to help you prepare for the future? Can
you give concrete examples? The researchers told students they
could answer the questions based on any teacher. The authors
developed semi-structured interviews in order to focus on themes
that would emerge rather than elicit specific answers about
perceived teacher support and communication. Participants completed a questionnaire that included their age,
grade level, and race/ethnicity. Students wrote in their age,
checked offtheir grade (9th–12th), gender (male or female)
and were asked their race/ethnicity (Latin/o, White, Asian,
Black, and Other). Each category had the option to specify. For example, many students that checked Latin/o, wrote
in Mexican-American. After students took the survey, they were asked if they were
willing to participate in a 30-min interview. Thirty students
(approximately 50% female, 50% male, 50% White, and 50%
Latina/o) were interviewed at a separate time and location for
the second phase of the study. The interviews took place either
afterschool or at lunch, in a quiet and uninterrupted location
at the school site. Parent consent and adolescent assent was
received. Two graduate student researchers, who were trained
and familiar with the study, facilitated the interviews. Again,
participants were reminded that their answers would remain
confidential. Students did not receive incentives to participate in
any stage of the research. Types of Possible Selves Instructions and questionnaire matrices were adopted from
Oyserman and James (2011). Participants were asked to write
in up to four expected (“What are your expectations for the
future?”) and four feared possible selves (“What are your fears
for the future?”). Analytical Procedures Using categories identified from previous studies (e.g., Oyserman
et al., 2004), the authors coded participants’ responses to the
possible selves questionnaire into six categories: achievement
(school
or
work
related
activities),
relationships
(social
interactions
with
others),
individual
(personality),
health
(physical wellness), lifestyle (material possessions), and risky
behavior (antisocial activity). Since students were able to mark
more than one response, the authors used counts (ranging
from 0 to 4) to measure each category. The authors of the
paper coded the first 20 participants’ responses of possible
selves and strategies together and then separately each coded
the remainder 65 entries. They then compared coding and had
an overall inter-rater agreement rate across the six categories
of types of possible selves and strategies, ranging between 92
and 97% (Saldana, 2009). Any discrepancies were resolved
through discussion. School Engagement Adolescents reported on an adapted version of a 3-item School
Engagement Scale (Gonzales et al., 2014). Participants responded
to the following items using a 4-point Likert scale ranging from
“I strongly disagree” to “I strongly agree:” “I like to do well in
school,” “It is very important to finish high school,” and “It is very
important to go to college.” The ratings were summed to create a
student engagement score. The scale had an alpha score of 0.87. School Engagement Researchers
have
also
explored
the
relationship
between
perceived teacher support and educational outcomes such
as academic
engagement,
effort,
expectations,
motivation,
and achievement. Findings consistently indicate a positive
relationship
between
teacher
support
and
a
myriad
of
advantageous
outcomes,
such
as
academic
achievement,
motivational outcomes, and educational aspirations (Wentzel,
1997, 1998; Wentzel et al., 2016; Roshandel and Hudley,
2018). For example, teachers can foster confidence in students
by serving as role models by sharing their own academic Four general education, social studies classrooms representing
the school demographics were identified and selected by the
school administration to participate in the study. The overall
teacher demographics of the school included 85% White, 12%
Latin/o, 2% Asian, and 1% Black, with 56% female. For this study, Frontiers in Education | www.frontiersin.org July 2020 | Volume 5 | Article 125 3 Possible Selves and Perceived Teacher Support Mireles-Rios and Roshandel Perceived Teacher Support and Communication
We measured perceived informational support and perceived
instrumental support (Malecki and Demaray, 2002; Roshandel
and Hudley, 2018). A composite was created for the four-item
measure of instrumental support (e.g., “My teachers explain
things that I don’t understand”) and the four-item measure of
informational support (e.g., “My teacher helps me solve problems
well by giving me information”). We added the single-item
question, “My teacher talks to me about my future plans.” Each
item was analyzed separately in order to capture the various types
of support teachers may provide and communicate. the demographics included three female teachers (two White,
one Latina) and one male teacher (White). Students were given
consent and assent forms, and those who returned the completed
forms, received the survey the next class period. Two graduate
student researchers and three undergraduate research assistants,
who were trained and familiar with the study, administered the
open-ended questionnaire during class time. All students were
given instructions about the questionnaire and were reminded
that their answers would remain confidential. Similar to previous
studies in this field, students took approximately 30 min to
complete the questionnaire (Elmore and Oyserman, 2012). The
University’s Institutional Review Board approved this study. Frontiers in Education | www.frontiersin.org Types of Possible Selves Reported All participants identified at least one expected and one feared
possible selves. Participants produced a total of 165 expected
possible selves and 243 feared possible selves. At least one of
each of the expected and feared possible selves focused on the
achievement category, comprising of 52% of the total expected
possible selves and 35% of the total feared possible selves. For
example, an achievement expected possible selves was “I will go to
college.” An achievement feared possible selves was “I am afraid
I won’t get into a good university.” For the other categories of
expected and feared possible selves and examples, see Table 2. Concrete and Abstract Strategies Measured
variables
Overall
Male
Female
White
Latina/o
M (SD)
M (SD)
M (SD)
M (SD)
M (SD)
School
engagement (scale
0–4)
2.8 (0.9)
2.8 (0.6)
2.9 (0.6)
3.0 (0.6)
2.7 (0.5)
Teacher support:
instrumental (scale
0–4)
3.1 (0.6)
3.1 (0.5)
3.1 (0.6)
3.1 (0.5)
3.1 (0.5)
Teacher support:
informational (scale
0–4)
2.9 (0.8)
2.8 (0.7)
3.2 (0.6)
3.1 (0.7)
2.9 (0.7)
Teacher
communication:
future (scale 0–4)
2.5 (0.8)
2.6 (0.8)
2.7 (0.7)
2.6 (0.8)
2.6 (0.7)
Expected PS types
Achievement
2.3 (0.8)
2.4 (0.7)
2.1 (0.8)
2.1 (0.8)
2.4 (0.8)
Relationship
0.25 (0.5)
0.20 (0.4)
0.29 (0.5)
0.36 (0.5)
0.20 (0.2)
Individual
0.29 (0.6)
0.13 (0.4)
0.41 (0.7)
0.36 (0.7)
0.20 (0.5)
Health
0.18 (0.4)
0.03 (0.2)
0.02 (0.2)
0.0 (0)
0.04 (0.2)
Lifestyle
0.51 (0.6)
0.53 (0.7)
0.48 (0.6)
0.49 (0.6)
0.54 (0.6)
Risky behavior
0.02 (0.5)
0.0 (0)
0.05 (0.2)
0.05 (0.2)
0.0 (0)
Feared PS types
Achievement
0.89 (0.8)
0.85 (0.8)
0.82 (0.8)
0.88 (0.9)
0.84 (0.8)
Relationship
0.03 (0.6)
0.23 (0.5)
0.36 (0.6)
0.26 (0.5)
0.33 (0.6)
Individual
0.22 (0.6)
0.21 (0.6)
0.25 (0.5)
0.13 (0.4)
0.31 (0.7)
Health
0.26 (0.5)
0.23 (0.5)
0.25 (0.5)
0.31 (0.6)
0.18 (0.4)
Lifestyle
0.55 (0.6)
0.59 (0.8)
0.48 (0.5)
0.46 (0.6)
0.60 (0.7)
Risky behavior
1.3 (0.9)
1.2 (0.9)
1.3 (0.9)
1.3 (0.8)
1.2 (0.9)
Frequency
expected PS
strategies
Achievement
2.0 (1)
2.2 (0.9)
1.9 (0.9)
1.9 (0.8)
2.1 (1)
Relationship
0.29 (0.5)
0.20 (0.5)
0.34 (0.6)
0.33 (0.5)
0.22 (0.4)
Individual
0.21 (0.5)
0.18 (0.4)
0.25 (0.6)
0.23 (0.5)
0.19 (0.5)
Health
0.26 (0.5)
0.28 (0.5)
0.21 (0.5)
0.26 (0.4)
0.24 (0.5)
Lifestyle
0.17 (0.4)
0.20 (0.4)
0.16 (0.4)
0.28 (0.5)
0.08 (0.3)
Risky behavior
0.03 (0.2)
0.03 (0.2)
0.05 (0.2)
0.05 (0.2)
0.02 (0.1)
Frequency feared
PS strategies
Achievement
1.0 (0.9)
1.1 (0.8)
0.91 (0.9)
0.89 (0.9)
1.1 (0.9)
Relationship
0.20 (0.4)
0.13 (0.3)
0.27 (0.5)
0.28 (0.5)
0.13 (0.3)
Individual
0.30 (0.4)
0.20 (0.5)
0.36 (0.6)
0.28 (0.6)
0.28 (0.5)
Health
0.30 (0.5)
0.38 (0.6)
0.23 (0.5)
0.38 (0.6)
0.22 (0.4)
Lifestyle
0.16 (0.4)
0.15 (0.4)
0.14 (0.3)
0.05 (0.2)
0.22 (0.5)
Risky behavior
0.92 (0.8)
1.0 (0.8)
0.91 (1)
0.85 (0.8)
1.1 (0.9)
Frequency of
expected concrete
strategies
0.9 (0.8)
1.1 (0.8)
0.7 (0.9)
0.95 (0.9)
0.87 (0.8)
Frequency of
expected abstract
strategies
2.1 (0.1)
1.9 (1)
2.2 (1)
2.1 (1)
2.1 (1)
Frequency of
feared concrete
strategies
1.3 (0.1)
1.4 (1)
1.2 (1)
1.0 (0.8)
1.5 (1)
Frequency of
feared abstract
1.6 (0.1)
1.6 (1)
1.5 (1)
1.7 (1)
1.4 (1) TABLE 1 | Means (SD) associated with measured variables. Concrete and Abstract Strategies conducting content analysis, created a category scheme by
coding five transcripts together. We used an inductive procedure,
adapting the category scheme to the content of the data. About
5% of the coding was discrepant, meaning that we agreed on
95% of the thought units in the text overall. We resolved the
discrepancies through discussion. All transcripts including the
training interviews were included in the analysis. Measured
variables
School
engagement (scale
0–4)
Teacher support:
instrumental (scale
0–4)
Teacher support:
informational (scale
0–4)
Teacher
communication:
future (scale 0–4)
Expected PS types
Achievement
Relationship
Individual
Health
Lifestyle
Risky behavior
Feared PS types
Achievement
Relationship
Individual
Health
Lifestyle
Risky behavior
Frequency
expected PS
strategies
Achievement
Relationship
Individual
Health
Lifestyle
Risky behavior
Frequency feared
PS strategies
Achievement
Relationship
Individual
Health
Lifestyle
Risky behavior
Frequency of
expected concrete
strategies
Frequency of
expected abstract
strategies
Frequency of
feared concrete
strategies
Frequency of
feared abstract
strategies Finally,
in
order
to
gain
a
better
understanding
of
adolescents’
possible
selves,
descriptive
statistics
were
analyzed to identify the types of possible selves and the
strategy
types
identified. Correlations
and
t-tests
were
run to distinguish any associations and group differences
between
variables. The
means
and
standard
deviations
associated
with
academic
motivation,
perceived
teacher
support and communication, types of possible selves and
strategies, and concrete and abstract strategies are presented
in Table 1. Types of Strategies for Possible Selves
Reported 2.0 (1)
2.2 (0.9)
1.9 (0.9)
1.9 (0.8)
2.1 (1)
0.29 (0.5)
0.20 (0.5)
0.34 (0.6)
0.33 (0.5)
0.22 (0.4)
0.21 (0.5)
0.18 (0.4)
0.25 (0.6)
0.23 (0.5)
0.19 (0.5)
0.26 (0.5)
0.28 (0.5)
0.21 (0.5)
0.26 (0.4)
0.24 (0.5)
0.17 (0.4)
0.20 (0.4)
0.16 (0.4)
0.28 (0.5)
0.08 (0.3)
0.03 (0.2)
0.03 (0.2)
0.05 (0.2)
0.05 (0.2)
0.02 (0.1) p
Similar to the types of possible selves, the strategies identified
by participants were categorized. The majority of participants
(95%) provided at least one strategy for each type of possible
selves. Achievement strategies were identified for the majority
of the possible selves, consisting of 54% of strategies for
expected selves and 32% for feared selves. Many students
used achievement strategies to address their various types
of fears. For example, a student with a risk behavior fear
(getting pregnant in high school or getting involved in a
gang, ID #77) reported an achievement strategy (focus on
school, ID #77). In addition, fears of being homeless, living
with parents, and living in poverty were also addressed with
strategies to stay in school, further their education, graduate
and go to college. For the other categories and examples of
strategies identified for expected and feared possible selves,
see Table 3. Concrete and Abstract Strategies We used Clinkinbeard and Murray (2012) definition of concrete
strategies to categorize how students would address their given
expectation or fear. A concrete strategy was one that could
be replicated by another person. For example, “taking detailed
notes” is a concrete response that others could repeat. All other
responses were coded as abstract (e.g., “do better in school”). These codes were dichotomously coded. All
audiotaped
interviews
were
transcribed
and
a
content analysis was used to discover patterns in the text
related to the frequency of occurrence of particular themes
(Krippendorff, 2018). The authors who had previous experience July 2020 | Volume 5 | Article 125 4 Possible Selves and Perceived Teacher Support Mireles-Rios and Roshandel TABLE 1 | Means (SD) associated with measured variables. Concrete vs Abstract Strategies Concrete vs Abstract Strategies
We also examined whether the strategy produced was concrete
or abstract. The majority (60%) of strategies identified by
participants for expected selves were abstract, whereas for
feared selves, the strategies identified were evenly distributed
among concrete (49%) and concrete (51%). See Table 3 for
specific examples. July 2020 | Volume 5 | Article 125 Frontiers in Education | www.frontiersin.org 5 Possible Selves and Perceived Teacher Support Mireles-Rios and Roshandel TABLE 2 | Types of possible selves by categories. Category type
Expected selves
Feared selves
Percentage
Examples
Percentage
Examples
Achievement
52
“I expect to join the Marine Corp Reserves” (ID #63)
35
“I am afraid of dropping out of school” (ID# 73)
Relationships
12
“I expect to be maintaining a great relationship with my
family and friends” (ID #6)
9
“I am afraid of becoming too serious/detached from
those I love” (ID# 68)
Individual
11
“I expect to be happy and fulfilled” (ID #11)
5
“I am afraid of being cocky or arrogant” (ID# 32)
Health
1.5
“I expect to stay active and healthy, stay in shape and
eat properly” (ID #37)
7
“I am afraid of being overweight” (ID# 9)
Lifestyle
22
“I want to be traveling the world” (ID #85)
16
“I am getting sucked into social media” (ID# 10)
Risky Behavior
1.5
“I expect to be drug-free” (ID #49)
28
“I am afraid of getting pregnant in high school” (ID# 39)
TABLE 3 | Types and examples of strategies for possible selves by categories. Concrete vs Abstract Strategies Category type
Expected selves
Feared selves
Total
percentage
Concrete Examples
Abstract Examples
Total
Percentage
Concrete Examples
Abstract examples
Achievement
54
“I’m enrolling in as many
computer science electives
as possible” (ID# 14)
“I’m keeping good grades”
(ID# 67)
32
“I currently have a job so I
can build my resume” (ID#
91)
“I am never going to give
up” (ID# 81)
Relationships
13
“I’m going to college to and
joining a business club to
network with people” (ID#
19)
“I’m planning on keeping in
touch with people” (ID# 50)
9
“By spending a lot of time
with my family and keeping
a strong bond” (ID# 68)
“I will try to not hang out
with bad friends” (ID# 27)
Individual
9
N/A
“I’m working hard to stay
happy and positive.” (ID#
48)
11
“I do my best not to
underestimate people or
come to conclusions based
on appearances” (ID# 32)
“I will stay true and pure to
myself” (ID# 23)
Health
13
“I’m going to gym and
exercise every day.” (ID# 6)
“I’m going to be healthy
and fit.” (ID# 2)
12
“I work out every day” (ID#
9)
“I will stay aware” (ID# 64)
Lifestyle
9
“I’m going to open a bank
and save $10 a week.” (ID#
55)
“I’m living life each moment
at a time.” (ID# 43)
6
“I read a lot of books” (ID#
10)
“Do everything in my power
to not fail in life” (ID# 69)
Risky behavior
2
N/A
“I’m not going to do drugs.”
(ID# 78)
30
“I make sure to focus on
school and not boys” (ID#
39)
“Don’t do anything bad”
(ID# 88) TABLE 2 | Types of possible selves by categories. Correlations Among the Measured
Variables also varied significantly with the number of concrete feared
strategies produced, t(82) = 2.23, p = 0.02, d = 0.19. Results also
indicated a significant difference between those who reported at
least one concrete strategy and those who did not. Those who
reported a concrete strategy reported more school engagement
compared to individuals who did not report a concrete strategy
(M = 2.6, SD = 0.62), t(83) = −1.83, p = 0.05, d = 0.49. Finally,
results of whether teachers did discuss their future or did not
significantly varied between those who reported at least one
concrete strategy and those who did not (M = 2.28, SD = 0.94),
t(83) = −2.37, p = 0.05, d = 0.37. In other words, the more
teachers communicated about the future, the more students’
reported concrete strategies. No other significant differences were
found between groups and concrete and abstract strategies. Correlations were analyzed among the demographic variables,
concrete
and
abstract
strategies,
and
perceived
teacher
support and communication variables (see Table 4). T-tests
were also conducted to examine group differences among
significant variables. Demographic Variables Results indicated a significant difference in GPA between White
students (M = 3.62, SD = 0.89) and Latina/o students (M = 2.96,
SD = 0.8), t(83) = 3.61, p = 0.001, d = 0.79. No other significant
differences were found between demographic variables. Concrete and Abstract Strategies Perceived Teacher Support and Communication
Results indicated significant differences in those who perceived
their teachers as providing more informational support between
males (M = 2.83, SD = 0.71) and females (M = 3.19, SD = 0.63), Perceptions of Teachers’ Communication About the
Future Perceptions of Teachers’ Communication About the
Future One of the most common themes that emerged was how teachers
communicate about students’ future in terms of going to college. Findings from the interviews indicated that most of their teachers
did not discuss adolescents’ future. One participant stated “We
don’t really talk about other things. Like the future doesn’t come
up. Sometimes she’ll mention college, but then we never talk
about it.” Other participants also discussed the college going
process, and most participants interviewed felt that one of the
reasons their teachers did not discuss their future was that they
did not necessarily know much about the process of getting into
college. For example, one participant said, “I think they just focus
on their class, what they’re teaching. I don’t think they really
know the college steps, just know what they’re going to teach
and that’s their subject.” Similarly, another participant said, “I
think she [the teacher] only knows science. We never talk about
anything else.” I think it’s just that, like, like, he really does help me and I can
come in at lunch and, like, he always says that we’re welcomed in
the classroom. Like he’ll actually be there for us, I think it’s just that
reassurance that he will actually help us, and, like, he’ll actually be
there for us, is what helps. (Interview #7) The teacher will say, “oh, you missed this quiz. You can take it,
like, on this day or during lunch or after school,” like, she gave
me options. And then, like, if I miss an assignment or something,
she’ll, like, tell me what I missed or check my syllabus and kind of,
like, explain it. (Interview #5) Students also reported that it was during outside of class time
that they would get more support and communication about
ways to strategize and problem solve. For example, one student
mentioned, Results also indicated that while across the board, students
did not express detailed communication about the future, only
the White students (9 out of 15) reported that their teachers
encouraged them to go to college. I, um, really struggled with coming to class sometimes, and
when we sat down at lunch he asked me to really think about,
um, why I have been slacking. Perceptions of Teachers’ Communication About the
Future Once I admitted that I didn’t
understand the work, we came up, um, we talked about some ways
I could ask questions to help me do the, um, the work and stuff. (Interview #22) Perceived Teacher Support and Communication She always answers questions and provides examples for each
lesson that she gives us and we write notes but she writes them
down while we write them, too and what to study. And sometimes
she gives us, um, booklets that we can write down little notes
like equations and stufffor the test. Um, that helps a lot, and
he answers questions very well and always gives, like, real life
examples. (Interview#4) Perceived Teacher Support and Communication The frequency of feared concrete strategies were significantly
different between White (M = 1.0, SD = 0.79) and Latina/o
(M = 1.52, SD = 1.13) students, t(83) = −2.42, p = 0.02, d = 0.54. Males (M = 1.41, SD = 0.98) and females (M = 1.23, SD = 0.97) July 2020 | Volume 5 | Article 125 Frontiers in Education | www.frontiersin.org 6 Possible Selves and Perceived Teacher Support Mireles-Rios and Roshandel TABLE 4 | Correlations among measured variables (n = 85). 1
2
3
4
5
6
7
8
9
10
11
1. Grade level
2. Gender
0.05
3. Race
0.07
−0.12
4. GPA
−0.03
0.07
−0.37**
5. Abstract strategies for expected selves
−0.05
0.15
−0.04
0.01
6. Concrete strategies for expected selves
−0.08
−0.25*
−0.5
−0.01
−0.42**
7. Abstract strategies for feared selves
−0.05
−0.02
−0.16
−0.03
−0.04
0.8
8. Concrete strategies for feared selves
−0.02
−0.07
0.26*
−0.07
0.29**
0.06
−0.38**
9. Academic engagement
−0.37**
0.05
−0.11
0.28**
−0.06
0.20
−0.05
0.03
10. Teacher support: instrumental
−0.36**
−0.12
0.07
0.07
0.14
0.12
−0.21
0.25**
0.24*
11. Teacher support: information
−0.16
0.26*
0.17
0.06
0.02
0.11
−0.14
0.08
0.25*
0.37**
12. Teacher support: future
−0.13
−0.63
0.14
−0.19
0.08
0.24*
−0.07
0.26*
0.14
0.22*
0.29**
*p < 0.05, **p < 0.01, and ***p < 0.001. They explain things well, um, they’ll write things in the board, and
they’ll make sure you have your notes out and make sure you’re
taking down your notes, um, they’ll be free to, like, e-mail with
you if you have questions or be in at lunch to see if you need any
help. I think there are some teachers like that here on campus. (Interview #12) t(81) = −2.46, p = 0.02, d = 0.54. That is, female students were
more likely than their male counterparts to perceive that their
teachers provided them with informational support. These quantitative results for this study give us a glimpse
into adolescents’ possible selves and the types of strategies they
believe will help them achieve an expected selves and avoid a
feared selves. We explicitly get to see students use of abstract and
concrete strategies. Lastly, we see how specific types of perceived
support from teachers is associated with the various components
of students’ possible selves. Interview Results The following themes emerged from the interviews. In addition, other participants mentioned how important it
was for them to have additional time with teachers. To many
students, offering time beyond regular class time signals a form of
caring. Through this time in the classroom, four students found
this space to help with academics. Two students mentioned, Various Forms of Support Another common trend that emerged was the different types of
support students perceived from teachers. For example, July 2020 | Volume 5 | Article 125 Frontiers in Education | www.frontiersin.org 7 Possible Selves and Perceived Teacher Support Mireles-Rios and Roshandel strategies significant for possible selves irrespective of being
expected or feared. Finally, having high expectations also emerged as an
important way that students perceived their teachers as
supportive. One student said, Further supporting past research, we found that adolescents
have a variety of possible selves regarding their future selves. Similar to previous studies, the types of possible selves produced
reaffirm the variety of goals and identities adolescents are
focusing on during this time. Participants from our study
identified more feared possible selves than expected possible
selves. This speaks to the uncertainty of adolescence during a
transitional period of becoming an autonomous self. In addition,
findings indicated that the majority of expected selves were
related to achievement goals, which support how possible selves
are more proximal in nature (e.g., students are currently in
school) (Oyserman et al., 2011). Sometimes I used to think he was just out to get us like the other
teachers. But then I realized, he really wanted us to do well. He
would push us, um, to think, and um, do things we’ve never tried. (Interview#20) Similarly, another student talked about the importance of
providing the expectation of a safe space for students to learn. He stated, When some kid tell them that they want to really learn and they
don’t kick out the students, or you know, actually talk to the
students that aren’t listening, you’re not supporting your other
students that actually want to learn. (Interview #6) We also found that regardless of the expectation or fear,
the majority of types of strategies students reported were
achievement focused. Although it may seem obvious to list
achievement strategies given that data was collected in a school
setting, it is important to note that even students, who were not
as academically engaged as their peers, still identified education
as a protective factor for achieving some non-academic possible
selves. This is important in our understanding of how to best
support students in the classroom, as adolescents recognize that
school may play a significant role in guiding students toward
creating plans to meet their future goals or avoid their fears. Various Forms of Support School may serve as the blueprint to action plans adolescents
develop (Oyserman et al., 2004, 2006). Based on these findings, it is apparent that students felt when
they teachers gave them specific ways of doing something (take
notes or ask questions) they felt they were not only supported but
also that they teacher cared and knew what they were doing. Relevancy and Relatability The final theme that emerged was how students reported the
importance of having teachers that can be relatable. For example, She’ll, um, just make sure everybody is doing their notes and she
we’ll make class fun, like, if kids are talking she’ll just pretend
to join in the conversation and be, like, “oh, that’s cool, what
happened next?” Like, she makes it really fun and enjoyable. (Interview #14) Findings also indicated the significance of concrete strategies
versus abstract ones. Specifically, we found that adolescents who
produced at least one concrete strategy were more engaged
and perceived their teachers as more supportive. This supports
previous studies that more specific action plans serve as a
motivational resource that are closely associated with changing
behaviors in the present that may impact adolescents’ futures
(Gollwitzer et al., 2004; Oyserman et al., 2004; Duckworth et al.,
2011; Gollwitzer and Oettingen, 2011; Anders and Olmstead,
2018). Therefore, if adolescents have specific action plans (i.e.,
concrete strategies) to meet their expected possible selves or avoid
their feared possible selves, they are more likely to have the
next steps to achieve their goals (Clinkinbeard and Zohra, 2012;
Anders and Olmstead, 2018). She teaches real life, I would say. Everything has an application,
like especially in the virtual enterprise or other classes I’ve taken
from her, like business applications and computer business. It’s
just they all have real-life applications, like from writing checks, to
making a balance sheet, to typing up a profit and loss statement. I feel like I can really use this information when I get out of here. (Interview #11) Such findings provide a glimpse into how high school
students perceive support from their teachers and the way they
communicate can impact their future goals. Furthermore, one contributing finding of this study is
that students who perceived their teachers as supportive by
communicating about their futures, provided more concrete
strategies to deal with their possible selves, indicating that
teachers may play a role in adolescents’ possible selves-strategy
elaboration. That is, teachers have the potential to guide
adolescents with the support of developing these concrete
strategies that will increase the likelihood of adapting current
behaviors to achieve the desired future goals. Our study found
that when teachers talked to students about their future, the
more concrete strategies they had to address their range of
possible selves. Frontiers in Education | www.frontiersin.org DISCUSSION that when teachers themselves provided more concrete forms
of support (e.g., accessible after class, teaching specific methods,
providing relevant examples) versus abstract forms of support
(e.g., available if needed, general comments), adolescents
reported they felt more supported and trusted the expertise
of their teachers. These results exemplify the types of support
that are necessary for teachers to provide in order to support
adolescents in the classroom and with their future goals. For
example, having a teacher available during lunch to work with a
student on specific methods to take notes to help them study for
a test is more beneficial than a teacher saying they are available if
a student needs it. These types of support model how teachers
can show students the importance of creating concrete versus
abstract strategies. The
association
between
perceptions
of
teacher
communication about the future and concrete strategies opens
up future research to recognize and explore this relationship. That is, when students know how to come up with achievement
strategies that are concrete, they are likely to address not only
their achievement expectations and fears but also their risk
behavior, health, individual, relationship and lifestyle possible
selves. This may be particularly useful given that all students
reported at least one risk behavior fear which was associated with
more abstract solutions. If teachers can help guide students into
developing concrete plans of action, they can indirectly address
these types of fears that may not emerge in the classroom setting. Given that concrete strategies associated with future-oriented
expectations and fears may be the most important pieces of
future planning and are likely linked to actual behavior change
(Gollwitzer et al., 2004; Oyserman et al., 2006; Clinkinbeard
and Murray, 2012), our findings that perceived teacher support
was associated with students concrete and abstract strategies
further upholds previous research that students’ perceptions
of teacher communication styles influences their motivation
(Lin et al., 2016). Previous work on the achievement and opportunity gap have
identified differences across gender and ethnicity in regards to
student achievement (Boykin and Noguera, 2011; Carter and
Welner, 2013). Our findings indicated that the only significant
difference with GPA was between Whites and Latinas/os. DISCUSSION While
it was expected that school engagement would be linked to GPA,
our findings indicated no differences between GPA and the other
variables and regardless of academic performance, adolescents
recognized that their current behavior in school plays a vital role
in their future goals. Therefore, teachers could use the context of
possible selves as a motivator to engage all students by making
content relatable and relevant to adolescents’ lives. Adolescents
can then be motivated to engage with their teachers and in turn
develop concrete strategies through communication and support. Even though previous research shows the importance and
benefits of having concrete strategies, the majority of students
identified abstract solutions for their expected selves. Given
that there were significant group differences in GPA between
White and Latina/o students, a possible solution for addressing
this apparent achievement gap might be to focus on fostering
teacher communication about the future and helping students
to develop more concrete plans for their future. Interview data
suggested that adolescents could benefit from their teachers
sharing concrete examples from their own experiences as a
way of providing more specific forms of support. Interestingly,
when it came to feared possible selves, adolescents reported
approximately 50% abstract and 50% concrete strategies to deal
with their fears. Perhaps adolescents’ motivation to avoid their
fears facilitates the development of more concrete strategies. These results also suggest that students in this study
recognized that their current behavior in school plays a vital role
in their future goals. Our research also indicates that youth are
reporting that doing well in school as a form of resisting and
coping with their fears, suggesting that they value education as
a tool for creating a stable future. This is particularly significant
because adolescents are able to move from concrete to abstract
thinking and planning, making it possible to think about their
futures (Piaget, 1964). In addition, as they are getting older they
are developing critical thinking skills. It has been argued that
critical thinking is a higher order mental function influenced
by social factors and performed within social contexts which
sometimes makes transferring what happens in a classroom
setting to a real world setting challenging (Lee, 2018). By teaching
students about concrete strategies, teachers could be providing
relevant context for developing critical thinking skills that may
help bridge this gap. DISCUSSION This study contributes to the possible selves research by
exploring adolescents’ possible selves and the role of perceived
teacher support as it is related to helping adolescents build
strategies for achieving their expected selves and/or avoiding
their feared selves. Based on our hypotheses, we found
that
when
adolescents
perceived
more
teacher
support,
they were more likely to have concrete strategies to deal
with their possible selves. Although we hypothesized that
greater perceived teacher communication about the future
will predict greater use of concrete over abstract strategies
for both expected and feared possible selves, we found
significant results for concrete strategies in relation to possible
selves more broadly. That is, we found the use of concrete Interview data provided us with deeper insight on how
these perceived teacher support variables were related to how
adolescents think about their future as well as the strategies
they develop. Specifically, results from the interviews showed July 2020 | Volume 5 | Article 125 Frontiers in Education | www.frontiersin.org 8 Possible Selves and Perceived Teacher Support Mireles-Rios and Roshandel times less likely to be encouraged by their teachers than their
White counterparts (Witenko et al., 2016). These findings may
be attribute to the mismatch demographics between students
and teachers, given that the majority of teachers were White
(Sleeter, 2001; Picower, 2009). Additionally, compared to females,
males had more concrete strategies. For example, females
perceived more informational support from teachers, compared
to males. Previous studies show that teachers spend more
time providing girls with more academic and informational
support or that girls value this type of support more than males
(Roshandel and Hudley, 2018). Teachers may also have different
approaches to different types of students, as previous studies
have indicated that teachers may have their own biases for
students’ future paths (Boykin and Noguera, 2011). Together
these findings indicate that based on individual differences,
students may have different needs for the types of support
they desire as well as different types of support they need in
terms of reaching a desired outcome (Roshandel and Hudley,
2018). Horowitz et al. (2020) suggest that in some school
contexts, teachers are able to provide the guidance needed for
students while other teachers are not. This calls for further
exploration around differences in race/ethnicity and gender
in the development of possible selves and perceptions of
teacher support. Frontiers in Education | www.frontiersin.org Implications for Teacher Communication
With Adolescents Many studies focusing on possible selves recommend the
need to understand the relationship between support systems
and the development of concrete strategies (Oyserman et al.,
2004; Oyserman et al., 2006; Clinkinbeard and Murray, 2012;
Clinkinbeard and Zohra, 2012; Zhu and Tse, 2015). This study
examines the associations with teachers, offering a new lens
in viewing teacher communication and the classroom context
as a space for “intervening” in the development of strategies
for adolescents to develop their future selves. For example,
by making teachers aware that achievement strategies are an
important coping mechanism for adolescents when dealing with
their fears, teachers can be more mindful about teaching students
how to set goals and create “action plans” even within content
area instruction. Hearing from the students provided insight into how they
viewed their teachers’ knowledge about the future and ways
that teachers can help support students in their development
of strategies applicable to their possible selves. Despite needing
a college degree to be a teacher, many students did not
think that teachers know the route to college. One suggestion
would be for teachers to share their journey to the profession
with their students so they can help understand the steps
needed for different types of careers. This could help students
develop their own future road maps. Additionally, students
mentioned the significance of teachers making time at lunch
and afterschool to help them with their work. This space
lends itself for opportunities for teachers to connect with
students. Strategies associated with future-oriented expectations
and fears may be the most important pieces of future
oriented visualization and planning that are linked to actual
behavior change (Ajzen, 1985; Oyserman and Markus, 1990;
Gollwitzer and Oettingen, 2011). This may be because of
a personal connection and relevancy to adolescents’ lives. Therefore, the findings from this study begin to shed light
on the role teacher’s play in supporting and shaping how all
adolescents can construct an action plan that will lead to the
desired outcomes. Given that preservice and in service teacher research focuses
on the development of 21st century skills (Urbani et al., 2017;
Roshandel and Hudley, 2018), our findings might suggest to
teachers that the use of possible selves and development of
strategies could be a way of teaching adolescents higher order
cognitive skills, such as critical thinking and creativity. DISCUSSION Interview data also revealed that Latina/o students were less
likely to discuss being encouraged by their teachers to attend
college than their White counterparts. Compared to White
students, Latina/o students had more concrete strategies to
address their possible selves. Similarly, a recent study examining
who encourages students to enroll in advanced classes, even
when controlling for a GPA, Latina/o students were over 5 By communicating with adolescents to think about their
future,
teachers
have
the
opportunity
to
help
with
the July 2020 | Volume 5 | Article 125 9 Possible Selves and Perceived Teacher Support Mireles-Rios and Roshandel development of concrete strategies to create tangible solutions to
deal with their fears and help develop their future expectations. This is particularly telling, as much of the research on teacher
support has focused on teachers working with younger students,
often because high school teachers specialize in content and not
necessarily the whole child (Noddings, 2012). Gehlbach et al. (2012) also discuss that early adolescence is a critical period
for the teacher-student relationship; however, in comparison to
elementary school, the interactions are less personal and frequent
(Eccles et al., 1993). Pianta and Allen (2008) argue that as
adolescents look to non-parental adults for social connections,
these relationships are perhaps the most important factor for
promoting positive youth development. Expanding the research
around possible selves to the student-teacher relationship in
secondary school may help students not only with their academic
fears but in other arenas in their lives. development but also the possible enhancing of student self-
efficacy in their own ability to generate an action plan. CONCLUSION Within possible selves literature, there is limited research
exploring the changes of possible selves within one life stage. As high school is a time of dramatic developmental changes, in
particular with the ability to plan and visualize their futures (Boyd
and Bee, 2012), this may serve as an optimal time for adolescents’
possible selves-strategy elaboration. This current study expands
research on the significant role that teachers can play to aid
adolescents in the development of concrete solutions. Through
instructional practices that can span across all content areas to the
development of personal “action plans” we can provide teachers
with practical ways to help students. Findings provide insight into
the way students perceive how their teachers communicate and
provide academic and emotional support which is associated with
their development of concrete strategies. Better understanding
the relationship of teacher communication about the future to the
provision of feedback for the future can have a significant effect
on the development of their future identities and cognitive skills. Limitations and Suggestions for Future
Research It is important to note the limitations of this study. Given
the small sample size, we were only able to explore how these
variables are associated. The brevity of the survey did not
allow us to get an in depth understanding the specific ways
perceived teacher support can aid in the development of concrete
strategy building for adolescents. Hearing from teachers about
their experiences around awareness and action taken around
addressing adolescents’ fears and strategy building would advance
ways to best support students and teachers. More research is
necessary to comprehend the relationship between sources of
support and the contexts in which strategies and behavior change
develops in adolescence. This also leads to the need for future
studies to explore how teacher communication may lead to
not only the development of meta-cognitive skills of strategy Implications for Teacher Communication
With Adolescents This
would allow students to make a personal connection to learning
a key skill set that may also lead to a potential personal behavior
change that will allow an adolescent to avoid their feared possible
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of Learning Research Report no. ETHICS STATEMENT RM-R took the lead on the manuscript preparation. SR collected
the data. RM-R and SR conducted the literature review,
the analysis, and the results and discussion. RM-R and SR
contributed to the article and approved the submitted version. The studies involving human participants were reviewed and
approved by University of California, Santa Barbara Office of
Research. Written informed consent to participate in this study
was provided by the participants’ legal guardian/next of kin. RM-R took the lead on the manuscript preparation. SR collected
the data. RM-R and SR conducted the literature review,
the analysis, and the results and discussion. RM-R and SR
contributed to the article and approved the submitted version. DATA AVAILABILITY STATEMENT The datasets generated for this study will not be made publicly
available. As per our human subjects’ agreement, all data collected
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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
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A new insect trackway from the Upper Jurassic—Lower Cretaceous eolian sandstones of São Paulo State, Brazil: implications for reconstructing desert paleoecology
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PeerJ
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How to cite this article Peixoto BCPM, Mángano MG, Minter NJ, dos Reis Fernandes LB, Fernandes MA. 2020. A new insect trackway
from the Upper Jurassic—Lower Cretaceous eolian sandstones of São Paulo State, Brazil: implications for reconstructing desert paleoecol-
ogy. PeerJ 8:e8880 http://doi.org/10.7717/peerj.8880 A new insect trackway from the Upper
Jurassic—Lower Cretaceous eolian
sandstones of São Paulo State, Brazil:
implications for reconstructing desert
paleoecology Bernardo de C.P. e M. Peixoto1,2, M. Gabriela Mángano3, Nicholas J. Minter4,
Luciana Bueno dos Reis Fernandes1 and Marcelo Adorna Fernandes1,2 1 Laboratório de Paleoicnologia e Paleoecologia, Departamento de Ecologia e Biologia Evolutiva, Universidade
Federal de São Carlos (UFSCar), São Carlos, São Paulo, Brazil
2 Programa de Pós Graduacão em Ecologia e Recursos Naturais, Centro de Ciências Biológicas e da Saúde, 1 Laboratório de Paleoicnologia e Paleoecologia, Departamento de Ecologia e Biologia Evolutiva, Universida
Federal de São Carlos (UFSCar), São Carlos, São Paulo, Brazil 3 Department of Geological Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada 3 Department of Geological Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
4 School of the Environment, Geography, and Geosciences, University of Portsmouth, Portsmouth, Hampshire,
United Kingdom ABSTRACT The new ichnospecies Paleohelcura araraquarensis isp. nov. is described from the Upper
Jurassic-Lower Cretaceous Botucatu Formation of Brazil. This formation records a
gigantic eolian sand sea (erg), formed under an arid climate in the south-central part
of Gondwana. This trackway is composed of two track rows, whose internal width
is less than one-quarter of the external width, with alternating to staggered series,
consisting of three elliptical tracks that can vary from slightly elongated to tapered
or circular. The trackways were found in yellowish/reddish sandstone in a quarry in the
Araraquara municipality, São Paulo State. Comparisons with neoichnological studies
and morphological inferences indicate that the producer of Paleohelcura araraquarensis
isp. nov. was most likely a pterygote insect, and so could have fulfilled one of the
ecological roles that different species of this group are capable of performing in dune
deserts. The producer could have had a herbivorous or carnivorous diet or been
part of the fauna of omnivores, being able to adopt herbivorous, carnivorous, and
saprophagous diets when necessary. In modern dune deserts, some species of pterygote
insects are detritivores (like Tenebrionidae), relying on organic matter that accumulated
among the sand grains of the dunes during dry periods with no plant growth. The
presence of additional burrows suggests that the Botucatu paleodesert would have
had a detritivorous fauna like this. Based on the interpretation of the ichnofossil
producers, it was possible to reconstruct the food web of this paleodesert. All the
omnivorous and herbivorous invertebrates and the herbivorous ornithopod dinosaurs
made up the primary consumers. These animals were, in turn, the food source for bigger
carnivorous or omnivorous animals unable to feed on detritus, like arachnids, possible
predatory insects, mammaliaforms, and theropod dinosaurs. The highest trophic level
was occupied by larger theropod dinosaurs and mammaliaforms, which, because of
their size, could prey upon a wide range of animals. The producer of Paleohelcura
araraquarensis isp. nov. could have been a primary consumer if it were an omnivorous Submitted 6 November 2019
Accepted 10 March 2020
Published 22 May 2020
Corresponding author
Bernardo de C.P. e M. Peixoto,
b.peixoto@protonmail.com
Academic editor
Claudia Marsicano
Additional Information and
Declarations can be found on
page 26
DOI 10.7717/peerj.8880
Copyright
2020 Peixoto et al. Distributed under
Creative Commons CC-BY 4.0 Submitted 6 November 2019
Accepted 10 March 2020
Published 22 May 2020
Corresponding author
Bernardo de C.P. e M. 1 Laboratório de Paleoicnologia e Paleoecologia, Departamento de Ecologia e Biologia Evolutiva, Universidade
Federal de São Carlos (UFSCar), São Carlos, São Paulo, Brazil
2 Programa de Pós Gradua¸cão em Ecologia e Recursos Naturais, Centro de Ciências Biológicas e da Saúde,
Universidade Federal de São Carlos (UFSCar), São Carlos, São Paulo, Brazil
3 Department of Geological Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
4 School of the Environment, Geography, and Geosciences, University of Portsmouth, Portsmouth, Hampshire,
United Kingdom 2 Programa de Pós Gradua¸cão em Ecologia e Recursos Naturais, Centro de Ciências Biológicas e da Saúde,
Universidade Federal de São Carlos (UFSCar), São Carlos, São Paulo, Brazil ABSTRACT Peixoto,
b.peixoto@protonmail.com
Academic editor
Claudia Marsicano
Additional Information and
Declarations can be found on
page 26
DOI 10.7717/peerj.8880
Copyright
2020 Peixoto et al. Copyright
2020 Peixoto et al. Distributed under
Creative Commons CC-BY 4.0 OPEN ACCESS detritivore or a herbivore, or a secondary consumer if it were produced by a predatory
insect or an omnivore relying on animal biomass. The description of this new trackway
expands the knowledge on the faunal composition of the Botucatu paleodesert and
provides insights into the ecological relationships in ancient deserts. The presence
of these arthropod trackways in Mesozoic eolian deposits helps to trace a continuity
between Paleozoic and post-Paleozoic desert ichnofaunas, further reinforcing a single
Octopodichnus—Entradichnus Ichnofacies for eolian deposits. detritivore or a herbivore, or a secondary consumer if it were produced by a predatory
insect or an omnivore relying on animal biomass. The description of this new trackway
expands the knowledge on the faunal composition of the Botucatu paleodesert and
provides insights into the ecological relationships in ancient deserts. The presence
of these arthropod trackways in Mesozoic eolian deposits helps to trace a continuity
between Paleozoic and post-Paleozoic desert ichnofaunas, further reinforcing a single
Octopodichnus—Entradichnus Ichnofacies for eolian deposits. Subjects Biodiversity, Ecology, Ecosystem Science, Entomology, Paleontology
Keywords Deserts, Erg, Palaeoecology, Trophic web, Gondwana, Ichnofacies, Botucatu
Formation, Paraná Basin Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 INTRODUCTION The Botucatu Formation, a stratigraphic unit of the Paraná Basin, is the testament of a
gigantic sand desert (erg) that existed from the Late Jurassic to the Early Cretaceous in the
south-central part of the supercontinent Gondwana, totaling an area of 1.5 × 106 km2,
encompassing parts of Brazil, Argentina, Uruguay, Paraguay, Namibia and South Africa
(Scherer & Goldberg, 2007). Ichnofossils are the only evidence of animal life in this ancient
desert because no animal body fossils have been found. Therefore, trace fossils play a central
role in understanding animal diversity and ecological relationships in this ancient erg. Eolian deposits have been traditionally considered of minor interest from an ichnologic
perspective. However, this situation has changed at an accelerated rate in recent years with
the publication of several papers on the topic (e.g., Ekdale, Bromley & Loope, 2007; Ekdale
& Bromley, 2012; Good & Ekdale, 2014; Krapovickas et al., 2016; Carmona, Ponce & Wetzel,
2018; Xing et al., 2018; Buatois & Echevarría, 2019; Marchetti et al., 2019a; Marchetti et al.,
2019b). A recent review emphasized the complex pattern of trace-fossil distribution in
eolian and related facies, the debate surrounding definition of an archetypal ichnofacies,
and delineation of macroevolutionary trends in desert environments (Krapovickas et al.,
2016). Documentation of trace fossils in desert successions is of paramount importance to
provide support to these models, and to help clarify the diagnostic characteristics of the
so-called Octopodichnus-Entradichnus Ichnofacies. Several trackways of tetrapods and arthropods have been recovered from eolian dune
deposits of different ages (McKeever, 1991, table 1). There has been contention over the
preservation potential of such trackways in ‘dry’ dune deposits and whether they had to have
been produced subaqueously (Brand, 1979; Brand & Tang, 1991; McKeever, 1991; Brand,
1992; Lockley, 1992; Loope, 1992). Moisture (Mckee, 1947; Sadler, 1993) and the presence of
clay minerals (Loope, 1986; McKeever, 1991) between sand grains have both been proposed
to play a role in trackway stabilization and preservation potential. Experiments have
demonstrated that the combination of the two can lead to enhanced survivorship of
arthropod trackways over those made in dry sand or sand with just surface moisture or the
presence of clay minerals alone (Davis, Minter & Braddy, 2007). Nevertheless, it has been Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 2/37 recently argued that special conditions are not necessarily needed to preserve such trace
fossils (see Davies & Shillito, 2018). Previous work Most of the previous studies in the Botucatu Formation focused on tetrapod trackways,
with only two publications dealing in detail with invertebrate trace fossils (Fernandes, Netto
& Carvalho de, 1988; Fernandes, Carvalho & Netto, 1990). Before these studies, invertebrate
trackways were only mentioned within the context of vertebrate ichnofaunas as the source
of food for the presumed mammaliaform producer of Brasilichnium elusivum (Leonardi,
1981, p. 803). Subsequently, Leonardi (1984, p. 54) illustrated invertebrate trackways
identical to those documented in this study, but whose repository is unknown. Arthropod
trackways from São Bento Quarry (Araraquara city—São Paulo State) were later illustrated
as well by Leonardi & Sarjeant (1986, p. 83), but no further information regarding a
repository was provided. Leonardi, Carvalho de & Fernandes (2007) reported trace fossils
of insects and arachnids in Araraquara (São Bento Quarry), but no illustrations were
provided. Fernandes (2005) identified arachnid tracks in slabs from São Bento Quarry, and
interpreted them as made by scorpions and spiders. Peixoto et al. (2016) documented new
findings of Taenidium serpentinum and Skolithos linearis, probably produced by insects. The occurrences of invertebrate ichnofossils are summarized in Table 1. The only plant fossils from the Botucatu Formation are conifer trunks, found in the
region of Araguari (Minas Gerais State), north of the Tringulo Mineiro, within the limits
of the sandstone occurrence area of this unit (Pires et al., 2011; Malaquias, Riff & Riff,
2017). Those trunks exhibit xylophagous marks assigned to termites (Isoptera) and beetles
(Coleoptera) (Riff, Kloster & Riff, 2017). With respect to the vertebrate trace-fossil record of the Botucatu Formation, there
are two ichnospecies of Brasilichnium produced by small mammaliaform organisms: one
demonstrating cursorial locomotion described as B. elusivum (Leonardi, 1981; Fernandes
& Carvalho, 2008), and the other one in hopping locomotion (D’Orazi Porchetti, Bertini &
Langer, 2017a), described as B. saltatorium (Buck et al., 2017b). There is also a record of a
burrow compatible with the Brasilichnium elusivum producer (Manes, Da Silva & Scheffler,
2017). There is some controversy in describing new ichnotaxa based on differences in
locomotion patterns instead of objective morphological attributes of the footprints alone
(Lockley, 2007; Minter, Braddy & Davis, 2007). Nevertheless, the presence of a hopping
behavior (e.g., B. saltatorium) is useful in indicating a biomechanical capability that
constrains eligible clades of possible producers, together with when this biomechanical
capability appeared. INTRODUCTION The aims of this study are to: (i) describe the new ichnospecies, Paleohelcura
araraquarensis isp. nov., which consists of trackways produced by pterygote insects walking
on sand dunes; (ii) discuss the implications of this record with respect to ecological
relationships within the Botucatu paleodesert; and (iii) assess its importance for our
understanding of eolian dune ichnofacies. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Notes.
SP, São Paulo State (Brazil); MG, Minas Gerais State (Brazil).
Ichnofossils descriptions were translated literally from the original sources. Modified from Leonardi, Carvalho de & Fernandes (2007). Ichnofossils descriptions were translated literally from the original sources. Modified from Leonardi, Carvalho de & Fernandes (2007) Previous work Trackways of mammaliaforms larger than the producer of Brasilichnium elusivum have
been described independently as Brasilichnium anaitti (D’Orazi Porchetti, Bertini & Langer, Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 3/37 3/37 Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Table 1
Occurrence of invertebrate ichnofossils from the Botucatu Formation and the first references describing them. Locality
Ichnofossil Description
Reference
Quarry 3–4 km from São Carlos (SP) (probably
Migliato or Araújo quarry)
‘‘Worm Tunnels’’
Pacheco & De Amaral (1913)
Sierra of Botucatu (SP)
‘‘Worm tubes.’’
Almeida (1954)
Pacaembú neighborhood, São Carlos (SP)
‘‘Worm trails.’’
Bjornberg & Tolentino (1959)
São Tomás Ranch quarry, Ibaté Municipality (SP)
‘‘Fossil tracks of conchostracans(?)’’
Paraguassu (1970)
Quarry near Araraquara (SP)
‘‘Vermiform trails and tracks of arthropods.’’
Leonardi (1980)
‘‘Arthropods trackways’’
(Leonardi, 1984)
‘‘Arthropod trails’’
(Leonardi & Sarjeant, 1986)
‘‘Invertebrate trackways and burrows’’
(Leonardi & Godoy, 1980)
(...)Ten rare forms of invertebrate
trails, mainly attributable to arthropods
(arachnids and insects, adults or larvae)(...)
(Leonardi, Carvalho de & Fernandes, 2007)
São Bento Quarry, Araraquara (SP)
Insects, scorpions and spiders
(Fernandes, 2005). Itagua¸cu Farm Quarry, São Carlos (SP)
‘‘Trails of vermiform invertebrates.’’
Leonardi & Godoy (1980)
Campo Minado Cave, Sierra of Itaqueri, Ipeúna
(SP)
Taenidium serpentinum and Skolithos linearis
Peixoto et al. (2016)
Sobradinho Farm, Araguari (MG)
Burrows of xylophagous termites and Coleoptera insects in
conifer wood. Riff, Kloster & Riff (2017)
Notes. SP, São Paulo State (Brazil); MG, Minas Gerais State (Brazil). Ichnofossils descriptions were translated literally from the original sources. Modified from Leonardi, Carvalho de & Fernandes (2007). 4/37 4/37 2017b) and as Aracoaraichnium leonardii (Buck et al., 2017a). These two ichnotaxa bear
several morphological similarities and were described from slabs reposited in different
scientific collections. In addition, theropod and ornithopod dinosaur trackways have
been recorded (Leonardi, 1979; Leonardi, 1980; Leonardi & Godoy, 1980; Leonardi, 1981;
Leonardi, 1984; Leonardi & Sarjeant, 1986; Leonardi, 1987; Leonardi et al., 2002; Fernandes,
2005; Leonardi, Carvalho de & Fernandes, 2007; Francischini et al., 2015). Also noteworthy
is the rare occurrence of an urolite, a biogenic mark interpreted as the result of the liquid
extrusion of urine from dinosaurs onto unconsolidated sediment (Fernandes, Fernandes &
Souto, 2004). Geological setting The Botucatu Formation is exposed in the Brazilian states of Mato Grosso, Mato Grosso do
Sul, Goiás, Minas Gerais, São Paulo, Paraná, Santa Catarina, and Rio Grande do Sul with the
same sedimentary system extending into Argentina, Uruguay, Paraguay, Namibia and South
Africa, covering an area over 1.5 × 106 km2 (Scherer & Goldberg, 2007). In São Paulo State,
the Botucatu Formation outcrops as a northeast-southwest strip (Fig. 1), with monotonous
deposits mostly consisting of yellowish to reddish, very fine- to coarse-grained sandstone,
mainly quartz arenite and subordinately subarkose. The quartz arenite is texturally and
mineralogically supermature, whereas the subarkose is texturally submature to mature and
mineralogically mature (Wu & Caetano-chang, 1992). The consensus is that the Botucatu
Formation represents a giant dry eolian depositional system (erg) based on the presence
of large to medium-sized cross-stratified sandstones (Scherer & Goldberg, 2007) (Fig. 2),
and on the basis of the mineralogical and textural maturity of the dominant deposits (Wu
& Caetano-chang, 1992). The landscape was dominated by linear, crescentic and some
star dunes, representing a hyperarid system, according the classification framework of
Mountney (2004), with winds predominantly coming from the north in the northern part
of the Paraná Basin (Scherer & Goldberg, 2007) where the study area is located (Araraquara
City). Fluvial/eolian sandstone of the Pirambóia Formation occurs below the Botucatu
Formation in the northern portion of the basin (State of São Paulo) (Milani et al., 2007, p. 287; Soares, Soares & Holz, 2008a, their fig2). The contact between these two formations
is still controversial (Giannini et al., 2004, p. 282; Soares, Soares & Holz, 2008b, their fig. 2; p.126) (Fig. 3). The Botucatu Formation is overlain by the magmatic extrusive rocks
of the Serra Geral Group (former Serra Geral Formation) (Milani et al., 2007; Fernandes
et al., 2018) (Fig. 3). Lenses of eolian sandstone (paleodunes) in the Serra Geral Group
indicate that the eolian depositional system was active during volcanism. The Botucatu
Formation and the Serra Geral Group have a concordant contact because the flow of lava
over the unconsolidated sand of the paleodunes created marks on the paleodune surfaces
(e.g., striations, crescentic ridges), formed breccias (peperites), and also preserved the relief
of the ancient dunes (Milani et al., 1998; Scherer, 2000; Scherer, 2002; Waichel et al., 2007;
Holz, Soares & Soares, 2008; Waichel, Scherer & Frank, 2008). Geological setting U–Pb baddeleyite/zircon dating for the lowest sub-unit of the Serra Geral Group that
makes concordant contact with Botucatu Formation in São Paulo State (Chapecó-type Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 5/37 Figure 1
The localization of Araraquara City, where the fossils were collected, and outcrop area of the
Botucatu and Pirambóia formations in the State of São Paulo, Brazil. Modified from Fernandes, Fernan-
des & Souto (2004). Full-size
DOI: 10.7717/peerj.8880/fig-1 Figure 1
The localization of Araraquara City, where the fossils were collected, and outcrop area of the
Botucatu and Pirambóia formations in the State of São Paulo, Brazil. Modified from Fernandes, Fernan-
des & Souto (2004). Full-size
DOI: 10.7717/peerj.8880/fig-1 Figure 1
The localization of Araraquara City, where the fossils were collected, and outcrop area of the
Botucatu and Pirambóia formations in the State of São Paulo, Brazil. Modified from Fernandes, Fernan-
des & Souto (2004). Full-size
DOI: 10.7717/peerj.8880/fig-1 dacites) yields an age of approximately 134 Ma (Janasi, Freitas & Heaman, 2011). This
radiometric date, together with the oldest paleomagnetic date of the Botucatu Formation
from southern Brazil (Tamrat & Ernesto, 2006) indicates a Late Jurassic to Early Cretaceous
age for this unit. dacites) yields an age of approximately 134 Ma (Janasi, Freitas & Heaman, 2011). This
radiometric date, together with the oldest paleomagnetic date of the Botucatu Formation
from southern Brazil (Tamrat & Ernesto, 2006) indicates a Late Jurassic to Early Cretaceous
age for this unit. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 MATERIALS & METHODS Paleontological material analyzed Paleontological material analyzed The ichnofossils described here were collected between 1997 and 2005, at São Bento quarry
(21◦49′07.6′′S48◦04′28.8′′W), in the municipality of Araraquara (São Paulo State). All the
ichnofossils here described were found during the commercial exploitation of the successive
layers of sandstone of the slipface of a single paleodune (that was more than 100 m long
and 20 m high) of the Botucatu Formation (Fig. 2. The full-size figures are in Figs. S1 and
S2). The paleodune slipface dips at 29◦in the S-SW direction. The São Bento quarry is currently inactive and is part of the Ouro Ichnofossiliferous Site,
in Araraquara (Leonardi et al., 2002), a region with several abandoned ichnofossiliferous
quarries. The sandstone slabs containing the ichnofossils here analyzed were collected from
sites A, C and D (sensu Fernandes, 2005) of the São Bento quarry and are: LPP-IC-0028,
LPP-IC-0029, LPP-IC-0030, LPP-IC-0031, LPP-IC-0032, LPP-IC-0033, LPP-IC-0034,
LPP-IC-0035. Sandstone extraction at the São Bento quarry was done without scientific
monitoring, and most of the fossils were in slabs ready to be cut, or already cut for
commercialization. Therefore, no data is available regarding orientation of specimens with
respect to the slopes. All of these slabs are deposited in the Paleoichnology Collection of
the Laboratório de Paleoicnologia e Paleoecologia (LPP) of the Federal University of São
Carlos (UFSCar), São Carlos campus. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 6/37 Figure 2
Representative drawing of the location of the work fronts of São Bento quarry. Photographs
1, 2, 3 and 4 are the work fronts rock outcrop in March 2004, during the commercial exploitation of the
Botucatu sandstone and collection of all ichnofossils here described. (A), (B), (C), (D), (E), (F), (G), (H),
(I), (J), (K), (L) are the relative location of the sites of occurrence of ichnofossils, as described by Fernan-
des (2005). The invertebrate ichnofossils occur in sites A, C, and D, in the work front 1. At present, the
quarry is inactive, and the outcrop looks different because of the further exploitation and weathering. Drawing is not in scale (modified from Fernandes, 2005). The full-size image is in the Supplemental Infor-
mation. Full-size
DOI: 10.7717/peerj.8880/fig- Figure 2
Representative drawing of the location of the work fronts of São Bento quarry. Photographs
1, 2, 3 and 4 are the work fronts rock outcrop in March 2004, during the commercial exploitation of the
Botucatu sandstone and collection of all ichnofossils here described. Paleontological material analyzed (A), (B), (C), (D), (E), (F), (G), (H),
(I), (J), (K), (L) are the relative location of the sites of occurrence of ichnofossils, as described by Fernan-
des (2005). The invertebrate ichnofossils occur in sites A, C, and D, in the work front 1. At present, the
quarry is inactive, and the outcrop looks different because of the further exploitation and weathering. Drawing is not in scale (modified from Fernandes, 2005). The full-size image is in the Supplemental Infor-
mation. Full-size
DOI: 10.7717/peerj.8880/fig-2 Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Full-size
DOI: 10.7717/peerj.8880/fig-2 The electronic version of this article in Portable Document Format (PDF) will
represent a published work according to the International Commission on Zoological
Nomenclature (ICZN), and hence the new names contained in the electronic version
are effectively published under that Code from the electronic edition alone. This
published work and the nomenclatural acts it contains have been registered in
ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life
Science Identifiers) can be resolved and the associated information viewed through
any standard web browser by appending the LSID to the prefix http://zoobank.org/. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 7/37 7/37 Figure 3
Simplified stratigraphic column showing the lithology, relative age and contact relationships
between the Pirambóia, Botucatu and Serra Geral stratigraphic units. Not in scale. Full-size
DOI: 10.7717/peerj.8880/fig-3
The LSID for this publication is: urn:lsid:zoobank.org:pub:53C73174-4645-40E1-BB7B-
75856AEAEAF5. The LSID for the here described Paleohelcura araraquarensis isp. nov. is: urn:lsid:zoobank.org:act:7D4303AE-BB63-4474-B79C-AD39AB144917. The online Figure 3
Simplified stratigraphic column showing the lithology, relative age and contact relationships
between the Pirambóia, Botucatu and Serra Geral stratigraphic units. Not in scale. Full-size
DOI: 10.7717/peerj.8880/fig-3 Figure 3
Simplified stratigraphic column showing the lithology, relative age and contact relationships
between the Pirambóia, Botucatu and Serra Geral stratigraphic units. Not in scale. Full-size
DOI: 10.7717/peerj.8880/fig-3 Figure 3
Simplified stratigraphic column showing the lithology, relative age and contact relationships
between the Pirambóia, Botucatu and Serra Geral stratigraphic units. Not in scale. Full-size
DOI: 10.7717/peerj.8880/fig-3 The LSID for this publication is: urn:lsid:zoobank.org:pub:53C73174-4645-40E1-BB7B-
75856AEAEAF5. The LSID for the here described Paleohelcura araraquarensis isp. nov. is: urn:lsid:zoobank.org:act:7D4303AE-BB63-4474-B79C-AD39AB144917. The online
version of this work is archived and available from the following digital repositories: PeerJ,
PubMed Central and CLOCKSS The LSID for this publication is: urn:lsid:zoobank.org:pub:53C73174-4645-40E1-BB7B-
75856AEAEAF5. The LSID for the here described Paleohelcura araraquarensis isp. nov. is: urn:lsid:zoobank.org:act:7D4303AE-BB63-4474-B79C-AD39AB144917. The online
version of this work is archived and available from the following digital repositories: PeerJ,
PubMed Central and CLOCKSS Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Trackway measurements The methodology and terminology of Trewin (1994), Braddy (2001) and Minter, Braddy &
Davis (2007) for arthropod trackway description have been adopted herein (Fig. 4). For
the description of preservation, the classification proposed by Seilacher (1964) is followed. For measurements of Paleohelcura araraquarensis isp. nov., four series on each side of the
trackway were selected on slabs LPP-IC-0028, LPP-IC-0029, LPP-IC-0032, LPP-IC-0035. For those slabs with more continuous trackways, eight series on slab LPP-IC-0030 and
nine series on slab LPP-IC-0031 were measured. The measured series are indicated in Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 8/37 Track width
Series
Track length
Internal width
External width
Track 1
Track 2
Track 3
Stride
Pace
Anterior
Posterior
Left
Right
Figure 4
Nomenclature and measurements used for the analysis of Paleohelcura araraquarensis isp. nov. Measurements of the trackway and track characteristics. Full-size
DOI: 10.7717/peerj.8880/fig-4 Figure 4
Nomenclature and measurements used for the analysis of Paleohelcura araraquarensis isp. nov. Measurements of the trackway and track characteristics. Full-size
DOI: 10.7717/peerj.8880/fig-4 the photographs in Figs. S3 and S4. The series measured were chosen on the basis of the
quantity and quality of the tracks on either side of the trackway. We attempted to select
the series to be measured with regular distances between them along the trackway. In the
slabs that show part and counterpart (LPP-IC-0029 with LPP-IC-0030 and LPP-IC-0031
with LPP-IC-0032), the same series were measured in the two slabs (i.e., negative epirelief
and positive hyporelief). LPP-IC-0029 and LPP-IC-0032 present shorter trackways than
their counterparts; therefore, only partial measurements were obtained. Measurements of
internal and external width were taken; as were the pace, stride, and lengths and widths of
the individual tracks (Fig. 4). Trewin (1994, p. 813) proposed using the ratio between the external width and the
internal width as a trackway parameter; however, such a relationship is not practical in the
case of Paleohelcura araraquarensis isp. nov. since there are sections of the trackways where
the internal width is zero, producing fractions with zero as divisor, and it is impossible to
divide by zero (Kaplan, 2000). Therefore, we adopt the inverse of the relation proposed
by Trewin (1994, p. 813), that is, internal width/external width (I/E), and we propose this
as a standard for the description of trackways. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Trackway measurements The ratio between the length and width
(L/W) of the tracks was also used to see if the tracks are circular (values close to one),
or elliptical/elongated (values greater than one). The measurements were taken using a
Vernier caliper. All measurements are listed in Table S1. The line drawing illustration of the
holotype of Paleohelcura araraquarensis isp. nov. was produced using the Inkscape vector
drawing program 0.92, whose license is free and open source (General Public License 3),
using a photograph as a model for the footprint contour. All the graphic elements were
produced with the aforementioned program. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 9/37 RESULTS AND DISCUSSION Ichnotaxonomy. Ichnogenus: Paleohelcura Gilmore, 1926 Type Ichnospecies: Paleohelcura tridactyla Gilmore, 1926. Type Ichnospecies: Paleohelcura tridactyla Gilmore, 1926. Emended Diagnosis: Trackways with external width greater than 20 mm, comprising two
parallel track rows with series of commonly three tracks, but there can be fewer or up to
four tracks per series. Series have alternating to staggered symmetry. Tracks vary from
slightly elliptical to tapered or circular and can be in a linear or triangular arrangement
within series. A medial impression may be present. Remarks: Historically, many arthropod trackway ichnotaxa were inadequately described
and illustrated, at times based on few and/or poorly preserved specimens (Trewin, 1994,
p. 821). Several trackway ichnotaxa with series of at most four tracks have been described,
resulting in potential junior synonyms. Analysis of trackways from the Permian of Germany
and southwestern United States (Minter, Braddy & Voigt, 2007; Minter & Braddy, 2009)
showed intergradations between several ichnotaxa, underscoring morphological and
preservational variations, due to small variations in locomotion or characteristics of the
substrate. Paleohelcura (Gilmore, 1926) was originally described from the lower Permian Coconino
Sandstone of western United States and subsequently described in other studies dealing
with the ichnology of this and other units (Toepelman & Rodeck, 1936; Brady, 1939; Brady,
1947; Brady, 1961; Alf, 1968; Sadler, 1993; Braddy, 1995; Lucas & Lerner, 2004; Morrissey &
Braddy, 2004; Voigt, Small & Sanders, 2005; Batchelor & Garton, 2013; Stoller, Rowland &
Jackson, 2013). Only Paleohelcura tridactyla is accepted as a valid ichnospecies, and four
forms are regarded as junior synonyms: P. dunbari (Brady, 1961), P. delicatula (Fischer,
1978), P. badensis Kozur, Loffler & Sittig (1994), and P.? lyonsensis (Toepelman & Rodeck,
1936), which was provisionally included in Paleohelcura when described. Sadler (1993) noted intergradations between P. tridactyla and P. dunbari, but retained
them as separate ichnospecies because she considered the two ichnotaxa as morphologically
different. On the contrary, it has been argued that the morphologic differences between
P. tridactyla and P. dunbari are minor, with intergradations between the two, and so P. dunbari should be regarded as a junior synonym of P. tridactyla (Minter, Braddy & Davis,
2007; Minter & Braddy, 2009). Paleohelcura delicatula is only known from a single specimen
that consists of comma-shaped tracks, opposite symmetry, and small size compared with P. tridactyla (Fischer, 1978). Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 RESULTS AND DISCUSSION New ichnotaxa should not ideally be erected on the basis of single
specimens and the characteristics presented are not reliable to erect a new ichnospecies. Therefore, we regard Paleohelcura delicatula as a junior synonym of Stiaria intermedia. Paleohelcura badensis is regarded as a junior synonym of Stiaria intermedia (Minter
& Braddy, 2009). Paleohelcura? lyonsensis has been considered a junior synonym of P. tridactyla (Braddy, 1995, p. 221). Gilmore (1927) erected Triavestigia niningeri, and Kozur,
Loffler & Sittig (1994) referred it to Paleohelcura as a distinct ichnospecies; however, the
holotype consists of an incomplete trackway, and the arrangement of the tracks within a Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 10/37 Table 2
Arithmetic mean of the measurements of the trackway. (I/E) Internal Width/External Width. LPP-IC-0033 and LPP-IC-0034 were not measured because of their poor preservation. Specimen
External width
mean (mm)
Internal width
mean (mm)
I/E Mean
Pace
mean (mm)
Stride
mean (mm)
LPP-IC-0028
22,80
2,43
0,11
12,73
12,80
LPP-IC-0029
23,43
2,70
0,11
10,78
11,30
LPP-IC-0030
22,75
1,68
0,07
5,49
10,92
LPP-IC-0031
23,22
1,41
0,06
6,13
11,75
LPP-IC-0032
22,10
1,95
0,09
10,90
11,40
LPP-IC-0035
23,55
3,11
0,10
11,18
11,27
Mean (mm)
22,97
2,21
0,09
9,53
11,57
Standard de-
viation/Mean
0,05
0,57
0,62
0,14
0,11
series suggests that it is a junior synonym of P. tridactyla (Braddy, 1995, p. 221; Minter &
Braddy, 2009). P l
h l
id
l i
i
il
S i
i i
di S
i h 1909 (W lk
1985 Mi Table 2
Arithmetic mean of the measurements of the trackway. (I/E) Internal Width/External Width. LPP-IC-0033 and LPP-IC-0034 were not measured because of their poor preservation. series suggests that it is a junior synonym of P. tridactyla (Braddy, 1995, p. 221; Minter &
Braddy, 2009). Paleohelcura tridactyla is similar to Stiaria intermedia Smith, 1909 (Walker, 1985; Minter
& Braddy, 2009). Stiaria was described from continental fine-grained sediment lenses
within andesites (Walker, 1985; Phillips & Smith, 2008, p. 5) from the Lower Devonian
Old Red Sandstone of Scotland, and was revised by Pollard & Walker (1984), and Walker
(1985), with the latter paper erecting a neotype, lectotype and paratypes not previously
assigned to this ichnotaxon. Both ichnogenera may possess a medial impression and linear
series with two to four tracks (Brady, 1947; Walker, 1985; Minter & Braddy, 2009). In fact,
Walker (1985) suggested that Paleohelcura should be regarded, at least in part, as a junior
synonym of Stiaria. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 RESULTS AND DISCUSSION Full-size
DOI: 10.7717/peerj.8880/fig-5
2018), and is endorsed here. Paleohelcura araraquarensis isp. nov. is placed in Paleohelcura
instead of Stiaria because it exhibits an external width greater than 20 mm. Figure 5
Holotype of Paleohelcura araraquarensis. isp. nov. (LPP-IC-0028). (A) Photograph of LPP-
IC-0028 slab showing the positive hyporelief of Paleohelcura araraquarensis isp. nov. (B) Representative
scheme of the holotype of Paleohelcura araraquarensis isp. nov. Lowercase letters c and d indicate exam-
ples of footprint orientation within the series. The producer walked from bottom to top. Full-size
DOI: 10.7717/peerj.8880/fig-5 2018), and is endorsed here. Paleohelcura araraquarensis isp. nov. is placed in Paleohelcura
instead of Stiaria because it exhibits an external width greater than 20 mm. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 RESULTS AND DISCUSSION Stiaria quadripedia is a similar ichnospecies and was also revised by Walker (1985),
but differs from Paleohelcura by presenting bifid or trifid tracks, which are possible to
delineate in finer-grained sediments. Stiaria intermedia consists of trackways with up to
three circular tracks, similar to Paleohelcura. In contrast, Stiaria quadripedia may have four
tracks, and is larger than S. intermedia. Size is not regarded as an appropriate ichnotaxobase
(Bertling et al., 2006), but analysis of the external widths of specimens assigned to Stiaria
intermedia and to Paleohelcura tridactyla has shown that they fall into two separate size
classes (Minter & Braddy, 2009). Whilst not separated by an order of magnitude, a working
model was proposed (Minter & Braddy, 2009) whereby Stiaria intermedia should be used
for trackways with an external width of less than 20 mm, and Paleohelcura for those with
an external width greater than 20 mm, like the trackways here described (see Table 2). The clarification of the ichnotaxonomic status of Stiaria intermedia and Paleohelcura
tridactyla remains to be achieved through examination of their holotypes and neotypes
(Minter & Braddy, 2009). In any case, Paleohelcura is a well-accepted ichnotaxon, which has
been recorded extensively. The working model proposed by Minter & Braddy (2009) has
been adopted in many papers (Lucas et al., 2005; Minter & Braddy, 2009; Fillmore, Lucas
& Simpson, 2010; Poschmann & Braddy, 2010; Batchelor & Garton, 2013; Getty et al., 2013;
Getty et al., 2017; Bernardi, Marchetti & Gobbi, 2018; Uchman, Gazdzicki & Blazejowski, Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 11/37 Figure 5
Holotype of Paleohelcura araraquarensis. isp. nov. (LPP-IC-0028). (A) Photograph of LPP-
IC-0028 slab showing the positive hyporelief of Paleohelcura araraquarensis isp. nov. (B) Representative
scheme of the holotype of Paleohelcura araraquarensis isp. nov. Lowercase letters c and d indicate exam-
ples of footprint orientation within the series. The producer walked from bottom to top. Full-size
DOI: 10.7717/peerj.8880/fig-5
2018), and is endorsed here. Paleohelcura araraquarensis isp. nov. is placed in Paleohelcura
instead of Stiaria because it exhibits an external width greater than 20 mm. Figure 5
Holotype of Paleohelcura araraquarensis. isp. nov. (LPP-IC-0028). (A) Photograph of LPP-
IC-0028 slab showing the positive hyporelief of Paleohelcura araraquarensis isp. nov. (B) Representative
scheme of the holotype of Paleohelcura araraquarensis isp. nov. Lowercase letters c and d indicate exam-
ples of footprint orientation within the series. The producer walked from bottom to top. Figures 5 and 6A. g
Horizon and type locality: São Bento Group, Botucatu Formation; Locality: Ouro;
municipality: Araraquara; São Paulo State (Fig. 1); São Bento quarry (Corpedras company)
(Fig. 2), geographical coordinates: 21◦49′07.6′′S 48◦04′28.8′′W, altitude: 670 m. Holotype: LPP-IC-0028: sandstone slab showing slightly curved trackway, preserved in
positive hyporelief over a length of 34 cm. Reposited in the Paleoichnology collection of
the Laboratório de Paleoecologia e Paleoicnologia (LPP) of the Federal University of São
Carlos (UFSCar) campus São Carlos-SP. Horizon and type locality: São Bento Group, Botucatu Formation; Locality: Ouro;
municipality: Araraquara; São Paulo State (Fig. 1); São Bento quarry (Corpedras company)
(Fig. 2), geographical coordinates: 21◦49′07.6′′S 48◦04′28.8′′W, altitude: 670 m. Holotype: LPP-IC-0028: sandstone slab showing slightly curved trackway, preserved in
positive hyporelief over a length of 34 cm. Reposited in the Paleoichnology collection of
the Laboratório de Paleoecologia e Paleoicnologia (LPP) of the Federal University of São
Carlos (UFSCar) campus São Carlos-SP. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 12/37 Figure 6
Photographs of some of the slabs bearing Paleohelcura araraquarensis isp. nov. (A) LPP-IC-
0028 (holotype). (B) LPP-IC-0029 and (C) LPP-IC-0030. The producer walked from bottom to top. Scale
bar: 10 cm. Full-size
DOI: 10.7717/peerj.8880/fig-6 Figure 6
Photographs of some of the slabs bearing Paleohelcura araraquarensis isp. nov. (A) LPP-IC-
0028 (holotype). (B) LPP-IC-0029 and (C) LPP-IC-0030. The producer walked from bottom to top. Scale
bar: 10 cm. Full-size
DOI: 10.7717/peerj.8880/fig-6 Full-size
DOI: 10.7717/peerj.8880/fig-6 Paratypes: Sandstone slabs: LPP-IC-0029 (negative epirelief) and its counterpart LPP-
IC-0030 (positive hyporelief); LPP-IC-0031 (negative epirelief) and its counterpart LPP-
IC-0032 (positive hyporelief); LPP-IC-0033 (negative epirelief) and its counterpart LPP-
IC-0034 (positive hyporelief); LPP-IC-0035 (negative epirelief) and with no apparent
counterpart slab. All slabs are reposited in the Paleoichnology collection of the Laboratório
de Paleoecologia e Paleoicnologia (LPP) of the Federal University of São Carlos (UFSCar)
campus São Carlos-SP. Etymology: It is dedicated to the city of Araraquara, São Paulo State, where these trackways
were found, along with most of the ichnofossils of the Botucatu Formation. Diagnosis: Trackways composed of two rows, whose internal width between the rows is
less than one-quarter of the external width; with alternating to staggered series, consisting
of up to three tracks with different sizes that may vary from slightly elongated to tapered
or circular in shape. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Figures 5 and 6A. Description: Due to the similarity between the size of the sandstone grains and the size of
the locomotory appendages of the producer, the tracks of Paleohelcura araraquarensis isp. nov. in all the analyzed slabs have little definition. The following slabs have counterparts:
LPP-IC-0029 (negative epirelief) and LPP-IC-0030 (positive hyporelief), Figs. 6B and
6C, respectively; LPP-IC-0031 (negative epirelief) and LPP-IC-0032 (positive hyporelief),
Figs. 7A and 7B, respectively; LPP-IC-0033 (negative epirelief) and LPP-IC-0034 (positive
hyporelief), Figs. 7C and 7D, respectively. The organization of the tracks in the series follows
a pattern with two, usually smaller, tracks grouped anteriorly and often more externally, and
a longer track more posteriorly and commonly internally positioned (Fig. 5C). In places,
the series adopt a linear configuration (Fig. 5D) that, despite showing some recurrence, is Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 13/37 Figure 7
Photographs of some of the slabs bearing Paleohelcura araraquarensis isp. nov. (A) LPP-IC-
0031. (B) LPP-IC-0032. (C) LPP-IC-0033. (D) LPP-IC-0034. (E) LPP-IC-0035. The producer walked from
bottom to top. Scale bar: 10 cm. Full-size
DOI: 10.7717/peerj.8880/fig-7
Table 3
Arithmetic mean of the measurements of each track in the series. (L/W) Length /Width. LPP-IC-0033 and LPP-IC-0034 were not mea-
sured because of their poor preservation. Figure 7
Photographs of some of the slabs bearing Paleohelcura araraquarensis isp. nov. (A) LPP-IC-
0031. (B) LPP-IC-0032. (C) LPP-IC-0033. (D) LPP-IC-0034. (E) LPP-IC-0035. The producer walked from
bottom to top. Scale bar: 10 cm. Full-size
DOI: 10.7717/peerj.8880/fig-7 Figure 7
Photographs of some of the slabs bearing Paleohelcura araraquarensis isp. nov. (A) LPP-IC-
0031. (B) LPP-IC-0032. (C) LPP-IC-0033. (D) LPP-IC-0034. (E) LPP-IC-0035. The producer walked from
bottom to top. Scale bar: 10 cm. Full-size
DOI: 10.7717/peerj.8880/fig-7 Table 3
Arithmetic mean of the measurements of each track in the series. (L/W) Length /Width. LPP-IC-0033 and LPP-IC-0034 were not mea-
sured because of their poor preservation. Figures 5 and 6A. Specimen
Track 1 (Mean)
Track 2 (Mean)
Track 3 (Mean)
Length (mm)
Width (mm)
L/W
Length (mm)
Width (mm)
L/W
Length (mm)
Width (mm)
L/W
LPP-IC-0028
5,41
3,14
1,73
4,94
3,03
1,63
5,11
3,79
1,35
LPP-IC-0029
4,98
2,83
1,76
4,38
3,41
1,28
4,28
3,37
1,27
LPP-IC-0030
4,43
2,73
1,62
4,13
2,77
1,49
4,32
3,21
1,35
LPP-IC-0031
4,60
2,97
1,55
4,53
3,07
1,47
4,81
3,77
1,27
LPP-IC-0032
4,96
3,10
1,60
4,44
3,01
1,47
4,77
3,53
1,35
LPP-IC-0035
4,28
2,84
1,51
4,73
3,28
1,44
5,41
3,74
1,45
Mean (mm)
4,78
2,93
1,63
4,52
3,09
1,47
4,78
3,57
1,34
Standard de-
viation/Mean
0,19
0,17
0,17
0,15
0,15
0,15
not an appropriate feature for the diagnosis because it is a variation in the more consistent
triangular pattern shown in Fig. 6C. The arithmetic means of the measurements of the trackway parameters are summarized
in Tables 2 and 3. All measurements are in the Table S1. The average external width of the Specimen
Track 1 (Mean)
Track 2 (Mean)
Track 3 (Mean)
Length (mm)
Width (mm)
L/W
Length (mm)
Width (mm)
L/W
Length (mm)
Width (mm)
L/W
LPP-IC-0028
5,41
3,14
1,73
4,94
3,03
1,63
5,11
3,79
1,35
LPP-IC-0029
4,98
2,83
1,76
4,38
3,41
1,28
4,28
3,37
1,27
LPP-IC-0030
4,43
2,73
1,62
4,13
2,77
1,49
4,32
3,21
1,35
LPP-IC-0031
4,60
2,97
1,55
4,53
3,07
1,47
4,81
3,77
1,27
LPP-IC-0032
4,96
3,10
1,60
4,44
3,01
1,47
4,77
3,53
1,35
LPP-IC-0035
4,28
2,84
1,51
4,73
3,28
1,44
5,41
3,74
1,45
Mean (mm)
4,78
2,93
1,63
4,52
3,09
1,47
4,78
3,57
1,34
Standard de-
viation/Mean
0,19
0,17
0,17
0,15
0,15
0,15
not an appropriate feature for the diagnosis because it is a variation in the more consistent
triangular pattern shown in Fig. 6C. The arithmetic means of the measurements of the trackway parameters are summarized
in Tables 2 and 3. All measurements are in the Table S1. The average external width of the
trackways is 22.97 mm, and the average internal width is 2.21 mm, with absolute values
of the latter varying from 3.80 mm to 0 mm (i.e., no internal separation between series). The internal trackway width is, on average, approximately one-tenth of the outer trackway
width. The ratio between the length and width of the tracks (L/W) is always greater than
one, which indicates that they are elongated. It is rare in Paleohelcura araraquarensis isp. nov. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Figures 5 and 6A. for tracks to have lengths and widths with similar values, that is, with a circular shape. Specimen
Track 1 (Mean)
Track 2 (Mean)
Track 3 (Mean)
Length (mm)
Width (mm)
L/W
Length (mm)
Width (mm)
L/W
Length (mm)
Width (mm)
L/W
LPP-IC-0028
5,41
3,14
1,73
4,94
3,03
1,63
5,11
3,79
1,35
LPP-IC-0029
4,98
2,83
1,76
4,38
3,41
1,28
4,28
3,37
1,27
LPP-IC-0030
4,43
2,73
1,62
4,13
2,77
1,49
4,32
3,21
1,35
LPP-IC-0031
4,60
2,97
1,55
4,53
3,07
1,47
4,81
3,77
1,27
LPP-IC-0032
4,96
3,10
1,60
4,44
3,01
1,47
4,77
3,53
1,35
LPP-IC-0035
4,28
2,84
1,51
4,73
3,28
1,44
5,41
3,74
1,45
Mean (mm)
4,78
2,93
1,63
4,52
3,09
1,47
4,78
3,57
1,34
Standard de-
viation/Mean
0,19
0,17
0,17
0,15
0,15
0,15 not an appropriate feature for the diagnosis because it is a variation in the more consistent
triangular pattern shown in Fig. 6C. The arithmetic means of the measurements of the trackway parameters are summarized
in Tables 2 and 3. All measurements are in the Table S1. The average external width of the
trackways is 22.97 mm, and the average internal width is 2.21 mm, with absolute values
of the latter varying from 3.80 mm to 0 mm (i.e., no internal separation between series). The internal trackway width is, on average, approximately one-tenth of the outer trackway
width. The ratio between the length and width of the tracks (L/W) is always greater than
one, which indicates that they are elongated. It is rare in Paleohelcura araraquarensis isp. nov. for tracks to have lengths and widths with similar values, that is, with a circular shape. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 14/37 There were variations observed between track measurements in the negative epirelief
and their corresponding counterpart slab. This may have two causes: (i) subjectivity
may have caused variation in the measurement of tracks of different toponomy, one in
negative epirelief and another one in positive hyporelief (i.e., methodological bias); or (ii)
it would suggest that arthropod tracks are susceptible to another type of alteration, after the
production of the footprint and the lithification of the substrate, generated by the splitting
of the layers in to part and counterpart slabs (i.e., taphonomic bias). In this situation,
tracks can lose parts, become smaller, or retain the sediment of the counterpart slab,
thereby modifying their size. Figures 5 and 6A. As such, the measurements taken from an ichnofossil may
not correspond precisely to the size of the tracks left by the animal when the substrate was
unconsolidated. It was not possible to take accurate track measurements of the counterpart
slabs LPP-IC-0033 and LPP-IC-0034 (Figs. 7C and 7D) due to the poor preservation of the
tracks, but they are included as paratypes because they represent part of the variation that
Paleohelcura araraquarensis isp. nov. can exhibit, whether due to preservation, taphonomic
process or produced by the disaggregation of the layers. The paratypes (LPP-IC-0029,
LPP-IC-0030, LPP-IC-0031, LPP-IC-0032, LPP-IC-0033, LPP-IC-0034) did not present
significant differences in relation to the holotype (LPP-IC-0028). Specimens in LPP-IC-0028, LPP-IC-0029, LPP-IC-0030, LPP-IC-0031, LPP-IC-0032,
LPP-IC-0035 show sediment displacement associated with the tracks (Fig. 8). In slabs with
positive hyporelief (LPP-IC-0028, LPP-IC-0030, LPP-IC-0032), the displacement appears
as a faint depression attached to the track. This displacement indicates the direction
of movement, being located on the opposite side from the direction of movement
of the animal, generated by the effort that the locomotory appendage applied to the
unconsolidated substrate to generate propulsion. However, we cannot rule out the
possibility that the displacement could have been generated by sliding of the animal
caused by the slope of the dune; this interpretation is less likely because the orientation of
the displacement is the same in all the specimens that exhibit it. Therefore, we consider
that the displacement was more likely to have been generated by the propulsion of the
animal over the sand. The commercial extraction of the sandstone at the São Bento quarry was undertaken
without scientific monitoring, and most of the fossils were in slabs ready to be cut, or
already cut for commercialization. Thus, the fossils were rescued and there was no record
of the orientation of the slabs in relation to the slope or whether the holotype and the
paratypes could be parts of the same large but fragmented individual trackway. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Comparisons The main difference between Paleohelcura araraquarensis isp. nov. and Paleohelcura
tridactyla, and its junior synonyms (revised in Remarks section of the ichnogenus
Paleohelcura) is the ratio of the internal width to the external width. Paleohelcura
araraquarensis isp. nov. has an internal width equal to or less than one-quarter of the
external width (on average one-tenth of the external width). The internal width in other
ichnospecies of Paleohelcura is greater than one-quarter of the external width, even in
Paleohelcura with narrow internal widths like some of from the lower Permian Robledo Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Figure 8
Specimen LPP-IC-0035 of Paleohelcura araraquarensis isp. nov. in negative epirelief exhibit-
ing deformation in the sediment by the effort of locomotion of the animal. Red arrows: strain by loco-
motion effort; White arrow: the direction of movement of the animal. The light source is at the top of the
photo.
Full-size
DOI: 10.7717/peerj.8880/fig-8 g
y
y
g
p
y
y
Series within Lithographus and P. araraquarensis isp. nov. share two usually smaller tracks
that are grouped anteriorly and commonly more externally, and a longer track that is
positioned more posteriorly and internally. We suggest that this characteristic may reflect
the pterygote insect leg arrangement (see Trace-fossil producer section). The main morphological characteristic of Lithographus are its linear tracks with
varied orientations, which differs from the rounded to elliptical tracks of Paleohelcura
araraquarensis isp. nov., whose long-axes are subparallel to the midline of the trackway. It
is, therefore, more reasonable to assign the trackways described here to Paleohelcura, and
to establish a new ichnospecies for forms with a narrow internal width. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Figure 8
Specimen LPP-IC-0035 of Paleohelcura araraquarensis isp. nov. in negative epirelief exhibit-
ing deformation in the sediment by the effort of locomotion of the animal. Red arrows: strain by loco-
motion effort; White arrow: the direction of movement of the animal. The light source is at the top of the
photo.
Full-size
DOI: 10.7717/peerj.8880/fig-8 Figure 8
Specimen LPP-IC-0035 of Paleohelcura ara
ing deformation in the sediment by the effort of loco
motion effort; White arrow: the direction of movemen
photo. Figure 8
Specimen LPP-IC-0035 of Paleohelcura araraquarensis isp. nov. in negative epirelief exhibit-
ing deformation in the sediment by the effort of locomotion of the animal. Red arrows: strain by loco-
motion effort; White arrow: the direction of movement of the animal. The light source is at the top of the
photo. Full-size
DOI: 10.7717/peerj.8880/fig-8 Figure 8
Specimen LPP-IC-0035 of Paleohelcura araraquarensis isp. nov. in negative epirelief exhibit-
ing deformation in the sediment by the effort of locomotion of the animal. Red arrows: strain by loco-
motion effort; White arrow: the direction of movement of the animal. The light source is at the top of the
photo. Full-size
DOI: 10.7717/peerj.8880/fig-8 Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Mountains and Coconino Sandstone of the USA that have external widths of similar or
lower absolute values to P. araraquarensis isp. nov. (Minter & Braddy, 2009, their fig. 30;
31), and those from the Permian Lyons Sandstone of Colorado (USA) (Toepelman &
Rodeck, 1936, their fig. 1). The triangular series arrangement and ellipsoidal tracks shown by Paleohelcura
araraquarensis isp. nov. (Fig. 5C) slightly resemble Lithographus and some of its junior
synonyms. Lithographus comprises trackways with series of up to three tracks with alternate
to staggered symmetry but differs from Paleohelcura in that the tracks are linear and have
varied orientations with respect to the midline of the trackway (Minter & Braddy, 2009). Several ichnogenera were synonymized with Lithographus by Minter & Braddy (2009). The triangular series arrangement and ellipsoidal tracks shown by Paleohelcura
araraquarensis isp. nov. (Fig. 5C) slightly resemble Lithographus and some of its junior
synonyms. Lithographus comprises trackways with series of up to three tracks with alternate
to staggered symmetry but differs from Paleohelcura in that the tracks are linear and have
varied orientations with respect to the midline of the trackway (Minter & Braddy, 2009). Several ichnogenera were synonymized with Lithographus by Minter & Braddy (2009). Series within Lithographus and P. araraquarensis isp. nov. share two usually smaller tracks
that are grouped anteriorly and commonly more externally, and a longer track that is
positioned more posteriorly and internally. We suggest that this characteristic may reflect
the pterygote insect leg arrangement (see Trace-fossil producer section). Trace-fossil producer The photographs
are frames of a video courtesy of R.E. Ritzmann showing a cockroach (Blaberus discoidalis) in ventral view
walking on an oiled glass plate (Video S1). Full-size
DOI: 10.7717/peerj.8880/fig-9 1º step
2º step
3º step
4º step
5º step
6º step
Series
{
Footprint being marked
Already marked footprint
A
C
B Series
{
C Series
{
C 1º step
2º step
3º step
4º step
5º step
6º step
Footprint being marked
Already marked footprint
A
B A C B Figure 9
Illustration simulating the marks left by a cockroach while walking (ventral view). (A) The
photographs show the advancement of a cockroach and the marks that would be left by its feet. The red
footprints on the photograph indicate footprints that have just been produced; magenta footprints indi-
cate already produced footprints. (B) Footprints produced in each step, broken down by color. (C) Asso-
ciation between the footprints within the series and the pairs of feet that produced them. The photographs
are frames of a video courtesy of R.E. Ritzmann showing a cockroach (Blaberus discoidalis) in ventral view
walking on an oiled glass plate (Video S1). Full-size
DOI: 10.7717/peerj.8880/fig-9 p. 2); therefore, neoichnological studies provide strong grounds to exclude scorpions as
producers of P. araraquarensis isp. nov. Neoichnological studies with cockroaches produced trackways with elongated tracks
because they walk on tarsal segments (tarsomeres) (Davis, Minter & Braddy, 2007). Therefore, segmented tarsi appear to be an important feature to generate tracks similar
to those of the ichnogenus Lithographus and its junior synonyms. Within the Hexapoda,
Protura, Diplura, Monura and Collembola possess undivided tarsi (Kristensen, 1998,
p. 289; Bitsch & Bitsch, 2000, p. 140), probably producing tracks similar to those of an
arachnid because they also do not have segmented tarsi. Only the true insects (Zygentoma,
Archaeognatha, and Pterygota) have segmented tarsi (Kristensen, 1998, p. 289; Bitsch
& Bitsch, 2000; Gorb & Beutel, 2001, p. 534). The neoichnology of representatives of
Zygentoma and Archaeognatha revealed that they produced circular/elliptical to elongated
tracks (Getty et al., 2013), which resemble those produced by arachnids. In this case, it
is due to the low mobility of the tarsomeres, which lead to the digitigrade posture (on
the pretarsus). Trace-fossil producer Although series arrangement and track shape are variable, the most common pattern
identified is useful for making neoichnological comparisons. An alternating tripod gait is a
relatively robust locomotion pattern for Hexapoda (Wöhrl, Reinhardt & Blickhan, 2017a). Arachnids, although possessing four pairs of locomotory appendages, can produce series
with three tracks, either by adopting a hexapedal gait or for taphonomic reasons (Davis,
Minter & Braddy, 2007; Schmerge, Riese & Hasiotis, 2013). Trackways comprising series
with alternating symmetry on either side of the medial line, and with a maximum of three
tracks per series, indicate that the animal maintained at least three feet on the ground while
walking (Fig. 9). Therefore, we restrict the discussion of the producer to the Arachnida and
Hexapoda. The trackways made by spiders do not resemble Paleohelcura araraquarensis isp. nov. because they have larger internal width, circular tracks, and a different series arrangement
Davis, Minter & Braddy, 2007, their Fig. 9). When scorpions leave series with three tracks
they resemble P. araraquarensis isp. nov., but with a different arrangement of tracks within
the series. In P. araraquarensis isp. nov., there are two tracks anteriorly positioned, and
a usually longer track more posteriorly and internally positioned. In scorpion trackways,
there are two tracks more posteriorly positioned and one track that lays anteriorly, and
the former are usually longer and commonly most internally positioned (Fig. 10; Davis,
Minter & Braddy, 2007, their fig. 7–8). In addition, even small scorpions, with comparable
size to the animal that made Paleohelcura araraquarensis isp. nov., leave trackways with
a large internal width, differing from the narrow internal width of P. araraquarensis isp. nov. (Fig. 10). Modern scorpions are very similar to Paleozoic scorpions (Polis, 1990, Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 17/37 1º step
2º step
3º step
4º step
5º step
6º step
Series
{
Footprint being marked
Already marked footprint
A
C
B
Figure 9
Illustration simulating the marks left by a cockroach while walking (ventral view). (A) The
photographs show the advancement of a cockroach and the marks that would be left by its feet. The red
footprints on the photograph indicate footprints that have just been produced; magenta footprints indi-
cate already produced footprints. (B) Footprints produced in each step, broken down by color. (C) Asso-
ciation between the footprints within the series and the pairs of feet that produced them. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Trace-fossil producer The increased mobility of the tarsomeres in Pterygota is linked to the
evolutionary pressure to climb and walk on a variety of new substrates due to their ability
to fly and the necessity to hold onto leaves and plant stems (Gorb & Beutel, 2001, p. 533). Most Pterygota walk on tarsomeres (Manton, 1972; Zollikofer, 1994, p. 98; Frazier et al.,
1999; Boggess et al., 2004; Davis, Minter & Braddy, 2007; Gladun & Gorb, 2007; Clemente &
Federle, 2008; Wöhrl, Reinhardt & Blickhan, 2017b); cockroach video in Video S1 of this
publication, courtesy of R.E. Ritzmann). As observed in neoichnological experiments
on cockroaches (Davis, Minter & Braddy, 2007), they all probably produce elongated or
elliptical tracks. Nevertheless, it is not possible to assume this for all pterygote insects
without more neoichnological experiments because there are species that walk on a few
distal tarsomeres and on their pretarsus (Niederegger & Gorb, 2003; Gladun & Gorb, 2007; Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 18/37 C
B
A
Figure 10 Scorpion Tityus serrulatus. tracks in sand (A and B) and Paleohelcura araraquarensis isp. nov. holotype LPP-IC-0028 (C). Scale bar: 1 cm. The arrow indicates the direction of the animal move-
ment. (A) and (B) photographs courtesy of Ravi Sampaio, 2015. Full-size
DOI: 10.7717/peerj.8880/fig-10 B C A Figure 10 Scorpion Tityus serrulatus. tracks in sand (A and B) and Paleohelcura araraquarensis isp. nov. holotype LPP-IC-0028 (C). Scale bar: 1 cm. The arrow indicates the direction of the animal move-
ment. (A) and (B) photographs courtesy of Ravi Sampaio, 2015. Full-size
DOI: 10.7717/peerj.8880/fig-10 Endlein & Federle, 2015), thereby probably producing elongated tracks, but less so than
those of full plantigrade insects. Trackways of some desert darkling beetles (Tenebrionidae) closely resemble Paleohelcura
araraquarensis isp. nov. in that they comprise a narrow internal width, elliptical tracks, and
series that usually exhibit the same arrangement of P. araraquarensis isp. nov. (Fig. 11). Despite being made by pterygote insects, they do not show strong linear tracks like the
cockroaches used in experiments by Davis, Minter & Braddy (2007) that made trackways
similar to Lithographus. Even with segmented tarsi, these Tenebrionidae leave elliptical
tracks just like in P. araraquarensis isp. nov. This is probably due to the small size of the
animal compared with the sand grain size, which diminishes the resolution of the tracks. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Trace-fossil producer The cockroaches used by Davis, Minter & Braddy (2007) were relatively large compared to
the grain size of the substrate, creating trackways of greater than 40 mm external width, and
so it is expected that the tracks reflected more faithfully the morphology of the locomotory
appendages. The series arrangement in the trackways of Tenebrionidae are also similar to those
observed in Paleohelcura araraquarensis isp. nov. and cockroach trackways, with two
usually smaller tracks grouped anteriorly and often more externally and a longer track
more posteriorly and often internally positioned. This arrangement could be related to the Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 19/37 A
B
Figure 11 Darkling beetles (Tenebrionidae) and their tracks from Morocco dunes. (A) Oblique view
of the trackway and the walking darkling beetle. (B) Perpendicular view of the trackway and the walk-
ing darkling beetle. The photos were not taken with scales, but these beetles are around 2 cm long. Pho-
tographs courtesy of Martin Harvey, 2004. Full-size
DOI: 10.7717/peerj.8880/fig-11 A A Figure 11 Darkling beetles (Tenebrionidae) and their tracks from Morocco dunes. (A) Oblique view
of the trackway and the walking darkling beetle. (B) Perpendicular view of the trackway and the walk-
ing darkling beetle. The photos were not taken with scales, but these beetles are around 2 cm long. Pho-
tographs courtesy of Martin Harvey, 2004. Full-size
DOI: 10.7717/peerj.8880/fig-11 positions of the legs and the role of each leg in the gait of pterygote insects. Therefore, we
consider that the producer of P. araraquarensis isp. nov. would probably be an insect from
the Pterygota rather than an arachnid, and suggest Recent Tenebrionidae as a plausible
analog, noteworthy for their high abundance and diversity in deserts, as a result of their
striking ability to adapt to hyperarid settings (Cloudsley-Thompson, 2001). This contrasts
with the interpretations of the producers of other ichnospecies of Paleohelcura, inferred
to have been made by scorpions and spiders (Davis, Minter & Braddy, 2007). Although a
beetle affinity is proposed, more neoichnological studies are necessary to discriminate other
potential producers since the diversity of pterygote insects is high (Clapham et al., 2016). In addition, it will enable greater understanding of the effect on trackway morphology of
the interaction among the size of an arthropod, the morphology of its limbs, the grain size,
and the moisture in the substrate. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Paleoautoecologic implications sharing some morphological characteristics
with trackways of some dune desert Tenebrionidae (see Trace-fossil producer section),
it is not possible to establish a confident link between the fossil tracks and the group
without eliminating other Pterygota as candidates through neoichnology or the discovery
of associated body fossils. Nevertheless, the existence of Taenidium isp. and Skolithos
linearis burrows (Fernandes, Netto & Carvalho de, 1988; Fernandes, Carvalho & Netto, Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Paleoautoecologic implications Looking at modern deserts, among Pterygota reaching similar size, the Coleoptera are one of
the most conspicuous and abundant animals in arid environments (Holm & Scholtz, 1980;
Cloudsley-Thompson, 2001; Whitford, 2002). Some, like Tenebrionidae, possess several
morphological, physiological, and mainly behavioral adaptations to deal with extremely
dry and hot environments (ultra-psammophilous) (Cloudsley-Thompson, 2001). Beyond
their remarkable incidence in deserts, these animals play an ecological role in dune deserts
of consuming detritus among sand grains and being able to rely on this biomass even when
there is no primary production during dry periods in hyperarid deserts (Seely & Louw,
1980; Southgate, Masters & Seely, 1996). Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 20/37 The producer of Paleohelcura araraquarensis isp. nov. being a pterygote insect could
predominately have been a herbivore, carnivore, or an omnivore. Omnivory is important
in desert food webs since primary productivity is limited by moisture availability (Polis,
1991). Such an animal would be able to feed on the organic matter that accumulated in the
paleodunes, but also be capable of adopting herbivorous, saprophagous, or carnivorous
diets when appropriate. Tenebrionidae are a notable example of this in modern dune deserts
(Koch, 1961; Holm & Scholtz, 1980; Robinson & Seely, 1980; Seely & Louw, 1980; Cloudsley-
Thompson, 2001). If it had been carnivorous, the producer of Paleohelcura araraquarensis
isp. nov. could be a predatory insect, like some species of Carabidae (Coleoptera) living in
dunes of the Negev desert (Filser & Prasse, 2008). Despite Paleohelcura araraquarensis isp. nov. sharing some morphological characteristics
with trackways of some dune desert Tenebrionidae (see Trace-fossil producer section),
it is not possible to establish a confident link between the fossil tracks and the group
without eliminating other Pterygota as candidates through neoichnology or the discovery
of associated body fossils. Nevertheless, the existence of Taenidium isp. and Skolithos
linearis burrows (Fernandes, Netto & Carvalho de, 1988; Fernandes, Carvalho & Netto,
1990; Fernandes, 2005; Peixoto et al., 2016) shows that the Botucatu paleodesert played host
to a community of omnivorous detritivorous insects just like modern dune deserts, where
they comprise most of the animal biomass on the dune slip-face (e.g., Namib dune desert:
Seely & Louw, 1980). P. araraquarensis isp. nov. could be produced by an insect of this
community like omnivorous Tenebrionidae, which produces similar trackways in modern
dune deserts. Despite Paleohelcura araraquarensis isp. nov. Paleoecology of the Botucatu desert The trace-fossil record of the Botucatu Formation may provide indirect evidence on the
abundance and diversity of organisms that inhabited these eolian dunes. Through the
interpretation of the phylogenetic affinity and probable nutrient source of the producers, it
is possible to make broad inferences about the ecological relationships of these organisms
in the context of the Late Jurassic—Early Cretaceous Botucatu desert. Modern desert food
webs are complex due to a high frequency of omnivory, generating highly connected food
webs, with species interacting with many predators and prey (Polis, 1991). Omnivorous
insects comprise most of the animal biomass on the dune slip-face of modern deserts (e.g.,
Namib dune desert; Seely & Louw, 1980). A similar situation is envisaged for the Botucatu
paleodesert. Taenidium isp. and Skolithos linearis were most likely produced by insects
feeding on detritus among the sand grains (Figs. 12G to 12E), like Tenebrionidae in modern
dune deserts. Those with compatible size could produce Paleohelcura araraquarensis isp. nov. when walking on the sand. Insects feeding on living plant material as omnivores or
herbivores could also produce P. araraquarensis isp. nov. (Fig. 12H to 12E). Accordingly,
relying on plant material, the detritivorous and herbivorous invertebrates are regarded as
part of the primary consumers in the trophic web of the Botucatu paleodesert. The geomorphological characteristics of an erg create habitats with abiotic characteristics
that determine the productivity, biomass and diversity found in the interdune, and the
windward and slip-face subenvironments of the dune field (Seely & Louw, 1980; Southgate, Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 21/37 Figure 12 Reconstruction of the Botucatu paleodesert food web based on the interpretation of the
probable producers of the ichnofossil of Botucatu Formation. The dashed arrows represent the ecologi-
cal relations and the flow of energy in the Botucatu paleodesert. (A) Large mammaliaform. (B) Theropod
dinosaurs. (C) Small mammaliaform. (D) Arachnids and possible insects. (E) Insects. (F) Ornithopod di-
nosaurs. (G) Detritus in the sand and blown by the wind. (H) Plants. On the left, the possible ecological
roles that the Paleohelcura araraquarensis isp. nov. producer could have played in the Botucatu desert are
shown. This scheme was made with modifications of artworks of different authors: Carivorous Insect from
Rafael Pasini; Scorpion in (D) from Gareth Monger; (A) and (C) Mammaliaforms from Ceri Thomas; (B)
Theropod dinosaur from Frederic Wierum; (F) Ornithopod dinosaur from Nobumichi Tamura. Paleoecology of the Botucatu desert All art-
works are under the CC-BY-SA 4.0 (https://creativecommons.org/licenses/by/4.0/). Full-size
DOI: 10.7717/peerj.8880/fig-12 Figure 12 Reconstruction of the Botucatu paleodesert food web based on the interpretation of the Figure 12 Reconstruction of the Botucatu paleodesert food web based on the interpretation of the
probable producers of the ichnofossil of Botucatu Formation. The dashed arrows represent the ecologi-
cal relations and the flow of energy in the Botucatu paleodesert. (A) Large mammaliaform. (B) Theropod
dinosaurs. (C) Small mammaliaform. (D) Arachnids and possible insects. (E) Insects. (F) Ornithopod di-
nosaurs. (G) Detritus in the sand and blown by the wind. (H) Plants. On the left, the possible ecological
roles that the Paleohelcura araraquarensis isp. nov. producer could have played in the Botucatu desert are
shown. This scheme was made with modifications of artworks of different authors: Carivorous Insect from
Rafael Pasini; Scorpion in (D) from Gareth Monger; (A) and (C) Mammaliaforms from Ceri Thomas; (B)
Theropod dinosaur from Frederic Wierum; (F) Ornithopod dinosaur from Nobumichi Tamura. All art-
works are under the CC-BY-SA 4.0 (https://creativecommons.org/licenses/by/4.0/). Full-size
DOI: 10.7717/peerj.8880/fig-12 Masters & Seely, 1996). Even though the slip-face occupies a small area of the erg, when
compared to the windward or interdune areas (in the case of ergs with well-spaced dunes),
and is a region of low plant growth, it has a high concentration of biomass per unit area
in the form of detritus, concentrated by the wind mainly at the base of the dunes (Seely
& Louw, 1980; Southgate, Masters & Seely, 1996). This detritus originates from adjacent
subenvironments, more conducive to plant growth where there is moisture (Seely & Louw,
1980; Southgate, Masters & Seely, 1996), and could even be carried by the wind from distant
locations, where the climatic regimes allow greater primary productivity (Robinson &
Seely, 1980). During prolonged periods of drought, with low primary productivity, the
dune slip-faces maintain high concentrations of biomass as detritus is deposited by the
wind, providing food for detritivores and letting them survive until periods of increased
availability of moisture and biomass, when they can proliferate (Southgate, Masters & Seely,
1996). Masters & Seely, 1996). Paleoecology of the Botucatu desert Therefore, we can infer that the Botucatu paleodesert landscape could have had some
shrubs, similar to modern arid dune deserts (e.g., Namib Desert: Fig. 13). The presence of
plants makes it reasonable to infer that some invertebrates in the area may not have relied
exclusively on detritus to survive but could also have been herbivorous (Fig. 12: flowing
energy from H to E). of the Botucatu Formation as ‘‘dwarf’’ when compared with the fauna of the older and
paleoenvironmentally wetter Guará Formation. It is possible that this could be due to
the lower primary productivity of the arid Botucatu paleodesert that would only support
viable populations of smaller herbivorous dinosaurs. We infer that the presence of these
small-sized herbivorous dinosaurs, less than 68 cm in height to the pelvic girdle, is evidence
that there was localized plant growth at least for some period in the region (Fig. 12H to F). This is supported by the fact that the home range of modern herbivorous animals increases
with increasing body size (e.g., ungulates: Ofstad et al., 2016), so these small ornithopods
with limited home range should not have fed far from where their tracks were found. Therefore, we can infer that the Botucatu paleodesert landscape could have had some
shrubs, similar to modern arid dune deserts (e.g., Namib Desert: Fig. 13). The presence of
plants makes it reasonable to infer that some invertebrates in the area may not have relied
exclusively on detritus to survive but could also have been herbivorous (Fig. 12: flowing
energy from H to E). Contrasting with most ‘‘dwarf’’ trackway producers, a trackway of a large dinosaur (3.6
m high and 5 m long) interpreted as that an ornithopod has been documented as well
(Fernandes, 2005; Fernandes & Carvalho, 2007; Francischini et al., 2015). Since the trackway
of only one animal was found and there are no trackways of associated predators, it is likely
to infer that there was no settled population of this animal in this location at the time. The
large ornithopod dinosaur that produced this trackway probably had an extensive home
range, encompassing habitats with different primary productivity to support their large
body size, and this trackway of an anomalously large animal for the environment would be
the record of an animal crossing a less suitable area, like modern desert-dwelling elephants
(Viljoen, 1989) and giraffes (Fennessy, 2009; Flanagan et al., 2016). Peixoto et al. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Paleoecology of the Botucatu desert Even though the slip-face occupies a small area of the erg, when
compared to the windward or interdune areas (in the case of ergs with well-spaced dunes),
and is a region of low plant growth, it has a high concentration of biomass per unit area
in the form of detritus, concentrated by the wind mainly at the base of the dunes (Seely
& Louw, 1980; Southgate, Masters & Seely, 1996). This detritus originates from adjacent
subenvironments, more conducive to plant growth where there is moisture (Seely & Louw,
1980; Southgate, Masters & Seely, 1996), and could even be carried by the wind from distant
locations, where the climatic regimes allow greater primary productivity (Robinson &
Seely, 1980). During prolonged periods of drought, with low primary productivity, the
dune slip-faces maintain high concentrations of biomass as detritus is deposited by the
wind, providing food for detritivores and letting them survive until periods of increased
availability of moisture and biomass, when they can proliferate (Southgate, Masters & Seely,
1996). Ornithopods are known to be primary consumers in several Cretaceous ecosystems
(Barrett, 2014), therefore, they likely assumed this role in the Botucatu paleodesert as well
(Fig. 12F). Based on trackways, Francischini et al. (2015) interpreted the dinosaur fauna Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 22/37 Figure 13 Kahani Dunes in the arid Namib Desert showing some plant growth (Stipagrostis sabuli-
cola). Photography courtesy of Oliver Halsey, 2016. Full-size
DOI: 10.7717/peerj.8880/fig-13 Figure 13 Kahani Dunes in the arid Namib Desert showing some plant growth (Stipagrostis sabuli-
cola). Photography courtesy of Oliver Halsey, 2016. Full-size
DOI: 10.7717/peerj.8880/fig-13 of the Botucatu Formation as ‘‘dwarf’’ when compared with the fauna of the older and
paleoenvironmentally wetter Guará Formation. It is possible that this could be due to
the lower primary productivity of the arid Botucatu paleodesert that would only support
viable populations of smaller herbivorous dinosaurs. We infer that the presence of these
small-sized herbivorous dinosaurs, less than 68 cm in height to the pelvic girdle, is evidence
that there was localized plant growth at least for some period in the region (Fig. 12H to F). This is supported by the fact that the home range of modern herbivorous animals increases
with increasing body size (e.g., ungulates: Ofstad et al., 2016), so these small ornithopods
with limited home range should not have fed far from where their tracks were found. Paleoecology of the Botucatu desert (2020), PeerJ, DOI 10.7717/peerj.8880 23/37 The next trophic level includes animals from various phylogenetic groups that feed
on detritivores and herbivores, therefore, they are carnivores and omnivores (secondary
consumers). In the Botucatu Formation, this would include arachnids such as scorpions
and spiders (Fig. 12D), the producer of Paleohelcura araraquarensis isp. nov. if it had been
a predator or an omnivore feeding on animal biomass, small mammaliaform organisms
that produced Brasilichnium elusivum and B. saltatorium (Fig. 12C), and small theropod
dinosaurs (Fig. 12B). Due to the high connectivity of the trophic web, there would be
interactions among the representatives of this level. Trophic interactions would have been
limited by the ability of an animal to prey upon another, which is linked to the size of the
prey. Therefore, the animals grouped here probably preyed on smaller animals from the
same trophic level or even eggs and juveniles of larger animals. The existence of predatory
arachnids in the Botucatu paleodesert does not preclude the existence of predatory insects
because this coexistence is observed in modern dune deserts (e.g., Negev: Filser & Prasse,
2008). Brasilichnium elusivum is the most common vertebrate trackway recorded in the
Botucatu Formation (Leonardi, Carvalho de & Fernandes, 2007) and is a typical ichnotaxon
in other Mesozoic paleodeserts around the world (Krapovickas et al., 2016; Xing et al.,
2018). Lizards and snakes that could have occupied the same trophic level are common
inhabitants of all modern hot deserts (Cloudsley-Thompson, 1991; Whitford, 2002, p. 129),
but no records of these types of animals have been found in the Botucatu Formation. Snakes appeared during the Middle Jurassic (Caldwell et al., 2015), and may not have
been widespread in desert ecosystems during the time of the Botucatu paleodesert (Late
Jurassic—Early Cretaceous). There are a few records of lizard-like tracks in paleodeserts
from the Permian (Loope, 1984; Haubold et al., 1995; Lockley & Hunt, 1995; Lockley et al.,
1998). Tracks assigned to lizards (‘‘lacertoids’’) generally show digit impressions with an
inturned ‘‘comb’’ of curved digits, and pes tracks that are longer than wide (Hunt & Lucas,
2006), however, these were not found in the Botucatu Formation. The top trophic level is hard to define due to the trophic generalism of desert animals. Paleoecology of the Botucatu desert This level is represented by animals that, because of their size, would be able to prey
on non-detritivorous animals, like the producer of Brasilichnium elusivum, arachnids and
small herbivorous dinosaurs (ornithopods), as well as the small detritivorous animals. Such
organisms would include the larger theropod dinosaurs reported from trackways in the
Botucatu Formation (Francischini et al., 2015) (Fig. 12B), and mammaliaform producers
of Brasilichnium anaitti, which are larger than the producers of Brasilichnium elusivum and
B. saltatorium (Fig. 12A). Implications for the definition of the Octopodichnus-Entradichnus
Ichnofacies of eolian environments Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Implications for the definition of the Octopodichnus-Entradichnus
Ichnofacies of eolian environments Two invertebrate eolian ichnofacies were defined independently, the Octopodichnus
Ichnofacies of Hunt & Lucas (2007) and the Entradichnus Ichnofacies of Ekdale, Bromley &
Loope (2007). Subsequently, they were both integrated as the Octopodichnus-Entradichnus
Ichnofacies (Buatois & Mángano, 2011, p. 78; Krapovickas et al., 2016; Buatois & Echevarría,
2019). In particular, Krapovickas et al. (2016) suggested that this ichnofacies is characterized Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 by (1) low to rarely moderate trace fossil diversity, (2) dominance of simple sub-superficial
dwelling traces produced mostly by coleopterans, orthopterans and arachnids, with
horizontal (e.g., Palaeophycus) and/or vertically oriented dwelling burrows (e.g., Skolithos,
Digitichnus); (3) superficial locomotion traces produced by arthropods, especially arachnids
(e.g., Octopodichnus, Paleohelcura); and (4) subordinate simple (Planolites) and meniscate
(e.g., Taenidium, Entradichnus) feeding burrows. The Chelichnus Ichnofacies of Hunt &
Lucas (2007) is considered the archetypal vertebrate ichnofacies of eolian environments. The combined analysis of sedimentary facies and variations in the occurrence, abundance,
and diversity of trace fossils may allow differentiation among hyper-arid, arid and semi-arid
deserts (Krapovickas et al., 2016). Like ichnofacies from other continental environments, arid eolian environments display
recurrence in the characteristics of their ichnocoenoses, which represent behavioral
convergence as a response to abiotic features of habitat and substrate (Buatois & Mángano,
2011). There is also recurrence of certain tracemakers in arid eolian environments, such as
scorpions and spiders, which can produce Paleohelcura and Octopodichnus (Brady, 1947;
Davis, Minter & Braddy, 2007), both common ichnotaxa in arid deserts (Krapovickas et al.,
2016). The Octopodichnus Ichnofacies is dominated by arthropod trackways and was based on
the study of Permian ichnoassemblages, illustrated by the Coconino Sandstone (Hunt &
Lucas, 2007). In contrast, the Entradichnus Ichnofacies is characterized as dominated by
simple shallow vertical and horizontal burrows, as well as meniscate trace fossils, having
been based on the study of Jurassic examples, in particular the Navajo Sandstone (Ekdale,
Bromley & Loope, 2007). It has been noted that the diverging characterization of these
ichnofacies was the result of the disparate databases (Buatois & Mángano, 2011; Krapovickas
et al., 2016). Undoubtedly, the apparent contrasting nature of Paleozoic and post-Paleozoic
eolian ichnofaunas has been detrimental to a unifying approach to ichnofacies definition. In this regard, the presence of arthropod trackways in Mesozoic eolian deposits (like P. araraquarensis isp. Implications for the definition of the Octopodichnus-Entradichnus
Ichnofacies of eolian environments nov.) helps to trace a continuity between Paleozoic and post-Paleozoic
desert ichnofaunas, further reinforcing the notion of a single Octopodichnus-Entradichnus
Ichnofacies for eolian deposits. Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 CONCLUSIONS Paleohelcura araraquarensis isp. nov. is characterized by elliptical tracks and a narrow
internal width to the trackway. Despite being included in Paleohelcura, an ichnogenus
usually attributed to arachnids, Paleohelcura araraquarensis isp. nov. was most likely
produced by a pterygote insect on the basis of neoichnological observations. The producer
of Paleohelcura araraquarensis isp. nov. could have occupied one of the ecological roles
that different species of Pterygota are capable of performing in modern dune deserts. It could have been a herbivore, or a carnivore (like carnivorous Carabidae in some
modern dune deserts) or been part of the fauna of omnivores, being able to adopt
herbivorous, carnivorous, and saprophagous diets when opportune. As an omnivore,
like the abundant Tenebrionidae beetles in some modern dune deserts, it could have been Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 capable of relying on organic particles that accumulated among the sand grains of the dunes
during dry periods with no plant growth. The presence of Taenidium isp. and Skolithos
linearis burrows suggests that the Botucatu paleodesert would have had a detritivorous
fauna like modern dune deserts, with the producers of these burrows having a compatible
size with the plausible producers of Paleohelcura araraquarensis isp. nov. Based on the interpretation of the ichnofossil producers, it was possible to reconstruct
the food web of this paleodesert, in which the producer of Paleohelcura araraquarensis
isp. nov. could have been a primary consumer if it were a herbivorous or an omnivorous
detritivorous insect, or a secondary consumer if it had been produced by predatory
insects or omnivores relying on animal biomass. To date, Paleohelcura araraquarensis
isp. nov. is only known from the Botucatu Formation, but the presence of such types
of arthropod trackways in Mesozoic eolian deposits helps to trace a continuity between
Paleozoic and post-Paleozoic desert ichnofaunas, further reinforcing the notion of a single
Octopodichnus-Entradichnus Ichnofacies for eolian deposits. ACKNOWLEDGEMENTS We thank R.E. Ritzmann for providing high-speed videos of cockroaches walking and
authorizing their use (for Fig. 9 and in Video S1); Dr. Stanislav N. Gorb for helpful
discussions regarding insect and arachnid foot morphology; Silvina de Valais for provided
photos of holotype and other specimens of Hexapodichnus casamiquelai; Emma Rainforth
for sharing her thesis; Martin Harvey, Oliver Halsey, Ravi Sampaio for sharing their
photographs for use in this publication; Ceri Thomas, Frederic Wierum, Gareth Monger,
Nobumichi Tamura, Rafael Pasini for letting us modify and publish their drawings in this
paper under CC-BY-SA 4.0; Ana Maria Ribeiro for letting us modify and publish the Fig. 1 map published in Revista Brasileira de Paleontologia. We are also grateful to the PeerJ
reviewers Verónica Krapovickas and Anthony Shillito, and the PeerJ editor Claudia A. Marsicano for their comments that helped to improve this paper. Funding This work was funded by the National Council for Scientific and Technological
Development (CNPq) of Brazil with a Master Degree grant to Bernardo de Campos
Pimenta e Marques Peixoto (process No. 133988/2015-5) and a Ph.D. Degree grant
(process number 141220/2018-0) to Bernardo de Campos Pimenta e Marques Peixoto. PeerJ’s Article Processing Charge (APC) was funded by the PROAP-CAPES. There was
no additional external funding received for this study. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contributions • Bernardo de C.P.e M. Peixoto conceived and designed the experiments, performed the
experiments, analyzed the data, prepared figures and/or tables, authored or reviewed
drafts of the paper, and approved the final draft. • M. Gabriela Mángano and Nicholas J. Minter performed the experiments, analyzed the
data, authored or reviewed drafts of the paper, and approved the final draft. • Luciana Bueno dos Reis Fernandes performed the experiments, prepared figures and/or
tables, authored or reviewed drafts of the paper, and approved the final draft. • Marcelo Adorna Fernandes conceived and designed the experiments, performed the
experiments, analyzed the data, authored or reviewed drafts of the paper, and approved
the final draft. Grant Disclosures The following grant information was disclosed by the authors:
National Council for Scientific and Technological Development (CNPq). PROAP-CAPES. 26/37 Peixoto et al. (2020), PeerJ, DOI 10.7717/peerj.8880 Competing Interests Competing Interests The authors declare there are no competing interests. Data Availability The following information was supplied regarding data availability: The full resolution photographs of the quarry and the work fronts are available in Figs. S1 and S2. Indications of the series measured are available in Figs. S3 and S4. All the
measurements taken are available in Table S1. The cockroach video used to produce Fig. 9
is available in Video S1. All the slabs with the ichnofossils (LPP-IC-0028, LPP-IC-0029, LPP-IC-0030, LPP-IC-
0031, LPP-IC-0032, LPP-IC-0033, LPP-IC-0034, LPP-IC-0035) are housed in Laboratório
de Paleoicnologia e Paleoecologia, Departamento de Ecologia e Biologia Evolutiva,
Universidade Federal de São Carlos (UFSCar), São Carlos, São Paulo, Brazil. New Species Registration The following information was supplied regarding the registration of a newly described
species: PublicationLSID: urn:lsid:zoobank.org:pub:53C73174-4645-40E1-BB7B-75856AEAEAF Paleohelcura araraquarensis isp. nov. LSID: urn:lsid:zoobank.org:act:7D4303AE-BB63-
4474-B79C-AD39AB144917 Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.8880#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
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Comparing adherence to MDR-TB treatment among patients on self-administered therapy and those on directly observed therapy: non-inferiority randomized controlled trial
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© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Comparing adherence to MDR‑TB treatment
among patients on self‑administered therapy
and those on directly observed therapy:
non‑inferiority randomized controlled trial Clara Wekesa1* , Christine Sekaggya‑Wiltshire1, Stella Zawedde Muyanja1, Ivan Lume1,
Maria Sarah Nabaggala1, Rosalind Parkes‑Ratanshi1,2 and Susan Adakun Akello3 Abstract Background Adherence is key to the treatment success of multi-drug resistant tuberculosis (MDR-TB) and pre‑
vention of community transmission. Directly observed therapy (DOT) is the recommended approach for the man‑
agement of patients with MDR-TB. Uganda implements a health facility-based DOT approach where all patients
diagnosed with MDR-TB report to the nearest private or public health facility for daily observation of ingesting their
medicines by a health care provider. Directly observed therapy is very costly for both the patient and health care
system. It follows the assumption that MDR TB patients have a history of poor adherence to TB treatment. But only
21% of MDR-TB patients notified globally and 1.4–12% notified in Uganda had been previously treated for TB. The shift
to all oral treatment regimen for MDR-TB provides an opportunity for the exploration of self-administered therapy for
this group of patients even with use of remotely operated adherence technology. We are conducting a non-inferiority
open-label randomized controlled trial to compare adherence to MDR-TB treatment among patients on self-adminis‑
tered therapy (measured by Medication Events Monitoring System (MEMS) technology) with a control group on DOT. Methods We plan to enrol 164 newly diagnosed MDR-TB patients aged ≥ 8 years from three regional hospitals based
in rural and urban Uganda. Patients with conditions that affect their dexterity and ability to operate the MEMS-oper‑
ated medicine equipment will not be eligible to participate in the trial. Patients are randomized to either of the two
study arms: self-administered therapy with adherence being monitored using MEMS technology (intervention arm)
or health facility-based DOT (control arm) and will be followed up monthly. Adherence is measured by the number
of days the medicine bottle is open to access medication as recorded by the MEMS software in the intervention arm
and treatment complaint days as recorded in the TB treatment card in the control arm. The primary outcome is the
comparison of adherence rates between the two study arms. Discussion The evaluation of self-administered therapy for patients with MDR-TB is important to inform cost-effec‑
tive management strategies for these patients. The approval of all oral regimens for the treatment of MDR-TB provides
an opportunity for innovations such as MEMS technology to support sustainable options for MDR-TB treatment
adherence support in low-resource settings. Trials Trials Trials Wekesa et al. Trials (2023) 24:326
https://doi.org/10.1186/s13063-023-07314-z Open Access Abstract *Correspondence:
Clara Wekesa
clara.wekesa@gmail.com
Full list of author information is available at the end of the article *Correspondence:
Clara Wekesa
clara.wekesa@gmail.com
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. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 10 Wekesa et al. Trials Wekesa et al. Trials (2023) 24:326 Trial registration Pan African Clinical Trials Registry, Cochrane #PACTR202205876377808. Retrospectively registered
on 13 May 2022
Keywords Multi-drug-resistant tuberculosis, Medication events monitoring system technology, Directly observed
therapy, Differentiated care, TB treatment adherence, TB treatment outcomes, Resource-limited settings, Sub-Saharan
Africa, TB drug PK levels Trial registration Pan African Clinical Trials Registry, Cochrane #PACTR202205876377808. Retrospectively registered
on 13 May 2022
Keywords Multi-drug-resistant tuberculosis, Medication events monitoring system technology, Directly observed
therapy, Differentiated care, TB treatment adherence, TB treatment outcomes, Resource-limited settings, Sub-Saharan
Africa, TB drug PK levels Keywords Multi-drug-resistant tuberculosis, Medication events monitoring system technology, Directly observed
therapy, Differentiated care, TB treatment adherence, TB treatment outcomes, Resource-limited settings, Sub-Saharan
Africa, TB drug PK levels Administrative information
Title
Comparing adherence to MDR-TB
treatment among patients on self-
administered therapy and those
on Directly Observed Therapy:
Non Inferiority Randomized
Controlled Trial (WISECAP Study)
Trial registration
Pan African Clinical Trials Registry,
Cochrane #PACTR202205876377808
on 13th May 2022
Protocol version
23.09.21 Version 1.2
Name and contact information
for the trial sponsor
This Trial is under funding by Janssen
Global Public Health, a division of
Janssen Pharmaceutica NV, under
grant number 1550786
Turnhoutseweg 30, B-2340 Beerse,
Belgium
Names, affiliations, and roles
of protocol contributors
Clara Wekesa1, Christine Sekaggya
Wiltshire1, Stella Zawedde Muyanja1,
Ivan Lume 1, Maria Sarah Nabagalla1,
Rosalind Parkes-Ratanshi1,2, Susan
Adakun Akello3
1. Infectious Diseases Institute, Mak‑
erere University
2. Roles and responsibilities:
committees Department of Public Health,
Cambridge University
3. TB unit, Mulago National Referral
Hospital
SA, CS, SZ, RPR and CW conceived
the study, participated in the pro‑
posal and protocol development. SN wrote up the statistical methods
of the study and IL contributed to
development of the study tools. All
authors read and approved the final
manuscript. R l
f
Th f
di
b d did
k Administrative information Background and rationale Multi-drug-resistant tuberculosis (MDR-TB), which is
resistance to both isoniazid and rifampicin, poses a chal-
lenge to TB control programmes globally because of the
risk of worse treatment outcomes including death com-
pared to drug-sensitive TB [1]. In 2020, 71% (2.1 of 3
million) of patients found to have pulmonary TB tested
positive for rifampicin resistance, a ten percent incre-
ment from the previous year [2]. Treatment success for
drug-resistant TB remains sub-optimal, below the World
Health Organization (WHO) target of 90%, increasing
risk for disease relapse and continued community trans-
mission. In Uganda, for the period between July 2019
and June 2020, 513 patients tested positive for rifampicin
resistance and or MDR-TB (RR/MDR-TB) and the treat-
ment success rate in the same period was 77.9% [3]. Global mortality resulting from MDR-TB remains high,
and more so among people co-infected with HIV/AIDS. Mortality rate among people living with HIV (PLHIV) is
20% in comparison to 15% among mono-infected MDR-
TB patients on treatment [4]. Observational studies
done in Uganda have reported a similar mortality rate of
16–18% among MDR-TB patients on treatment [4, 5].ff In an effort to counter the effects of non-adherence,
including poor treatment outcomes and transmission of
resistance strains, directly observed therapy (DOT) was
adopted as a strategy, where patients are observed as they
take their daily dose by either a community health care
worker, family member or health care worker. The obser-
vation may occur at a health facility when the patient
reports for their daily dose or may entail the health pro-
vider visiting the patient at their home premise. Uganda
implements a health facility-based DOT approach where
all patients diagnosed with MDR-TB report to the near-
est private or public health facility for DOT. Although
treatment success rates on patients treated using the
DOT strategy in Uganda have gradually improved from
70.6% in 2018 to 77.9% in 2020, these rates are still below
the 90% target set by the WHO. Factors that contribute
to low treatment success rates with DOT include lack of
financial resources to make daily clinic visits, poor road
infrastructure, long distances to nearest health facility,
insufficient and overworked health care workers, and
overburdened facilities [6, 7]. Therefore, innovations that Role of sponsor The funding body did not take part
in the conception of the experiment
and will be independent of the study
conduct, analysis and interpretation
of the results. The funding body did
not participate in the write-up of this
manuscript. The Data Monitoring Board and the
Trial Steering Committee will make
recommendations concerning the
conduct of the trial based on periodi‑
cally shared data progress reports
and interim results. The independent
Trial Monitor will make recommen‑
dations of the adherence to study
protocol and operating procedures
as well as ensuring participant safety
during the course the study. Wekesa et al. Trials (2023) 24:326 Wekesa et al. Trials (2023) 24:326 Page 3 of 10 outcomes between MDR-TB patients on self-adminis-
tered therapy and standard DOT can support remote monitoring of MDR-TB treatment
should be explored to reduce time and financial costs. A
systematic review on strategies to improve adherence to
MDR-TB treatment demonstrated that community DOT
had varied effectiveness and that barriers to achieving
adherence are patient-based; there is therefore a need to
develop personalized interventions [8].h Methods We used the SPIRIT reporting guidelines in reporting
our study methods [11]. Trial designh This study will be conducted as a multi-centre open-
label randomized clinical trial with two parallel arms
and 1:1 enrolment ratio (82 patients in the intervention
arm and 82 controls). We shall enrol a total of 164 par-
ticipants newly diagnosed with pulmonary MDR-TB or
rifampicin-resistant TB (RR-TB) from 3 TB tertiary care
centres from 2 regions of the country. The Medication Events Monitoring System (MEMS)
(AARDEX®) is a portable smart medication device
designed to monitor treatment adherence by transmit-
ting electronic information on the number of times the
medicine cap is opened, specifying dates and times. This
information is taken to correlate with actual times that
patients ingest their medication and can be used to vali-
date the accuracy of self-reported adherence. The device
is operated by a long-lasting battery and is able to trans-
mit software information in real time and store infor-
mation for many months following battery outage. The
information on patient non-adherence as captured by
MEMS technology has been shown to accurately corre-
late with treatment outcomes among people living with
HIV [9]. In addition, the device has also shown to out-
perform self-report or pill counts and is acceptable to
patients who found it easy to open and carry it on their
person [9, 10]. Explanation for choice of comparators For the purposes of this study, we shall be using patients
on standard of care (DOT), as the comparator group. This comprises patients with MDR-TB who have to
report daily to a DOTS facility to be observed as they
ingest their medication. DOT is the standard of care for
MDR-TB patients and presently the only alternative for
patient follow-up. Category 1—A bedaquiline-based injection-shorter
treatment regimen (STR) (9 to 12 months) reserved
for MDR-TB/RR-TB patients who have not been pre-
viously treated with second-line drugs and in whom
resistance to fluoroquinolones and second-line
injectable agents has been excluded. Drug options for
shorter treatment regimen include an intensive phase
of 4 months (extended to 6 months in case of delayed
sputum smear conversion) containing high-dose gati-
floxacin or moxifloxacin, kanamycin, prothionamide,
clofazimine, high-dose isoniazid, pyrazinamide, and
ethambutol followed by a continuation phase of 5
months containing gatifloxacin or moxifloxacin, clo-
fazimine, ethambutol and pyrazinamide [12]. Diagnosis and management of MDR‑TB Diagnosis of MDR-TB is based on Gene Xpert® testing of
sputum for identification of resistance to rifampicin (RR-
TB) followed by culture and drug susceptibility testing
(DST) for phenotypic characterization; however, treat-
ment is started prior to the receipt of culture and sensitiv-
ity results due to long turnaround time of 10–16 weeks. Treatment options for MDR-TB/RR-TB currently include: Study setting
Th
d This study will be conducted in Mulago National Referral
Hospital in the central region and Lira regional referral
hospital in the northern region. These hospitals regis-
ter the highest number of MDR-TB patients in Uganda. Both these hospitals are public government hospitals not
for profit. The hospitals house specialized TB wards and
clinics, operating a weekly TB clinic to follow up patients
with drug-sensitive and drug-resistant TB on therapy and
to initiate therapy for newly diagnosed patients. Being
referral hospitals, these facilities receive patients referred
from health facilities within a radius of up to 30km and in
some cases even further. We plan to conduct an open-label randomized non-
inferiority clinical trial among patients receiving MDR-
TB treatment in Uganda to compare adherence rates
among those using self-administered therapy with
MEMS technology vs DOT, which is the current standard
of care in Uganda for MDR-TB. We will correlate adher-
ence information with serum drug concentrations and
compare determined treatment outcomes between the
two groups. Specific objectivesh The primary objective of the study is to determine if
adherence to MDR-TB treatment among patients on
self-administered therapy (measured by MEMS technol-
ogy) is non-inferior to that among patients on directly
observed therapy (DOT). The secondary objectives of the
study include determining the correlation between MDR-
TB drug serum concentrations and adherence as meas-
ured by MEMS technology and comparing treatment Wekesa et al. Trials (2023) 24:326 Wekesa et al. Trials (2023) 24:326 Wekesa et al. Trials (2023) 24:326 Page 4 of 10 Category 2—The longer treatment regimens (18 to
20 months) based on core backbone agents but indi-
vidualized depending on age, fluoroquinolone resist-
ance, and site of disease. patterns of missed medication and brainstorming for
solutions with the patient (Table 1). Determination of TB treatment outcomes At the end of therapy treatment (9 months for partici-
pants that qualify for shorter MDR-TB/RR-TB treatment
regimen and 18 months for those selected for the longer
MDR-TB/RR-TB treatment regimen), clinical and labora-
tory information is routinely compiled by each MDR-TB
centre and forwarded to the National TB Program (NTP)
review committee for discussion on consensus on treat-
ment outcome based on a specified criterion (Table 2). TB treatment outcomes for this study will be as deter-
mined by the review committee. Adherence to interventions Participants on the intervention arm will be given
monthly study visits in which the adherence software
shall be read to determine information on use of the
medicine cap and box. Based on the information dis-
played, study personnel provide tailored adherence coun-
selling to the participants. For participants below the age
of 18 and under guardianship, their caretakers will be
included in the adherence counseling sessions. Study outcomes
O
i Our primary outcome is the adherence rates among
MDR-TB patients on self-administered therapy vs
DOT. Our secondary outcomes include (1) drug con-
centrations and adherence data for patients on self-
administered therapy vs DOT; (2) proportion of
patients with sputum conversion at 2, 8 and 11 months
in each study arm; and (3) MDR-TB treatment out-
comes (cure, treatment completion, loss to follow-up
or death) by study arm. Intervention arm For this trial, we shall use two kinds of MEMS-oper-
ated devices, the MEMS cap and the MEMS Electronic
DosePak® (EDP). At enrolment, patients on the self-
administering arm will be provided with both devices;
the MEMS cap will be placed over a medicine bottle
containing bedaquiline. The concomitant medication
will be packaged in a convenient pack containing all tab-
lets to be swallowed each day and placed into the medi-
cation box with the EDP® technology Patients will be
instructed on how to use the MEMS devices. Each time
a patient opens up the cap over the bedaquiline bottle
or the lid of the medicine box, the time and date of this
event will be immediately captured. Adherence data from
the MEMS devices will be downloaded by the study team
every month during the routine visits. The devices will
provide information on specific calendar dates and times
when the devices were opened. Information on the cal-
endar days with event markings to signify the date and
time when medication containers were opened will be
displayed on a computer. The software will contain two
separate input calendars for both the MEMS cap and
the EDP box for each participant. This same adherence
data will be shared with primary clinician and used to
tailor adherence counselling given to patients, exploring Interventions: modifications Participants may withdraw from the study at any time at
their own request, or they may be withdrawn at any time
at the discretion of the investigator or sponsor for safety
or behavioural reasons, or the inability of the subject
to comply with the protocol required schedule of study
visits or procedures. Patients who are withdrawn at any
time of the study may be replaced. According to standard of care, patients with MDR-TB
are managed as in-patients at central MDR treatment
facilities (which include Mulago and Lira hospitals) in
the intensive phase of treatment after which they are
discharged and required to report to the central MDR
treatment facility monthly for review and drug refills. Following discharge, during the continuation phase,
patients are required to report daily to the nearest private
or public facility registered under DOT programme [13]. Monthly reviews during routine care include safety labo-
ratory investigations including a full blood count, liver
and renal function tests, sputum culture, DST and adher-
ence counseling. Eligibility criteria We shall enrol consented patients with newly diagnosed
pulmonary MDR-TB and RR-TB patients aged ≥8 years,
initiated on oral MDR-TB therapy (category 1 or 2). Patients with any condition that inhibits the use of the
MEMS device, e.g. dexterity or debilitating arthritis, will
be excluded from participation. Control: DOT No evidence of clinical deterioration
Treatment completed (success)
1. A patient who has culture converted and
2. Received treatment for a total duration of 9–11 months and
3. Has less than 3 consecutive negative TB cultures at least 30 days apart
and
4. No evidence of clinical deterioration
Loss to follow-up
A patient with treatment interrupted for 2 consecutive months and more
Treatment failure
1. A patient who failed to culture convert by month 5 (shorter regimen) or
month 6 (longer regimen) or
2. In the initial 6 months of treatment more than 2 of 5 cultures are positive
or
3. Treatment stopped due to adverse events
4. Permanent discontinuation of two Group A drugs (BDQ, LZD, or LFX) in
the first 6 months of the modified shorter regimen or
5. More than 2 new drugs added because of poor clinical response or
6. Panel decision to terminate any further DR-TB treatment
Died
A patient who died for any reason during treatment Definition Time from initiation of treatment to the time point where the patient has
two consecutive negative cultures
1. A patient who has culture converted and
2. Received treatment for a total duration of 9–11 months and
3. Has at least 3 consecutive negative TB cultures at
least 30 days apart and
4. No evidence of clinical deterioration
1. A patient who has culture converted and
2. Received treatment for a total duration of 9–11 months and
3. Has less than 3 consecutive negative TB cultures at least 30 days apart
and
4. No evidence of clinical deterioration
A patient with treatment interrupted for 2 consecutive months and more
1. A patient who failed to culture convert by month 5 (shorter regimen) or
month 6 (longer regimen) or
2. In the initial 6 months of treatment more than 2 of 5 cultures are positive
or
3. Treatment stopped due to adverse events
4. Permanent discontinuation of two Group A drugs (BDQ, LZD, or LFX) in
the first 6 months of the modified shorter regimen or
5. More than 2 new drugs added because of poor clinical response or
6. Panel decision to terminate any further DR-TB treatment
A patient who died for any reason during treatment Time to culture conversion Treatment completed (success) Died A patient who died for any reason during treatment Recruitment In order to enrol the required sample size in the stipu-
lated time frame of the study, we shall enrol from sites
with high volume numbers of MDR-TB in the country,
which include Mulago National Referral Hospital and
Lira Regional Referral Hospital. We shall also endeav-
our to enrol all eligible participants at each clinic day at
both sites. Randomization and allocation of intervention for the patients using MEMS technology is ≥ 70%, with
study power of 0.9, precision of 0.05, and 20% loss to
follow-up rate. We therefore require a sample size of 164
patients (82 in each arm) to demonstrate a 19% incre-
ment in MDR-TB treatment success. The intervention
(MEMS technology) approach is considered non-inferior
(19%) to the control (DOT) approach if the lower limit of
the 95%CI of the difference between the two groups (i.e. MEMS group minus DOT group) is > −10%. Blinded randomization will be performed by the study
team (medical officer or study nurse) at the time of enrol-
ment using an automated randomization sequence within
the Redcap data capture tool. The randomization will be
done in real time on the Redcap tool as it will be Inter-
net enabled. Participants will be randomized to either
intervention arm (arm 1; self-administered therapy) or
control arm (arm 2; DOT). Stratified randomization will
be performed according to age group and gender. Due to
the nature of the intervention and organization of patient
care, it will not be possible to blind either participants or
study staff to the study arm allocation Control: DOT Device: MEMS CAP without LCD display and EDP® technology on medicine box. Standard MEMS devices record the time and date when the bottle is opened
The MEMS technology automatically complies drug dosing history information by electronically recording
the date and time of each opening of the medication cap and or box
Behavioural: Adherence counselling. Patients will receive adherence counselling as scheduled by their
primary care health providers Patients report daily to the nearest health
unit to be observed by a health care pro‑
vider as they ingest their medication
Behavioural: Adherence counselling. Patients will receive adherence counsel‑
ling as scheduled by their primary care
health providers Table 2 Description of MDR-TB treatment outcomes
Outcome
Definition
Time to culture conversion
Time from initiation of treatment to the time point where the patient has
two consecutive negative cultures
Cured
1. A patient who has culture converted and
2. Received treatment for a total duration of 9–11 months and
3. Has at least 3 consecutive negative TB cultures at
least 30 days apart and
4. No evidence of clinical deterioration
Treatment completed (success)
1. A patient who has culture converted and
2. Received treatment for a total duration of 9–11 months and
3. Has less than 3 consecutive negative TB cultures at least 30 days apart
and
4. No evidence of clinical deterioration
Loss to follow-up
A patient with treatment interrupted for 2 consecutive months and more
Treatment failure
1. A patient who failed to culture convert by month 5 (shorter regimen) or
month 6 (longer regimen) or
2. In the initial 6 months of treatment more than 2 of 5 cultures are positive
or
3. Treatment stopped due to adverse events
4. Permanent discontinuation of two Group A drugs (BDQ, LZD, or LFX) in
the first 6 months of the modified shorter regimen or
5. More than 2 new drugs added because of poor clinical response or
6. Panel decision to terminate any further DR-TB treatment
Died
A patient who died for any reason during treatment Table 2 Description of MDR-TB treatment outcomes
Outcome
Definition
Time to culture conversion
Time from initiation of treatment to the time point where the patient has
two consecutive negative cultures
Cured
1. A patient who has culture converted and
2. Received treatment for a total duration of 9–11 months and
3. Has at least 3 consecutive negative TB cultures at
least 30 days apart and
4. Sample size Based on records from the NTP, treatment success rate
for MDR-TB is 77.9%; from clinical trials, adequate
adherence has been defined as 76–80% adherence rate;
and from a meta-analysis, it has shown that 70% of
patients on MDR-TB therapy are adherent [14, 15]. Our
sample size calculation assumed that the adherence rate Page 5 of 10 Wekesa et al. Trials (2023) 24:326 Wekesa et al. Trials (2023) 24:326 Wekesa et al. Trials (2023) 24:326 Table 1 Description of study arms Allocation concealment mechanismh The designing of the randomization sequence within
the Redcap tool was performed by a biostatistician who
is blinded from the participant enrolment activities. The
variables used to design the randomization sequence
included gender and age group. At the stage of enrol-
ment, the randomization is conducted in real time on the
Internet-enabled Redcap tool by the member of the study
team conducting the enrolment. The study team have no
control over the randomization sequence. Control arm Patients randomized to the control arm will receive daily
DOT provided by a peripheral health facility close to
their residence. Under the DOT programme, patients Wekesa et al. Trials (2023) 24:326 Wekesa et al. Trials (2023) 24:326 Page 6 of 10 report to the nearest DOT provider facility daily and drug
intake is observed. On each visit, the TB card is ticked off
in correspondence with the number of days they made a
visit to the health centre and this card is retained at the
health facility. At the monthly visits, the TB card is taken
to the central MDR treatment facility for review by the
managing clinician. At this point, the study team will
record the number of doses taken/missed according to
the TB card. Participants will receive adherence counsel-
ling on all routine visits at the central facility (Table 1). daily), it was not feasible to blind study participants, pri-
mary care providers, the study team and or the data
manager. report to the nearest DOT provider facility daily and drug
intake is observed. On each visit, the TB card is ticked off
in correspondence with the number of days they made a
visit to the health centre and this card is retained at the
health facility. At the monthly visits, the TB card is taken
to the central MDR treatment facility for review by the
managing clinician. At this point, the study team will
record the number of doses taken/missed according to
the TB card. Participants will receive adherence counsel-
ling on all routine visits at the central facility (Table 1). Data collection plan All participants will be reviewed by the study team
monthly during routine visits (Table 3). On the ini-
tial visit, participants will be taken through a screen-
ing questionnaire to assess eligibility. Patients found
to be eligible will then be taken through the informed
consent process in the language of their preference. Enrolled participants will provide demographic infor-
mation including age and gender. At each monthly
study visit, participants will be taken through an
interviewer-led questionnaire to capture clinical his-
tory (reported adverse events, treatment outcomes)
and adherence information (adherence data down-
loads from MEMS devices, adherence data from TB
treatment cards, pills counts, and participant-reported
adherence on days of treatment missed). Adherence
data will be used to provide tailored counselling for
each participant to encourage treatment completion
and this data will be shared with the participant’s pri-
mary clinician. Laboratory assessments Blood sampling to measure the concentrations of
bedaquiline and linezolid will be conducted on months
2, 8 and 11 of treatment. Drug intake on the days for
blood sampling will be performed by DOT and blood
samples taken off prior to drug intake and 1-h post-drug
intake. The blood samples will be placed in cooler boxes Allocation implementationhfi The study team (medical officer and or study nurse) hav-
ing determined participant eligibility enter the partici-
pant gender and age into the automated randomization
sequence within the Redcap tool and click a button to
determine study allocation and or study arm in real time. * Drug susceptibility testing: sputum culture and sputum DST will be provided as standard of care by the health facility Statistical: outcomes
Adherence assessment
Th
i The study follow-up duration will be 24 months to cater
for patients on both short- and long-term oral MDR-
TB therapy. Treatment duration will be approximately
9 to 18 months: 9 months for participants that qualify
for shorter MDR-TB/RR-TB treatment regimen and 18
months for those selected for the longer MDR-TB/RR-TB
treatment regimen. The primary outcome is the overall adherence rates
among MDR-TB patients on self-administered therapy
and on DOT. For the participants on the intervention
arm, opening of the devices will be equated to a patient
swallowing their medication and adherence will be
derived from the number of days the device was opened
as a proportion of the total number of days in the entire
period of their MDR-TB treatment. Patients on the
intervention arm will be required to take no less than
70% of their medication to be considered as having good
adherence. For participants on the control arm (DOT),
adherence information will be obtained from their TB
treatment cards. Adherence rates will be calculated
from the number of days that they report to the DOTS
facility as a proportion of the total number of treatment
complaint days in the entire period of their MDR-TB
treatment. Participants who take their medication at
least 80% of the time (missing no more than 5 days each
month) will be considered as having good adherence. To ensure data quality, study information is captured
with an e-questionnaire in Redcap that has been designed
to send real-time notifications for inputted variables that
may be out of range. The tool also does not accept for
the person collecting data (medical officer and or study
nurse) to verify the tool, but is redirected to the study
coordinator and Data Manager to do the final verification
for correct and missing data. In addition, the entire study
team was trained afore on the use of the study Redcap
tool and on other study-related standard operating pro-
cedure manuals including the collection of blood speci-
mens for continued reference. In the event of a protocol
violation, retraining on study protocol and procedures
will be conducted. The primary analysis will be a comparison of overall
adherence in each arm. Adherence by self-administra-
tion (proportion of expected openings of the MEMS
devices) and DOT (proportion of DOT visits) will also
be compared at 2 months and 6 months in the interven-
tion and control arms. Retention of study participants in the Trial For all persons approached to participate in the study,
full information will be provided about the trial its
aims, study procedures and duration. Participants will
be given an opportunity to ask any questions they may
have at the time of consent and any other time of the
study. Data from participants that have withdrawn and
or been withdrawn from the study will be retained for
sub-analysis. All participants that have been withdrawn
from study participation will be replaced to ensure the
sample size is achieved. Data management Clinical data will be entered into a study-specific data-
base by trained designated study staff on a regular basis
from completed Case Record Forms (CRF) using Red-
cap. Information will primarily be captured using Red-
cap and CRFs will be printed out for filing and storage. Blinding (masking) Given the nature of the intervention (a visible medicine
box and the requirement not to report to a DOT facility Table 3 Description of study procedures
* Drug susceptibility testing: sputum culture and sputum DST will be provided as standard of care by the health facility Table 3 Description of study procedures Wekesa et al. Trials (2023) 24:326 Page 7 of 10 Case Record Forms and other source documents will
be kept in locked cabinets. No participant-identifying
information will be disclosed in any publication or at
any conference activities arising from the study. The
participant data captured in the Redcap tool will be
accessed in real time by the data manager, who will
provide data queries for the study team to address. In
addition, the Redcap data collection tool has been fash-
ioned to generate automated checks on entry of certain
variables that are out of range. Data management pro-
cedures are detailed in the standard operating manual. and transported to the laboratory on dry ice for samples
from Lira Hospital and in a cooler box for samples from
Mulago Hospital, processed and stored at −4 to −80
°C. Pharmacokinetic analysis will be performed at the
Infectious Diseases Institution (IDI) translation labora-
tory. Quantification of the drug concentrations will be
performed in batches using validated high-performance
liquid chromatography (HPLC) and mass spectrometry
at the Translation Laboratory of the Infectious Diseases
Institute, Makerere University using MS. Sputum smears and cultures will be performed as part
of routine care and testing conducted at the government
reference laboratory.h Statistical: outcomes
Adherence assessment
Th
i The chi-square test and 1-way
analysis of variance models will be used to compare out-
comes by randomization arm. We shall use independ-
ent samples t-test to test for statistical difference mean
adherence rates in the patient in the intervention and
control arms. We will use an intent-to-treat approach
(analysed all participants regardless of drug refills) to
compare adherence outcomes by randomization arm
using a generalized estimating equations model using
repeated measures with identity link, exchangeable cor-
relation structure and robust variance estimates. Data monitoring: formal committeeh Data monitoring: formal committeeh Harms All observed or volunteered adverse events (AEs) regard-
less of treatment group or suspected causal relationship
will be reported. During each study visit, the study doc-
tors will assess AEs that may have occurred since the pre-
vious visit. The severity of adverse events will be graded
according to the National Institute of Health Division of
AIDS (DAIDS) classification system for reporting adverse
experiences in adults [16]. Additional analyses and handling of missing data y
g
g
Interim data analysis will be conducted once 50% of the
overall sample size has been achieved with the purpose of
guiding the data monitoring board on recommendations
for the study. Missing data will be dealt with as missing
covariate and outcome data. As a generalized estimating
equations model will be used, missing outcome data will
be dealt with using a missing at random assumption. If >
3% of data for covariate data is missing, multiple impu-
tation will be used for the primary analysis. Given the
way the adherence rate is computed, we anticipate that
there may be a number of ways that the outcome meas-
ure could be missing. These include loss to follow-up and
or partial information collected at anyone study visit. A
further analysis will be performed in which missing pri-
mary outcome data will be imputed. Multiple imputation
will be also performed on missing covariate and outcome
data for all secondary clinical analyses in a similar way to
that performed for the primary analysis. Auditing
d An independent study monitor will be assigned to the
study. The study monitor will perform at least two study
audits annually to assess adherence to study protocol,
SOPs, observe for study deviations and integrity of con-
duct of the study. The study monitor will provide a writ-
ten report of their findings and recommendations after
each study visit. Secondary outcomes To assess correlation of specific MDR-TB drug serum
concentrations with observed adherence, bedaquiline Wekesa et al. Trials (2023) 24:326 Page 8 of 10 Page 8 of 10 Steering Committee. The DMB is chaired by Dr. Simon
Kasasa Senior Lecturer, in the Department of Epidemiol-
ogy & Biostatistics at Makerere University School of Public
Health in the College of Health Sciences, and other mem-
bers include Dr. Suzanne Kiwanuka, a review Coordinator
for Centre for Systematic Reviews on Human Resources
for Health; Dr Paul Otuba, National MDR-TB Coordina-
tor at the NTP; and Dr. Dathan Byonanebye, a lecturer in
the Department of Community Health and Behavioural
Sciences, Makerere University. The DMB is independent
from the sponsor and has no competing interests. and linezolid pre-dose concentrations (Ctrough) will be
used. To cater for those who may be adherent but have
low bedaquiline or linezolid concentrations, we will use
of cut-off value of bedaquiline Ctrough from previous stud-
ies, below which a participant will be classified as non-
adherence. Linezolid will be used to assess more recent
adherence than bedaquiline. We will compare time to sputum culture conversion
in the intervention and control arm using time to event
analysis. We shall also use chi-square testing to com-
pare treatment success rates in the intervention and
control arms. Treatment success will be evaluated using
a composite outcome that includes TB cure (for micro-
biologically diagnosed patients) and TB treatment com-
pletion (for clinically diagnosed TB). We will undertake
per-protocol and intention to treat analyses to compare
TB cure rates in the two study arms. We will determine
the proportions of patients retained in care of retention
in care at 2 and 6 months in the two study arms using
chi-square tests. Study outcomes by randomization arm
will be compared using a generalized estimating equa-
tions (GEE) model using repeated measures with iden-
tity link, exchangeable correlation structure and robust
variance estimates. Adjusted coefficients along with their
corresponding 95% CIs will be generated. Statistical sig-
nificance will be set at the P < .05 level and statistical
analyses performed using Stata version 14 (StataCorp,
College Station, TX, USA). Data monitoring: Interim analysishi The first interim analysis will be conducted when 50%
of the participants complete the study medication. Trial
monitoring is conducted independent of the investiga-
tors by internal monitors at the Infectious Diseases Insti-
tute. The Trial Steering Committee is composed of Stavia
Turyahabwe, program manager at the NTP in Uganda;
Angom Esther, Nurse in charge at Lira Regional Referral
Hospital; Dr Agnes Kiragga, a senior statistician with expe-
rience in HIV clinical trials and cohort studies; and Eliza-
beth Tindyebwa, a lay representative from the Friends’
Council (patient group) at the Infectious Disease Insti-
tute, Uganda. Since this trial is unblinded, the DMB will
have access to intervention assignment. They will ensure
that we protect the safety of the participants while assur-
ing study integrity. The DMB will periodically (every 6
months) review the data, including interim analyses, and
communicate any concerns to the study team and TSC. Data access Access to the database will be given to authorized per-
sonnel only (study investigators, project coordinator and
data manager) and a log of authorized personnel will be
stored in the trial master file. Dissemination plans Dissemination workshops will be held with study par-
ticipants, funders and other stakeholders to commu-
nicate the results of the study. The study results will be
presented to officials in charge of the National TB Pro-
gram in Uganda and at other national and international
conferences. We shall also aim to publish in high-impact,
peer-reviewed journals with a focus on open access. Full
anonymity of participants’ details will be maintained
throughout. We shall adhere to the International Com-
mittee of Medical Journal Editor (ICMJE) recommenda-
tions on the definition and roles of authorship. We shall
not procure the services of professional writers. Declaration of interestsh The authors have no competing interests to declare. Ethical considerationsh The independent Data Monitoring Board (DMB) will
make recommendations concerning the study to the Trial The study has received approvals from the Mulago
Hospital Research and Ethics Committee (Ref No: Wekesa et al. Trials (2023) 24:326 Wekesa et al. Trials (2023) 24:326 Page 9 of 10 MHREC2125), the Uganda National Council for Science
and Technology (Ref No: HS1796ES) and the National
Drug Authority (RefNo:476/NDA/DPS/08/2021). The
informed consent/assent document are in compliance
with GCP and local regulatory authorities. Participants
are taken through the consent process by the study team
(Medical Officer or Study Nurse). Assent is requested
from children between 8 and 17 years, and in addition,
consent is sought from their parents or legally authorized
representative. Informed consent is sought from partici-
pants aged 18 years and above and emancipated minors. Approval for protocol amendments shall be submitted to
the Mulago Hospital Research and Ethics Committee for
approval and then communicated to the National Drug
Authority and The Uganda National Council for Science
and Technology before implementation. Participants will
be free to withdraw at any point of the study without any
compromise to receiving their standard therapy. Children
aged 8–17 years that do not provide assent but whose
guardians have signed consent or vice versa will not be
included in the study. Discussionh The introduction of new strategies like the use of shorter
course regimen still does not address the operational limi-
tations of DOT, including the unguaranteed accuracy
of patient adherence information. In as much as MEMS
technology may not be absolutely accurate in estimating
patient adherence since opening the device may not imply
drug ingestion, it does provide a patient-centred solution,
relieving the burden on patients to meet an obligation of
frequent clinic visits and maintaining patient autonomy
[17]. MEMS technology also has the potential to meaning-
fully engage both caregivers and patients via its interactive
functions such as LCDs on drug balances, reminders and
real-time electronic display of patient adherence informa-
tion. The adherence data abstracted from the MEMS tech-
nology enables caregivers to understand their patients’
adherence patterns and using this information, tailor coun-
selling information, a better approach to improving adher-
ence. The limitations of the MEMS technology include its
inability to capture patient records on taking more than
the required dosing and pill dumping [18]. The MEMS
technology may also overestimate adherence by being
unable to differentiate other instances in which the medi-
cine devices are opened simply out of curiosity and altered
behaviour as a result of being observed. The device may
also be associated with inconvenience (on person carriage)
and device fatigue which can negatively impact adherence
[9]. Despite these limitations, this is one of the few studies
that may address some of the facility and patient-based fac-
tors associated with non-adherence to MDR-TB therapy. It
may also provide a cheaper and sustainable alternative for
effective long-term follow-up of patients with MDR-TB
with favourable outcomes including completion of therapy. The addition of pharmacokinetic data further strengthens
adherence assessment in addition to the devices, patient
self-reports and pill counts. We believe this study will dem-
onstrate how MEMS technology may be used to remotely
assess and therefore strengthen adherence in patients with
tuberculosis for favourable treatment outcomes. Confidentialityf All study staff will ensure the protection of participant
personal data and will not include participant names
on any forms, reports, publications, or in any other
disclosures. Protocol Version: 1.2 23.09.2021
MRHEC Ref No: MHREC2125
UNCST Ref No: HS1796ES
NDA Ref No: 476/NDA/DPS/08/2021
Start date: 15.11.2021 Protocol Version: 1.2 23.09.2021
MRHEC Ref No: MHREC2125
UNCST Ref No: HS1796ES
NDA Ref No: 476/NDA/DPS/08/2021
Start date: 15.11.2021 Protocol Version: 1.2 23.09.2021
MRHEC Ref No: MHREC2125
UNCST Ref No: HS1796ES
NDA Ref No: 476/NDA/DPS/08/2021
Start date: 15.11.2021 Approximate date for completion of recruitment: 31.01.2023
Participant enrolment is at 70% and expected completion
is January of 2023. Page 10 of 10 Page 10 of 10 Wekesa et al. Trials (2023) 24:326 Wekesa et al. Trials (2023) 24:326 Abbreviations
BDQ
Bedaquiline
CRF
Case Report Form
DAIDS
National Institutes of Health Division of AIDS
DMB
Data Monitoring Board
DOT
Directly observed therapy
DR-TB
Drug-resistant tuberculosis
DST
Drug susceptibility testing
EDP
Electronic DosePak®
GCP
Good Clinical Practice
GEE
Generalized estimating equations
HF-DOT
Health facility-based directly observed therapy
HIV
Human immunodeficiency virus
HPLC
High-performance liquid chromatography
IDI
Infectious Diseases Institute
LCD
Liquid crystal display
LFX
Levofloxacin
LZD
Linezolid
MDR-TB
Multi-drug-resistant tuberculosis
MEMS
Medications Events Monitoring System
MHREC
Mulago Hospital Research Ethics Committee
NTLP
National Tuberculosis and Leprosy Program
PK
Pharmacokinetics
PLHIV
People living with HIV
RR-TB
Rifampicin-resistant tuberculosis
SAE
Severe adverse event
SMS
Short Messaging Service
WHO
World Health Organization 5. Baluku JB, Nakazibwe B, Naloka J, Nabwana M, Mwanja S, Mulwana R,
et al. Treatment outcomes of drug resistant tuberculosis patients with
multiple poor prognostic indicators in Uganda: a countrywide 5-year
retrospective study. J Clin Tuberc Other Mycobact Dis. 2021;23:100221. g
g
y
y
retrospective study. J Clin Tuberc Other Mycobact Dis. 2021;23:1002 6. Fiseha D, Demissie M. Assessment of Directly Observed Therapy (DOT)
following tuberculosis regimen change in Addis Ababa, Ethiopia: a quali‑
tative study. BMC Infectious Diseases. 2015;15(1):405. 7. Queiroz EM, De-La-Torre-Ugarte-Guanilo MC, Ferreira KR, Bertolozzi
MR. Tuberculosis: limitations and strengths of directly observed
treatment short-course. Revista latino-americana de enfermagem. 2012;20(2):369–77. 8. Pradipta IS, Houtsma D, van Boven JFM, Alffenaar JC, Hak E. Interventions
to improve medication adherence in tuberculosis patients: a systematic
review of randomized controlled studies. NPJ Prim Care Respir Med. 2020;30(1):21. 9. Shellmer DA, Zelikovsky N. The challenges of using medication event
monitoring technology with pediatric transplant patients. Pediatr Trans‑
plant. 2007;11(4):422–8. 10. Schoenthaler A, Ogedegbe G. Patients’ perceptions of electronic
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Americans. Ann Pharmacother. 2008;42(5):647–52. 11. Chan AW, Tetzlaff JM, Gøtzsche PC, Altman DG, Mann H, Berlin JA, et al. SPIRIT 2013 explanation and elaboration: guidance for protocols of clini‑
cal trials. BMJ (Clinical research ed). 2013;346: e7586. 12. Competing interests The authors declare that they have no competing interests. Received: 9 December 2022 Accepted: 17 April 2023 Received: 9 December 2022 Accepted: 17 April 2023 •
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? Choose BMC and benefit from: Authors’ contributions 17. Alipanah N, Jarlsberg L, Miller C, Linh NN, Falzon D, Jaramillo E, et al. Adherence interventions and outcomes of tuberculosis treatment: a
systematic review and meta-analysis of trials and observational studies. PLoS medicine. 2018;15(7): e1002595. 17. Alipanah N, Jarlsberg L, Miller C, Linh NN, Falzon D, Jaramillo E, et al. Adherence interventions and outcomes of tuberculosis treatment: a
systematic review and meta-analysis of trials and observational studies. PLoS medicine. 2018;15(7): e1002595. 18. Lyimo RA, van den Boogaard J, Msoka E, Hospers HJ, van der Ven A,
Mushi D, et al. Measuring adherence to antiretroviral therapy in northern
Tanzania: feasibility and acceptability of the Medication Event Monitoring
System. BMC Public Health. 2011;11(1):92. Protocol Version: 1.2 23.09.2021
MRHEC Ref No: MHREC2125
UNCST Ref No: HS1796ES
NDA Ref No: 476/NDA/DPS/08/2021
Start date: 15.11.2021 Prasad R, Gupta N, Banka A. Shorter & cheaper regimen to treat
multidrug-resistant tuberculosis: a new hope. Indian J Med Res. 2017;146(3):301–3. 13. WHO consolidated guidelines on drug-resistant tuberculosis treatment. Geneva; World Health Organisation; 2019. 13. WHO consolidated guidelines on drug-resistant tuberculosis treatment. Geneva; World Health Organisation; 2019. 14. Vernon A, Fielding K, Savic R, Dodd L, Nahid P. The importance of adher‑
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1 Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations. 1 Infectious Diseases Institute, Makerere University, Kampala, Uganda. 2 Depart‑
ment of Public Health, Cambridge University, Cambridge, UK. 3 TB unit, Mulago
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burgh CR Jr. Management of drug-resistant tuberculosis. The Lancet. 2019;394(10202):953–66. 2. Organisation WH. Global Tuberculosis Report. World Health Organisation;
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English
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The Association of Palmitoylethanolamide with Luteolin Decreases Autophagy in Spinal Cord Injury
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Molecular neurobiology
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cc-by
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2
Department of Pharmacological and Physiological Science, Saint
Louis University School of Medicine, 1402 South Grand Blvd, St
Louis, MO 63104, USA 1
Department of Biological and Environmental Sciences, University of
Messina, Viale Ferdinando Stagno D’Alcontres, 31,
98166 Messina, Italy Rosalba Siracusa1 & Irene Paterniti1 & Giuseppe Bruschetta1 & Marika Cordaro1 &
Daniela Impellizzeri1 & Rosalia Crupi1 & Salvatore Cuzzocrea1,2 & Emanuela Esposito1 Received: 9 March 2015 /Accepted: 26 June 2015 /Published online: 5 July 2015
# The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Spinal cord injury (SCI) is a devastating condition
of the central nervous system (CNS) often resulting in severe
functional impairment and for which there are not yet restor-
ative therapies. In the present study, we performed a widely
used model of SCI to determine the neuroprotective propriety
of palmitoylethanolamide (PEA) and the antioxidant effect of
a flavonoid luteolin (Lut), given as a co-ultramicronized com-
pound co-ultraPEALut. In particular, by western blot analysis
and immunofluorescence staining, we investigated whether
this compound (at the dose of 1 mg/kg) was able to modulate
autophagy. Our results showed that treatment with co-
ultraPEALut after SCI reduced the expression of proteins pro-
moter of autophagy such as Beclin-1 and microtubule-
associated protein 1A/1B-light chain 3 (MAP-LC3). In con-
trast, this compound decreased the levels of mammalian target
of rapamycin (mTOR), p-Akt, and p-70S6K which are pro-
teins that inhibit autophagy. These data confirmed that the
protective role of co-ultraPEALut is associated with inhibition
of excessive autophagy and regulation of protein degradation. Therefore, treatment with co-ultraPEALut could be consid-
ered as a possible therapeutic approach in an acute traumatic
lesion like SCI. TRACTED Keywords Neuroprotection . Anti-inflammatory . Anti-oxidant . Autophagy . Spinal compression
Introduction
D ART The Association of Palmitoylethanolamide with Luteolin
Decreases Autophagy in Spinal Cord Injury &
sito1
otection . Anti-inflammatory . phagy Spinal compression
RTICLE & Marika Cordaro1 &
1,2 & Emanuela Esposito1
2015
rlink.com
Keywords Neuroprotection . Anti-inflammatory . Anti-oxidant . Autophagy . Spinal compression
Introduction
D ARTICLE Rosalba Siracusa1 & Irene Paterniti1 & Giuseppe Bruschetta1 & Marika Cordaro1 &
Daniela Impellizzeri1 & Rosalia Crupi1 & Salvatore Cuzzocrea1,2 & Emanuela Esposito1 Mol Neurobiol (2016) 53:3783–3792
DOI 10.1007/s12035-015-9328-6 Mol Neurobiol (2016) 53:3783–3792
DOI 10.1007/s12035-015-9328-6 Co-ultramicronization Process of PEA and Lut
E The co-ultramicronization process was performed using jet
mill equipment (Sturtevant, Inc., 348 Circuit Street Hanover,
MA, USA) with a chamber of 300 mm in diameter, operated
with Bspiral technology^ and driven by compressed air at 10
to 12 bars. Crashing was determined by the high number of
collisions that occurred among particles as a result of the high
level of kinetic (not mechanical) energy. This process is effec-
tive not only in reducing product particle size but also in
modifying crystalline structure. Scanning electron microscopy
showed an intimate intermixing of PEA and Lut, while anal-
ysis by differential scanning calorimetry and X-ray diffraction
indicated transformation into a crystalline form different from
the original two, definable as Ba higher energy content form.^
The composite showed the following particle size distribution:
96 % <10 μm, 80 % <5 μm, and 40 % <2 μm. Co-
ultraPEALut (Epitech Group s.r.l.) was dissolved in 10 % eth-
anol (Sigma-Aldrich, St Louis, MO, USA) and used at a con-
centration of 1 mg/kg in the in vivo study. ED ARTICLE In recent years, the pathophysiology of SCI has been a
focus of extensive studies; animal models have been proved
to be important tools in this field and are employed to inves-
tigate the mechanisms of primary and secondary injury. Pre-
vious studies have shown that autophagy plays a key role in
secondary injury in both animal models and human tissue by
causing progressive degeneration of the spinal cord [15, 16]. However, the molecular pathway of secondary injury and the
role of autophagy in the recovery of SCI remain unclear. Fol-
lowing acute spinal cord injury, an increase in mTOR expres-
sion and p70S6K activity also may be required for functional
improvement [17]. The involvement of autophagic mecha-
nism underlying spinal neuroprotective effects of
palmitoylethanolamide (PEA) and luteolin association was
never been investigated. Thus, we studied the autophagic
pathway involved in spinal cord compression and to explore
the effects of the treatment with a new co-ultramicronized
composite of N-PEA and luteolin (Lut) on autophagy expres-
sion. This formulation (co-ultraPEALut) is based on associa-
tion of anti-inflammatory PEAwith anti-oxidant Lut on a 10:1
mass basis. We have previously demonstrated that treatment
with co-ultraPEALut significantly reduced the inflammatory
process in an ex vivo model of spinal cord organotypic cul-
tures and in experimental mouse models of TBI [18, 7]. Introduction
AR Spinal cord injury (SCI) is defined as an acute traumatic lesion
of neural elements in the spinal canal (spinal cord and cauda
equina), resulting in a change, either temporary or permanent,
in normal motor, sensory, or autonomic function. Spinal cord
injury usually begins with a sudden, traumatic blow to the
spine that causes local segmental damage to the spinal cord,
which is called primary injury [1, 2]. The primary damage to
the tissue is followed by a second phase of tissue degenera-
tion, the secondary injury that can occur over weeks or even
months. In secondary injury, acute inflammation can develop
into a chronic process if feedback mechanisms fail to inhibit
amplification of the inflammatory response. Chronic inflam-
mation leads to a continuous influx of neutrophils, macro-
phages, lymphocytes, and eosinophils from the circulation,
causing more destruction and scarring of tissue [3]. Cell death
resulting from all of these mechanisms occurs through necrot-
ic and apoptotic phenomena or autophagy. ED A a poss b e t e apeut c
ike SCI. RETR Concern about autophagy has recently increased because
of its potential role in neurodegenerative diseases, such as
Parkinson’s and Alzheimer’s diseases, where autophagy may
be a protective mechanism [4]. Earlier studies also demon-
strated increased autophagic activity at lesion sites after cere-
bral hypoxia-ischemia (HI) injury [5], intracerebral hemor-
rhage (ICH) [6], and traumatic brain injury (TBI) [7, 8]. * Emanuela Esposito
eesposito@unime.it
RE 1
Department of Biological and Environmental Sciences, University of
Messina, Viale Ferdinando Stagno D’Alcontres, 31,
98166 Messina, Italy Autophagy, literally Bself-eating, is now recognized as an
intracellular catabolic mechanism for the degradation, elimi-
nation, and recycling of long-lived proteins and unwanted
organelles in a cell during development and under stress con-
ditions [9, 10]. Therefore, autophagy plays an important role Mol Neurobiol (2016) 53:3783–3792 3784 in homeostasis and also to a kind of cell death, which is known
as autophagic cell death [11, 12]. It begins with the formation
of double-membraned vesicle that subsequently engulfs cyto-
plasmic components, including cytosolic proteins and organ-
elles, to become autophagosomes (APs). APs fuse with lyso-
somes to form autolysosomes, and intra-autophagosomal
components are degraded by lysosomal hydrolases. Several
key molecular components participate in the initiation, pro-
gression, and completion of autophagy, like the mammalian
target of rapamycin (mTOR) that inhibits autophagy, whereas
Beclin 1 and LC3 promote it [8, 13]. Introduction
AR However, autophagy
seems to be a double-edged sword that protects cells when it
is moderately activated, yet its excessive activation can induce
autophagic cell death [14]. dark cycle. The animals were acclimatized to their environ-
ment for 1 week and had ad libitum access to tap water and
rodent standard diet. The study was approved by the Univer-
sity of Messina Review Board for the care of animals. All
animal experiments complied with regulations in Italy (D.M. 116192) as well as the EU regulations (O.J. of E.C. L 358/1
12/18/1986). SCI Mice were anesthetized with intraperitoneal administration of
ketamine and xylazine (2.6 and 0.16 mg/kg body weight, re-
spectively). A longitudinal incision was made on the midline
of the back, exposing the paravertebral muscles, as previously
described [18]. These muscles were dissected away, the spinal
cord was exposed via a four-level T5 to T8 laminectomy, and
SCI was produced by extradural compression at T6 to T7
level, using an aneurysm clip with a closing force of 24 g. Following surgery, 1.0 cm3 of saline was administered subcu-
taneously in order to replace the blood volume lost during the
surgery. During recovery from anesthesia, mice were placed
on a warm heating pad and covered with a warm towel. The
mice were individually housed in a temperature-controlled
room at 27 °C. Food and water were provided to the mice
ad libitum. During this time period, the animals’ bladders
were manually voided twice a day until the mice were able
to regain normal bladder function. In all injured groups, the
spinal cord was compressed for 1 min. Sham animals were
only subjected to laminectomy. Spinal cord tissues were taken
at 24 h after trauma. Tissue segments contained the lesion Therefore, the aim of this study was to demonstrate that
treatment co-ultraPEALut inhibited autophagy even after SCI,
having neuroprotective effect. RET having neuroprotective
Methods
Animals
RE g
p
Methods
Animals
CD1 mice (male 25–30 g; Harlan Nossan, Milan, Italy) were
housed in a controlled environment and provided with stan-
dard rodent chow and water. Mice were housed in stainless
steel cages in a room kept at 22±1 °C with a 12-h light, 12-h
RE Co-ultramicronization Process of PEA and Lut
E Fur-
thermore in this last model, we showed that co-ultraPEALut
reduced autophagy after TBI [7]. TRACTED The dose of co-ultraPEALut (1 mg/kg) used was based on
previous in vivo studies [18]. ED Animals CD1 mice (male 25–30 g; Harlan Nossan, Milan, Italy) were
housed in a controlled environment and provided with stan-
dard rodent chow and water. Mice were housed in stainless
steel cages in a room kept at 22±1 °C with a 12-h light, 12-h 3785 Mol Neurobiol (2016) 53:3783–3792 ImmunoResearch) for 1 h at room temperature. To ascertain
that blots were loaded with equal amounts of protein lysates,
they were also incubated in the presence of the antibody
against β-actin (1:5000; Santa Cruz Biotechnology). Signals
were detected with enhanced chemiluminescence detection
system reagent according to manufacturer’s instructions
(SuperSignal West Pico Chemiluminescent Substrate, Pierce). The relative expression of the protein bands was quantified by
densitometry with Bio-Rad ChemiDoc™XRS+software and
standardized to β-actin levels. Images of blot signals (8-bit/
600-dpi resolution) were imported to analysis software (Image
Quant TL, v2003). A preparation of commercially available
molecular weight markers made of proteins of molecular
weight 10 to 250 kDa was used to define molecular weight
positions and as reference concentrations for each molecular
weight. TICLE (1 cm on each side of the lesion), according to counts of
retrogradely labeled neurons at the origin of distinct descend-
ing motor pathways and to morphometric assessments of nor-
mal residual tissue at the injury epicenter, as previously de-
scribed [19]. Immunofluorescence Stainin
RT 4. SCI+co-ultraPEALut (10:1): mice were subjected to SCI
and administered co-ultraPEALut at the final dose of
1 mg/kg i.p. at 1 and 6 h after SCI (n=30). After deparaffinization and rehydration, detection of GFAP,
NeuN, Beclin-1, and MAPLC3-α was carried out after boiling
in 0.1 M citrate buffer for 1 min. Nonspecific adsorption was
minimized by incubating the section in 2 % (v/v) normal goat
serum in PBS for 20 min. Sections were incubated with mouse
monoclonal anti-NeuN (1:100, v/v Millipore), or with mouse
monoclonal anti-GFAP (1:100, v/v Santa Cruz Biotechnolo-
gy), or with polyclonal rabbit anti-Beclin-1 (1:100, v/v Santa
Cruz, Biotechnology), or with rabbit anti-MAPLC3α (1:100,
v/v Santa Cruz Biotechnology) antibody in a humidified
chamber for O/N at 37 °C. Sections were washed with PBS
and were incubated with secondary antibody FITC-
conjugated anti-mouse Alexa Fluor-488 antibody (1:2000
v/v Molecular Probes, UK) and with TEXAS RED-
conjugated anti-rabbit Alexa Fluor-594 antibody (1:1000 in
PBS, v/v Molecular Probes, UK) for 1 h at 37 °C. Sections
were washed and for nuclear staining 4′,6′-diamidino-2-
phenylindole (DAPI; Hoechst, Frankfurt; Germany) 2 μg/ml
in PBS was added. Sections were observed and photographed
at ×20 magnification using a Leica DM2000 microscope
(Leica). All images were digitalized at a resolution of 8 bits
into an array of 2560×1920 pixels. Optical sections of fluores-
cence specimens were obtained using a HeNe laser (543 nm), a
laser UV (361–365 nm), and an argon laser (458 nm) at a 1-
min, 2-s scanning speed with up to 8 averages; 1.5 μm sections
were obtained using a pinhole of 250. Contrast and brightness
were established by examining the most brightly labeled pixels
and applying settings that allowed clear visualization of struc-
tural details while keeping the highest pixel intensities close to
200. The same settings were used for all images obtained from
the other samples that had been processed in parallel. Digital
images were cropped and figure montages prepared using
Adobe Photoshop CS5 (Adobe Systems; Palo Alto, CA). ED ART Experimental Groups and Treatments Mice Were Randomly Allocated into the Following Groups Mice Were Randomly Allocated into the Following Groups 1. Sham+vehicle: mice were subjected to laminectomy, but
the aneurysm clip was not applied and treated intraperito-
neally (i.p.) with vehicle (n=30). 1. Sham+vehicle: mice were subjected to laminectomy, but
the aneurysm clip was not applied and treated intraperito-
neally (i.p.) with vehicle (n=30). 1. Sham+vehicle: mice were subjected to laminectomy, but
the aneurysm clip was not applied and treated intraperito-
neally (i.p.) with vehicle (n=30). 2. Sham+co-ultraPEALut (10:1)=same as the Sham+vehi-
cle group, but co-ultraPEALut was administered at the
final dose of 1 mg/kg i.p. at 1 and 6 h after laminectomy
(n=30). 2. Sham+co-ultraPEALut (10:1)=same as the Sham+vehi-
cle group, but co-ultraPEALut was administered at the
final dose of 1 mg/kg i.p. at 1 and 6 h after laminectomy
(n=30). rence concentration
e Staining
RTIC 3. SCI+vehicle: mice were subjected to laminectomy and
the aneurysm clip was applied (n=30). 3. SCI+vehicle: mice were subjected to laminectomy and
the aneurysm clip was applied (n=30). Western Blot Analysis Cytosolic and nuclear extracts were prepared with slight mod-
ifications of a published procedure [20]. Spinal cord tissue
from each mouse was suspended in extraction Buffer A con-
taining 0.2 mM PMSF, 0.15 mM pepstatin A, 20 mM
leupeptin, 1 mM sodium orthovanadate, homogenized at the
highest setting for 2 min, and centrifuged at 12,000 rpm for
4 min at 4 °C. Supernatants represented the cytosolic fraction. The pellets, containing enriched nuclei, were resuspended in
Buffer B containing 1 % Triton X-100, 150 mM NaCl, 10 mM
Tris–HCl pH 7.4, 1 mM EGTA, 1 mM EDTA, 0.2 mM PMSF,
20 mm leupeptin, and 0.2 mM sodium orthovanadate. After
centrifugation 10 min at 12,000 rpm at 4 °C, the supernatants
that we obtained were the nuclear proteins. Protein concentrations
were estimated by the Bio-Rad protein assay using bovine serum
albumin as standard. Briefly, samples were heated to 100 °C
for 5 min, and equal amounts of protein were separated on
18 % SDS-PAGE gel and transferred to nitrocellulose mem-
brane. Specific primary antibody, anti- anti-MAPLC3α
(1:500, Santa Cruz Biotechnology), Beclin-1 (1:500; Santa
Cruz Biotechnology), anti-p62 (1:1000; Cell Signaling),
anti-mTOR (1:1000; Cell Signaling), anti-p70S6K (1:1000;
Cell Signaling), and p-Akt (1:1000; Cell Signaling) were
mixed in 1× PBS, 5 % w/v nonfat dried milk, 0.1 % Tween-
20 (PMT), and incubated at 4 °C, overnight. After, mem-
branes were incubated with peroxidase-conjugated bovine
anti-mouse IgG secondary antibody or peroxidase-
conjugated goat anti-rabbit IgG (1:2000, Jackson
RETRACTED 3786 Mol Neurobiol (2016) 53:3783–3792 Materials For histopathological examination by standard hematoxylin and
eosin (H&E) staining, the mice were anesthetized with ketamine
and xylazine (2.6 and 0.16 mg/kg body weight respectively)
24 h after injury and then perfused transcardially with cold
PBS (0.1 M). Tissues were removed under magnified vision,
and segments containing the lesion (1 cm on each side of the
lesion) were collected in 4 % paraformaldehyde for proper fix-
ation and then dehydrated with graded ethanol and embedded in
paraffin wax. Sections of 5-μm thickness were cut into longitu-
dinal sections for the posterior area of the spinal cord, stained
with H&E, and studied using light microscopy (Dialux 22
Leitz). Representative images were shown. Blinded histologic
scoring (Fig. 1d) was performed on a 6-point scale: 0, normal; 1,
1–5 eosinophilic neurons within the gray matter area; 2, 5–10
eosinophilic neurons; 3, >10 eosinophilic neurons; 4, a small
infarction (less than one-third of the gray matter area); 5, a mod-
erate infarction (one-third to one-half of the gray matter area);
and 6, a large infarction (more than half of the gray matter area). Unless otherwise stated, all compounds were obtained from
Sigma-Aldrich, while co-ultraPEALut was a kind gift from
Epitech Group s.r.l. (Saccolongo, Italy). All other chemicals
were of the highest commercial grade available. All stock
solutions were prepared in non-pyrogenic saline (0.9 % NaCl,
Baxter, Milan, Italy) or 10 % dimethyl sulfoxide. E Effect of Co-ultraPEALut on Histological Parameters To evaluate the severity of the trauma at the level of the
perilesional area at 24 h after injury, the sections obtained
from each group were stained with hematoxylin and eosin
(H&E). Compared with the sham group (Fig. 1a, b, see mag-
nification higher a1 and b1, respectively), the SCI+vehicle
group showed the presence of edema and alteration of the
white matter (Fig. 1b, d, see magnification higher b1). Treat-
ment with co-ultraPEALut attenuated acute inflammation and
injury at 24 h after SCI (Fig. 1c, d, see magnification higher
c1). Effect of Co-ultraPEALut on Autophagy After SCI:
Expression of Beclin-1, p62, and MAP-LC3 Statistical Evaluation All values in the figures and the text are expressed as mean±
SEM. Results shown in the figures are representative of at
least three experiments performed on different in vivo exper-
imental days. In each experiment, we used five animals per
group, unless otherwise indicated. The results were analyzed
by one-way analysis of variance followed by a Bonferroni
post hoc test for multiple comparisons. A p value of less than
0.05 was considered significant. RTICL Fig. 1 Effects of co-ultraPEALut treatment on histological alterations of
the spinal cord tissue 24 h after injury. Compared with the Sham-surgery
mice (a, a1), significant histologic damage to the spinal cord was evident
in mice subjected to SCI and treated with vehicle (b, b1). In the
perilesional area of the SCI mice, edema was evident, as and alteration
of the white matter. A significant reduction of SCI-associated damage was
observed in the tissue samples collected from mice treated with co-
ultraPEALut (c, c1). These data are also visible in the histological score
(d). ***p<0.001 vs Sham; ###p<0.001 vs SCI
RETRACTED AR of the white matter. A significant reduction of SCI-associated damage was
observed in the tissue samples collected from mice treated with co-
ultraPEALut (c, c1). These data are also visible in the histological score
(d). ***p<0.001 vs Sham; ###p<0.001 vs SCI
ED AR ED AR RETRAC of the white matter. A significant reduction of SCI-associated damage was
observed in the tissue samples collected from mice treated with co-
ultraPEALut (c, c1). These data are also visible in the histological score
(d). ***p<0.001 vs Sham; ###p<0.001 vs SCI of the white matter. A significant reduction of SCI-associated damage was
observed in the tissue samples collected from mice treated with co-
ultraPEALut (c, c1). These data are also visible in the histological score
(d). ***p<0.001 vs Sham; ###p<0.001 vs SCI Fig. 1 Effects of co-ultraPEALut treatment on histological alterations of
the spinal cord tissue 24 h after injury. Compared with the Sham-surgery
mice (a, a1), significant histologic damage to the spinal cord was evident
in mice subjected to SCI and treated with vehicle (b, b1). In the
perilesional area of the SCI mice, edema was evident, as and alteration Mol Neurobiol (2016) 53:3783–3792 3787 Results autophagy. To compare the level of Beclin-1 protein between
the injured and the treated spinal cord section, western blot
analysis was performed. We showed that Beclin-1 expression
dramatically increased in the spinal cord tissue (T6 to T8 tract)
collected from mice subjected to SCI compared to the sham
group, while the treatment with co-ultraPEALut significantly
reduced SCI-induced Beclin-1 expression (Fig. 2a, see densi-
tometry analysis in a1). For more accurate quantification of
co-ultraPEALut effects on autophagy process, we examined
levels of p62 by western blot analysis. We detected that p62
expression increased after SCI, whereas the treatment with co-
ultraPEALut significantly reduced levels of this protein
(Fig. 2b, see densitometry analysis in b1). p62 also interacts
with a central component of the machine autophagy, autoph-
agic marker MAP-LC3, and carries the altered proteins to
degradation by autophagy. Therefore, by western blot analy-
sis, we showed that the ratio of LC3 I to LC3 II increased in
untreated animals 24 h after injury, though this ratio signifi-
cantly diminished to near sham levels of expression following
co-ultraPEALut treatment (Fig. 2c, see densitometry analysis
in c1), suggesting a reversal of autophagic activity upregula-
tion seen following SCI. ARTICLE Effect of Co-ultraPEALut on Autophagy After SCI:
Expression of Beclin-1, p62, and MAP-LC3 To investigate the mechanisms of autophagy after SCI, we
examined levels of proteins involved in the regulation and
formation of autophagosomes. Beclin-1 has a central role in
autophagy because it interacts with various cofactors inducing Fig. 2 Effect of co-ultraPEALut about proteins promoting autophagy
after SCI. Western blot analysis for Beclin-1, p62, and LC3 showed an
increase of levels of these proteins in SCI group compared to the Sham
group, whereas the treatment with co-ultraPEALut significantly reduced
SCI-induced Beclin-1, p62, and LC3 expression (a, a1; b, b1; c, c1
respectively). The data are representative of at least three independent
experiments. a **p<0.01 vs Sham; ##p<0.01 vs SCI. b **p<0.01 vs
Sham; #p<0.05 vs SCI. c *p<0.05 vs Sham; #p<0.05 vs SCI
RETRACTED A ED D RACTED D Fig. 2 Effect of co-ultraPEALut about proteins promoting autophagy
after SCI. Western blot analysis for Beclin-1, p62, and LC3 showed an
increase of levels of these proteins in SCI group compared to the Sham
group, whereas the treatment with co-ultraPEALut significantly reduced SCI-induced Beclin-1, p62, and LC3 expression (a, a1; b, b1; c, c1
respectively). The data are representative of at least three independent
experiments. a **p<0.01 vs Sham; ##p<0.01 vs SCI. b **p<0.01 vs
Sham; #p<0.05 vs SCI. c *p<0.05 vs Sham; #p<0.05 vs SCI Mol Neurobiol (2016) 53:3783–3792 3788 Effect of Co-ultraPEALut on Beclin-1 and MAP-LC3
Expression by Immunofluorescence Staining upstream signal in the regulation of autophagy which is pos-
itively regulated by PI3K/Akt and results in the inhibition of
autophagy. Therefore, by western blot analysis, we detected
the activity of Akt/mTOR signaling pathways in our model
in vivo. Our results showed that p-Akt and mTOR levels de-
creased significantly after SCI, compared with the Sham
group, while co-ultraPEALut treatment increased p-Akt and
mTOR expression, inhibiting autophagy (Fig. 5a, c, see den-
sitometry analysis in a1 and b1, respectively). E To better understand the Beclin-1 and MAP-LC3 expression in
a specific population of cells, including neurons and astrocytes,
the spinal cord sections (T6 to T8 tract) at 24 h after compres-
sion were double-stained for Beclin-1 (red) or MAP-LC3 (red)
and various cell type markers, such as NeuN (green) for neurons
and GFAP (green) for astrocytes (Figs. 3 and 4). In the double
staining, the expression of Beclin-1 was observed in NeuN and
GFAP-labeled cells of the injured SCI group (Fig. 3g–i and j–l,
respectively) compared to Sham (Fig. 3a–c and d–f, respective-
ly), whereas a treatment with co-ultraPEALut not only de-
creased significantly these proteins expression but also the dou-
ble staining is less observed in both NeuN and GFAP (Fig. 3m–
o and p–r, respectively). Similarly, at 24 h after SCI, double
staining of LC3 revealed a strong co-localization with NeuN
and GFAP in SCI group (Fig. 4g–i and j–l, respectively) com-
pared to the Sham group (Fig. 4a–c and d–f, respectively), while
treatment of co-ultraPEALut decreased significantly these pro-
tein expressions (Fig. 4m-o and p-r respectively). Moreover, a downstream target of mTOR signal a p70S6K
activity was assessed by western blot analysis. p70S6k activ-
ity was suppressed in mice after SCI, whereas the co-
ultraPEALut treatment enhanced activation of p70S6K signal-
ing (Fig. 5c, see densitometry analysis in c1). CLE y
y
)
i
h
i
l i j
i
RTICL Discussion The CNS is sensitive to mechanical injuries causing perma-
nent functional deficits such as the case of patients who have
SCI. The mechanical forces imparted to the spinal cord cause
immediate tissue disruption, with direct axonal and neuronal
injury, inducing the death of neurons. Moreover, neurons con-
tinue to die for hours or days after SCI, as a consequence of
excitotoxicity, vascular dysfunction, and, in particular, be-
cause of the ensuing inflammatory response [21]. Inflamma-
tory responses are a major component of secondary injury and
ED ART Effect of Co-ultraPEALut on PI3K/Akt/mTOR Pathway
After SCI
D Treatment with the Co-ultraPEALut reduced Beclin-1
expression (m–r). All images were digitalized at a resolution of 8 bits
into an array of 2048×2048 pixels
D AR Fig. 4 Co-localization of NeuN/LC3 and GFAP/LC3 after SCI. Results
are shown for (a–f) Sham group, (g–l) mice with SCI, and (m–r) mice
with SCI treated with co-ultraPEALut. Double-stained sections at 24 h
post-injury indicated that LC3 expression increased in both neurons and
astrocytes respectively. Orange arrows indicate co-localizations (g–l) and
D still unknown. The most plausible explanation is that catabo-
lism through autophagy is predominantly survival-supporting,
but that an imbalance in cell metabolism, where autophagic
cellular consumption exceeds the cellular capacity for synthe-
sis, promotes cell death [28]. ED play a central role in regulating the pathogenesis of acute and
chronic SCI [22]. Functional recovery after SCI is often poor
and, as yet, there are no nonsurgical therapies that have been
successfully translated for use in the clinic. Therefore, there is
a great need for drugs that will reduce post-traumatic,
inflammation-related spinal cord damage because it has been
reported that reducing inflammation decreases secondary de-
generation and the functional deficit after SCI. In a previous
study, we showed that co-ultraPEALut treatment (1 mg/kg)
exerted beneficial effects in ex vivo model using spinal cord
organotypic slice cultures and in in vivo model of spinal cord
compression injury in mice. Briefly, we found that co-
ultraPEALut had anti-inflammatory, antioxidant, and neuro-
protective properties [18]. ETRACTED Therefore, the aim of the present study was to explore new
therapeutic approaches promoting survival of cells. In partic-
ular, we wanted to investigate whether the new compound co-
ultraPEALut (at the dose of 1 mg/kg) was able to modulate
autophagy promoting functional recovery after SCI. In the first step, using H&E staining, we evaluated the
effect of co-ultraPEALut on spinal cord at the level of the
perilesional area at 24 h after injury. Histological analysis
showed that mice, after SCI, presented edema and alteration
of the white matter, while mice treated with co-ultraPEALut
showed an attenuation of acute inflammation and injury after
SCI. Furthermore, recent studies demonstrated that another pos-
sible consequence of SCI is energy depletion, and this elicits
autophagy [15, 16, 23]. Autophagy is a cellular catabolic deg-
radation response to starvation or stress, whereby cellular pro-
teins, organelles, and cytoplasm are engulfed, digested, and
recycled to sustain cellular metabolism [9, 24]. Effect of Co-ultraPEALut on PI3K/Akt/mTOR Pathway
After SCI
D The PI3K/Akt/mTOR pathway is an intracellular signaling
pathway important in regulating the cell cycle. mTOR is
ED Fig. 3 Co-localization of Neun/Beclin-1 and GFAP/Beclin-1 after SCI. Results are shown for a–f Sham group, g–l mice with SCI, and m–r mice
with SCI treated with co-ultraPEALut. Double-stained sections at 24 h
post injury indicated that Beclin-1 expression increased in both neurons
and astrocytes respectively. Orange arrows indicate co-localizations (g–l)
and revealed a high co-localization between NeuN/Beclin1 and GFAP/
Beclin-1 double staining. Treatment with the co-ultraPEALut
reduced Beclin-1 expression (m–r). All images were digitalized at a
resolution of 8 bits into an array of 2048×2048 pixels
RETRACTED D TRA and revealed a high co-localization between NeuN/Beclin1 and GFAP/
Beclin-1 double staining. Treatment with the co-ultraPEALut
reduced Beclin-1 expression (m–r). All images were digitalized at a
resolution of 8 bits into an array of 2048×2048 pixels Fig. 3 Co-localization of Neun/Beclin-1 and GFAP/Beclin-1 after SCI. Results are shown for a–f Sham group, g–l mice with SCI, and m–r mice
with SCI treated with co-ultraPEALut. Double-stained sections at 24 h
post injury indicated that Beclin-1 expression increased in both neurons
and astrocytes respectively. Orange arrows indicate co-localizations (g–l) Mol Neurobiol (2016) 53:3783–3792 3789 Fig. 4 Co-localization of NeuN/LC3 and GFAP/LC3 after SCI. Results
are shown for (a–f) Sham group, (g–l) mice with SCI, and (m–r) mice
with SCI treated with co-ultraPEALut. Double-stained sections at 24 h
post-injury indicated that LC3 expression increased in both neurons and
astrocytes respectively. Orange arrows indicate co-localizations (g–l) and
revealed a high co-localization between NeuN/LC3 and GFAP/LC3
double staining. Treatment with the Co-ultraPEALut reduced Beclin-1
expression (m–r). All images were digitalized at a resolution of 8 bits
into an array of 2048×2048 pixels
D ARTICLE FAP/LC3 after SCI. Results
e with SCI, and (m–r) mice
uble-stained sections at 24 h
creased in both neurons and
ate co-localizations (g–l) and
revealed a high co-localization between NeuN/LC3 and GFAP/LC3
double staining. Treatment with the Co-ultraPEALut reduced Beclin-1
expression (m–r). All images were digitalized at a resolution of 8 bits
into an array of 2048×2048 pixels
D ARTICLE revealed a high co-localization between NeuN/LC3 and GFAP/LC3
double staining. Treatment with the Co-ultraPEALut reduced Beclin-1
expression (m–r). All images were digitalized at a resolution of 8 bits
into an array of 2048×2048 pixels
D ARTICLE CLE revealed a high co-localization between NeuN/LC3 and GFAP/LC3
double staining. Effect of Co-ultraPEALut on PI3K/Akt/mTOR Pathway
After SCI
D Finally, we wanted to evaluate
on Akt/mTOR/p70S6K pathway
considered a central regulatory p
lation involved in regulating cell
entiation, and survival [33, 34]
regulator of autophagy directly s
protein is a central signal integrat
point with upstream Akt and dow
two most important mediators [8
blot analysis, we detected mTO
phorylation of Akt after SCI. O
Akt and mTOR levels decreased
co-ultraPEALut treatment increa
teins. Moreover, p70S6k activity
SCI, whereas the co-ultraPEALu
tion of p70S6K signaling. These
tective role of co-ultraPEALut is
excessive autophagy via Akt/mT
ulation of protein degradation. o-ultra PEALut about proteins inhibiting autophagy
blot analysis for mTOR, p-Akt, and p70S6K showed
of these proteins in SCI group compared to the Sham
reatment with co-ultraPEALut significantly increased
SCI-induced mTOR, p-Akt, and p70S6
respectively). The data are representat
experiments. a **p<0.01 vs Sham; #
Sham; ##p<0.01 vs SCI. c ***p<0.00
ETRACTED ARTIC TED ARTIC Fig. 5 Effect of co-ultra PEALut about proteins inhibiting autophagy
after SCI. Western blot analysis for mTOR, p-Akt, and p70S6K showed
a decrease of levels of these proteins in SCI group compared to the Sham
group, whereas the treatment with co-ultraPEALut significantly increased
ACTE SCI-induced mTOR, p-Akt, and p70S6K expression (a, a1; b, b1; c, c1
respectively). The data are representative of at least three independent
experiments. a **p<0.01 vs Sham; #p<0.05 vs SCI. b **p<0.01 vs
Sham; ##p<0.01 vs SCI. c ***p<0.001 vs Sham; ##p<0.01 vs SCI
E Finally, we wanted to evaluate the effect of co-ultraPEALut
on Akt/mTOR/p70S6K pathway that in autophagy has been
considered a central regulatory pathway of the protein trans-
lation involved in regulating cell proliferation, growth, differ-
entiation, and survival [33, 34]. mTOR kinase is a master
regulator of autophagy directly suppressing autophagy. This
protein is a central signal integrator that functions as a check-
point with upstream Akt and downstream p70S6K being the
two most important mediators [8]. Therefore, using western
blot analysis, we detected mTOR expression and the phos-
phorylation of Akt after SCI. Our results indicated that p-
Akt and mTOR levels decreased significantly after SCI, while
co-ultraPEALut treatment increased expression of these pro-
teins. Moreover, p70S6k activity was suppressed in mice after
SCI, whereas the co-ultraPEALut treatment enhanced activa-
tion of p70S6K signaling. These data confirmed that the pro-
tective role of co-ultraPEALut is associated with inhibition of
excessive autophagy via Akt/mTOR/p70S6K signals and reg-
ulation of protein degradation. Effect of Co-ultraPEALut on PI3K/Akt/mTOR Pathway
After SCI
D Constitutive,
basal autophagy also has an important homeostatic function,
by maintaining protein and organelle quality control. Al-
though most evidence supports a role for autophagy in sus-
taining cell survival, paradoxically, cell death resulting from
progressive cellular consumption has been attributed to unre-
strained autophagy [25–27]. The mechanisms that regulate the
mutually opposed survival and death roles for autophagy are
RET In the second step, we studied the effects of this new com-
pound on autophagy markers after SCI. At first, we examined
the levels of proteins that promote autophagy such as Beclin-1
and MAP-LC3. Beclin-1 is a protein that participates in the
regulation of autophagy and has an important role in develop-
ment, tumorigenesis, and neurodegeneration [29]. Beclin-1
functions as a scaffold for the formation of the PI3K complex,
one of the first components recruited during the development
of autophagosomes. During autophagy initiation and
autophagosome formation, Beclin-1 binds microtubule- Mol Neurobiol (2016) 53:3783–3792 3790 Fig. 5 Effect of co-ultra PEALut about proteins inhibiting autophagy
after SCI. Western blot analysis for mTOR, p-Akt, and p70S6K showed
a decrease of levels of these proteins in SCI group compared to the Sham
group, whereas the treatment with co-ultraPEALut significantly increased
SCI-induced mTOR, p-Akt, and p70S6K expression (a, a1; b, b1; c, c1
respectively). The data are representative of at least three independent
experiments. a **p<0.01 vs Sham; #p<0.05 vs SCI. b **p<0.01 vs
Sham; ##p<0.01 vs SCI. c ***p<0.001 vs Sham; ##p<0.01 vs SCI
ACTED ARTICLE ICLE n 1A/1B-light chain 3 I (LC3I) that is con-
mbrane-bound form (LC3II), which then inter-
iquitin-binding protein p62/sequestosome 1
62 accumulates when autophagy is inhibited
vels can be observed when autophagy is in-
be used as a marker to study autophagic flux
y, we observed an increase of the expression
3, and p62 after SCI that dramatically de-
atment with co-ultraPEALut. Furthermore,
sess whether Beclin-1 and MAP-LC3 were
cific cells. Therefore, the spinal cord sections
pression were double-stained for Beclin-1 or
various cell type markers, such as NeuN for
AP for astrocytes. By immunofluorescence
served a strong co-localization of Beclin-1
un and GFAP. These results showed that after
on of these two proteins increases in neurons
while treatment with co-ultraPEALut reduces
lin-1 and LC3 in both cell types. Effect of Co-ultraPEALut on PI3K/Akt/mTOR Pathway
After SCI
D associated protein 1A/1B-light chain 3 I (LC3I) that is con-
verted to its membrane-bound form (LC3II), which then inter-
acts with the ubiquitin-binding protein p62/sequestosome 1
[30–32]. Since p62 accumulates when autophagy is inhibited
and decreased levels can be observed when autophagy is in-
duced, p62 may be used as a marker to study autophagic flux
[32]. In this study, we observed an increase of the expression
of Beclin-1, LC3, and p62 after SCI that dramatically de-
creased after treatment with co-ultraPEALut. Furthermore,
we wanted to assess whether Beclin-1 and MAP-LC3 were
expressed in specific cells. Therefore, the spinal cord sections
at 24 h after compression were double-stained for Beclin-1 or
MAP-LC3 and various cell type markers, such as NeuN for
neurons and GFAP for astrocytes. By immunofluorescence
staining, we observed a strong co-localization of Beclin-1
and LC3 with Neun and GFAP. These results showed that after
SCI, the expression of these two proteins increases in neurons
and astrocytes, while treatment with co-ultraPEALut reduces
the levels of Beclin-1 and LC3 in both cell types. RETRAC associated protein 1A/1B-light chain 3 I (LC3I) that is con-
verted to its membrane-bound form (LC3II), which then inter-
acts with the ubiquitin-binding protein p62/sequestosome 1
[30–32]. Since p62 accumulates when autophagy is inhibited
and decreased levels can be observed when autophagy is in-
duced, p62 may be used as a marker to study autophagic flux
[32]. In this study, we observed an increase of the expression
of Beclin-1, LC3, and p62 after SCI that dramatically de-
creased after treatment with co-ultraPEALut. Furthermore,
we wanted to assess whether Beclin-1 and MAP-LC3 were
expressed in specific cells. Therefore, the spinal cord sections
at 24 h after compression were double-stained for Beclin-1 or
MAP-LC3 and various cell type markers, such as NeuN for
neurons and GFAP for astrocytes. By immunofluorescence
staining, we observed a strong co-localization of Beclin-1
and LC3 with Neun and GFAP. These results showed that after
SCI, the expression of these two proteins increases in neurons
and astrocytes, while treatment with co-ultraPEALut reduces
the levels of Beclin-1 and LC3 in both cell types. RETRAC Mol Neurobiol (2016) 53:3783–3792 3791 antioxidant gamma-glutamylcysteinyl ethyl ester. J Cereb Blood
Flow Metab 28(3):540–550 We conclude that co-ultraPEALut treatment at the dose of
1 mg/kg is capable to improve neurobehavioral functions, and
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creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appro-
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suggest that SCI induces autophagic cell death at the lesion
site; therefore, co-ultraPEALut treatment inhibiting autophagy
in in vivo model of SCI has protective effect. In conclusion,
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The authors would like to thank Maria Antonietta
Medici for her excellent technical assistance during this study and Mr
Francesco Soraci for his secretarial and administrative assistance and
Miss Valentina Malvagni for her editorial assistance with the manuscript. This study was supported by PON 01_02512. 13. Park JM, Tougeron D, Huang S, Okamoto K, Sinicrope FA (2014)
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https://openalex.org/W320603215
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https://www.nature.com/articles/srep10471.pdf
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English
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Hand Washing Induces a Clean Slate Effect in Moral Judgments: A Pupillometry and Eye-Tracking Study
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Scientific reports
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cc-by
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Hand Washing Induces a Clean
Slate Effect in Moral Judgments:
A Pupillometry and Eye-Tracking
Study received: 31 December 2014
accepted: 16 April 2015
Published: 21 May 2015 Kai Kaspar1,2, Vanessa Krapp2 & Peter König2,3 Physical cleansing is commonly understood to protect us against physical contamination. However,
recent studies showed additional effects on moral judgments. Under the heading of the “Macbeth
effect” direct links between bodily cleansing and one’s own moral purity have been demonstrated. Here we investigate (1) how moral judgments develop over time and how they are altered by hand
washing, (2) whether changes in moral judgments can be explained by altered information sampling
from the environment, and (3) whether hand washing affects emotional arousal. Using a pre-post
control group design, we found that morality ratings of morally good and bad scenes acquired more
extreme values in the control group over time, an effect that was fully counteracted by intermediate
hand washing. This result supports the notion of a clean slate effect by hand washing. Thereby, eye-
tracking data did not uncover differences in eye movement behavior that may explain differences
in moral judgments. Thus, the clean slate effect is not due to altered information sampling from
the environment. Finally, compared to the control group, pupil diameter decreased after hand
washing, thus demonstrating a direct physiological effect. The results shed light on the physiological
mechanisms behind this type of embodiment phenomenon. The fundamental goal of physical cleansing is to protect us against physical contamination and, hence,
to foster physiological health. However, empirical studies showed that physical cleansing also touches
psychological aspects. Already, Shakespeare described in his drama that Lady Macbeth repeatedly experi-
enced the desire to wash her hands after she incited her husband to kill the King of Scotland. Accordingly,
Zhong and Liljenquist1 found that moral impurity actually elicits the desire for cleansing-related prod-
ucts. This so-called “Macbeth effect” stimulated further research on the relationship between physical
and moral purity. In a follow-up study, they demonstrated that washing one’s hands after recalling an
unethical deed of the past reduced the motivation to volunteer, thus indicating that hand washing already
restored a suitable moral self-image and, hence, reduced the desire to compensate the unethical deed
by voluntary help. More recently, Reuven et al.2 replicated this finding, while the effect was stronger in
participants with obsessive-compulsive disorder. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 www.nature.com/scientificreports/ www.nature.com/scientificreports/ this Macbeth effect was accompanied by an active cortical network that comprises sensorimotor brain
areas, thus indicating an embodiment of the moral-purity metaphor. Also, Lobel et al.5 found that more
money was donated to charity before rather than after bathing for religious purification. Finally, Lee and
Schwarz6 found that the Macbeth effect seems to be specific with respect to the motor modality that is
primarily involved in the moral transgression. The desire for mouthwash was high after communicat-
ing a lie via voice mail, while the desire for hand sanitizer was higher after lying in an email. All these
observations demonstrate a direct link between one’s own bodily cleanliness and moral purity. Physical
cleansing seems to have the power to restore the perception of one’s own moral self that, in turn, may
reduce or even eliminate the Macbeth-like cognitions and behavior.f g
Research on embodied cognition provides a simple explanation for this effect: Williams et al.7 stated
that “early sensorimotor experiences serve as the foundation for the later development of more abstract
concepts and goals” (p. 1257). In early childhood, we learn the concept of physical purity and how to
achieve this goal. This knowledge serves as the scaffold for the later development of conceptually related
abstract ideas such as moral purity. Accordingly, Lakoff and Johnson8 proposed in their work on concep-
tual metaphors that abstract concepts are built upon earlier experiences that are often body-related. As
a consequence, established associations between the physical and abstract cognitive concept emerge that
are assumed to be stable across the whole lifespan9 and are presumably grounded on the same neuro-
anatomical foundation10. Similarly, according to Rozin’s body-to-soul-preadaptation theory of disgust11,
a neuronal circuitry, developed for the processing of the originally food-related emotion of disgust, was
preadapted for an extension to other threats beyond the rejection of food, such as moral threats. Indeed,
Schnall et al.12 found that the feelings of disgust increased the severity of moral judgments relative to
controls. Correspondingly, Moll et al.13 found that strongly overlapping brain regions were activated by
pure disgust (without moral connotations) and the moral emotion of indignation. The strong conceptual
and neuroanatomical relationship between moral and physical purity might explain why physical cleans-
ing can affect moral reasoning and vice versa.f gf
g
Importantly, the psychological effect of physical cleansing is not limited to the moral domain. www.nature.com/scientificreports/ Recent
studies showed that washing one’s hands can wash off traces of the past in general. Lee and Schwarz14
found that washing can reduce post-decisional dissonance effects and Xu et al.15 showed that the expe-
rience of bad luck and good luck can be washed away. Kaspar16 found that hand washing after failure
in an anagram task did not only increase optimism but also hampered future performance. The author
assumes that hand washing may have “lowered participants’ need to work harder in order to restore
their perception of competence” (p. 71). Finally, Florack et al.17 found that hand washing can reduce
decision preferences that are biased by ownership. Given these findings, the Macbeth effect may only be
one signature of a more general “clean slate effect,” as termed by Lee and Schwarz14. Correspondingly,
Xu et al.15 concluded that hand washing removes both desirable as well as undesirable traces of the past. Similarly, Florack et al.17 stated that “the physical action of hand washing resets the cognitive system to a
more neutral state” (p. 288). Hence, hand washing apparently does not only help to wash away our sins1
but also has the power to generally attenuate the effects of past events.f p
g
yf
p
However, in addition to the idea of a general clean slate effect, it has been shown that hand washing, as
well as the mere activation of the cleanliness concept, can alter moral judgments. This aspect is crucial, as
it includes the question of how cleanliness treatments work in a specific context. Zhong et al.18 reported
that study participants who had either actually cleansed their hands or merely visualized themselves in a
clean versus dirty state rated critical social issues, such as pornography and smoking, to be more morally
wrong. Also, participants who were primed for cleanliness reported a higher moral self-image that medi-
ated the effect of the cleanliness priming on moral judgments. This result indicates that the attribution
of physical purity to oneself generalizes to the impression of moral purity as well. Consequently, when
we feel to be morally clean, this may license harsher moral judgments. y
y
j
g
In contrast, Schnall et al.19 found that certain moral actions were judged less severe, in contrast to a
control condition, when the concept of physical cleanliness was activated either by a sentence unscram-
bling task or by washing one’s hands after experiencing disgust. Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 Hand Washing Induces a Clean
Slate Effect in Moral Judgments:
A Pupillometry and Eye-Tracking
Study In a study by Gollwitzer and Melzer3, inexperienced
video game players felt higher moral distress after playing a video game that included violence against
humans (versus objects), and they selected more hygiene products in a subsequent product choice task
than frequent video game players. Moreover, in a study by Denke et al.4, participants enacted a sequence
of scenarios that included either a moral deed (telling the truth) or an immoral one (lying). Immediately
after a scenario ended, they rated the desirability of different products, while only cleansing-related
products were rated as more desirable after an immoral act than after a moral one. At the same time, 1Social and Media Psychology, Department of Psychology, University of Cologne, Cologne, Germany. 2Neurobiopsychology, Institute of Cognitive Science, University of Osnabrück, Osnabrück, Germany. 3Department
of Neurophysiology and Pathophysiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Correspondence and requests for materials should be addressed to K.K. (email: kkaspar@uni-koeln.de) Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 1 1 The Present Study y
In contrast to previous studies on cleanliness effects, we applied a pre-post control group design. Hitherto,
it is unclear how moral judgments evolve across several issues of similar morality. Smith et al.22 found
that increasing the familiarity of information about a person leads to less systematic or analytic process-
ing of this information, thus resulting in more stereotyped judgments and less individualized judgments. Correspondingly, we assume that stereotyping in the moral domain would be reflected by more extreme
judgments when judging numerous morally good and bad actions in a sequence. In such a situation,
the notion of a clean slate effect suggests that hand washing in the meantime would counter increasing
stereotyping, thus leading to less extreme judgments after hand washing due to a reset of the cognitive
moral system. In contrast, the notion of an enhanced moral self by cleansing one’s hands would predict
even more severe moral judgments. However, previous studies have mainly focused on immoral actions
and, hence, the question arises as to whether the effect of hand washing would also generalize to mor-
ally good issues. Indeed, according to the idea of moral consistency, moral self-regard may motivate to
behave in a way that helps to maintain a high moral self5. In this sense, hand washing may also lead to
more extreme judgments in the case of morally good actions in order to perpetuate one’s high moral self
by emphasizing the praiseworthiness of moral actions.f y
p
g
p
Moreover, we asked whether hand washing modulates information sampling or whether it affects
moral judgements at a later stage of the judgment process. Two extreme scenarios are conceivable. The
act of hand washing could alter sampling information from the environment. The unchanged process of
moral judgement would act on a different set of sampled information and potentially reach a different
verdict. Alternatively, the act of hand washing could be neutral with respect to the sampling of informa-
tion, but the process of moral judgement would evaluate identical information differently. Only the latter
case would exemplify a direct link between hand washing and moral judgments. In fact, we propose to
interpret the effects of washing in terms of information sampling and weighting. The hitherto reported
effects showed that the act of cleansing did not literally wash away what had already happened in the
past. Instead, washing led to a specific (re-)weighting of the available information. The Present Study For example, hand
washing did not undo an unethical deed that participants had previously committed1, did not change
previous decisions14, and did not substitute a failure experience by success16, but it changed the weighting
of unethical deeds, reduced the need to devaluate non-chosen options, and increased one’s optimism
to be more successful in the future, respectively. In each of these cases, we can assume that the effect
was based on a selective sampling of information. In fact, as highlighted by Lindskog et al.23, “several
accounts of human judgment and decision making assume that people sample information from memory
prior to making a judgment or decision” (p. 783). However, we do not only selectively sample informa-
tion from memory but also from the environment, while the current information sampling depends on
the individual’s attention, capacity, and processing goals24. Thus, the effects of hand washing may base
on sampling biases on both stages. Following Smith et al.22, we expected that increasing the familiarity
of information (induced by a long sequence of moral vs. immoral scenes) would lead to a less systematic
processing of information and result in more stereotyped (i.e., extreme) moral judgments (see above). If hand washing affects information sampling from memory (e.g., when classifying a specific scene as
very immoral compared to social norms stored in memory), it should not show an effect on the visual
exploration of moral and immoral scenes. If, however, hand washing affects information sampling from
the environment, changes on the level of eye movements should be traceable. According to the notion of
a clean slate effect, washing should reset one’s cognitive state to a more neutral one17 and, hence, increase
peoples’ openness for each individual scene so that they are more motivated to visually explore it before
making a moral judgment. Correspondingly, Ybarra25 stated that “being cognitively open refers to the
tendency to seek out and integrate additional information into a current judgment” (p. 431). In fact,
when people repeatedly observe visual scenes, their viewing activity decreases, while higher interest in
visual scenes elicits a more active exploration of the scenes26. In contrast, if washing elicits the impression
of one’s own moral cleanliness, even more severe moral judgments are conceivable, which could result
from an additionally lowered engagement in stimulus exploration.f y
g g
p
Finally, does hand washing affect the physiological level or is its impact limited to purely disembodied
cognitive processes? www.nature.com/scientificreports/ Zhong et al.18 proposed a simple expla-
nation for the seemingly contradicting results; in their study, the activation of the cleanliness concept
apparently restored one’s own moral self-image, and in the work of Schnall et al.19, the state of cleanliness
was perhaps attributed to the judged target but not to the self of the judging person. Hence, it seems
to be crucial who is viewed as clean. Johnson et al.20 failed to replicate the results of Schnall et al.19,
but in a more recent replication study, Huang21 showed that less severe moral judgments only occurred
when study participants invested low effort in the sentence unscrambling task. We suppose that low task
involvement facilitated the attribution of cleanliness to the judged targets.f j
g
g
To conclude, activating cleanliness cognitions with a priming procedure showed mixed effects that
are presumably signatures of different attribution processes. In contrast, washing one’s own hands makes
the attribution of a clean state to oneself indubitable. Thereby, two mechanisms have been posited so
far: on the one hand, washing may lead to an increase in one’s perceived moral purity and, hence, makes
judgments about others’ moral transgressions more severe. On the other hand, washing may lead to a
general clean slate effect, thus attenuating the impression of past events and setting the cognitive system
to a more neutral state. The present study contrasts these two potential mechanisms in the moral domain
by changing the experimental paradigm and methodology. 2 www.nature.com/scientificreports/ Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 Methods
All h The display resolution was 1280 × 960 pixels, the refresh rate was 85 Hz,
and the distance to the screen was set at 80 cm without a headrest to facilitate normal viewing conditions. The computer running the experiment was connected to the host computer (Pentium 4, Dell Inc., Round
Rock, TX, USA) with EyeLink software via a local network. yt
An EyeLink II system (SR Research, Ontario, Canada) recorded subjects’ pupil size and eye move-
ments at a sampling rate of 500 Hz and compensated for head movements. In order to calibrate the sys-
tem, the subjects made saccades to a grid of 13 fixation spots on the screen, which appeared one by one
in a random order. The tracking of the eye, which provided the lower validation error, started as soon
as this value was below the 0.5° visual angle. A fixation spot appeared after each trial in the middle of
the screen in order to control for slow drifts in measured eye movements. In the cases of an error larger
than 1°, calibration and validation were repeated. Procedure. Upon arrival, the subjects were introduced into the study procedure and had to sign a
consent form. As a recent study revealed the strong impact of mood on how people observe complex
images31, the subjects initially rated their current mood on four 5-point Likert-items to assess their cur-
rent happiness, alertness, calmness, and listlessness32. This measurement enabled us to check whether
potential differences in the dependent variables between the washing and non-washing group were con-
taminated by pre-experimental differences in subjects’ mood. No difference was revealed, all t(38) ≤ 1.29,
p ≥ 0.206. Afterward, the subjects sat down in front of a screen. The experimenter instructed them to
freely observe each of the following images and to subsequently rate the morality of the depicted scene by
a key press. Then, the eye-tracker was calibrated and validated. Each of the 72 scenes was presented for
six seconds in accordance to previous studies (e.g.,26,31,33) and were followed by a screen that asked to rate
the morality of the previous scene on a 9-point scale from 1 (very immoral) to 9 (very moral). The next
scene was presented after a corresponding key press. After the subjects had observed half of the scenes
(block 1), a break was introduced. Methods
All h All the participants gave written informed consent to participate in this study. We performed the study in
accordance with the Declaration of Helsinki and national guidelines of the German Psychological Society. The experimental methods were approved by the Ethical Committee of the University of Osnabrück
(Germany). Participants. Forty subjects (16 males), who were naive to the purpose of the study, participated for
course credits. Their average age was 22.25 years (SD = 3.85). We selected the number of subjects accord-
ing to previous studies in the field that reported significant differences between a real cleansing condition
and a control condition in which no hand cleansing was applied. Across four studies,14 (Study 1;15 Study 1
and 2;19 Study 2), a sample size of n = 20 per condition was found to be sufficient. All subjects had
normal or corrected-to-normal visual acuity and passed the Ishihara Test for Color Blindness29. They
were randomly assigned to one of the two experimental conditions (washing group vs. no washing group). Material and Apparatuses. The subjects observed 72 complex images, 36 immoral scenes, and 36
moral scenes. The immoral scenes were selected in accordance to the morality ratings of different nega-
tive social issues18, and the moral scenes were selected according to the notion that morally good actions
are those that bring about a great amount of happiness and the least amount of physical or psychological
pain for other persons or the environment. The scenes depicted human behavior that is either morally bad
(e.g., physically beating someone up, polluting the environment, stealing something, taking illegal drugs)
or good (e.g., helping old people, handing a gift, caring for children, laughing together). The images were
partially borrowed from the International Affective Picture Set (IAPS,30) and partially borrowed from
the internet. All images had a resolution of 1280 × 960 and were full-colored. Two independent raters
categorized all images and completely agreed. Inter-rater agreement was additionally validated by a sam-
ple of 20 subjects (not included in the eye-tracking session) who observed all images in a randomized
order and rated each image on a 9-point scale from 1 (very immoral) to 9 (very moral). On average, all
36 immoral scenes were rated ≤ 3.95 (SD = 1.79), and all 36 moral scenes were rated ≥ 5.60 (SD = 1.57). Images were presented on a 21-in. Samsung SyncMaster 1100 DF 2004 CRT Monitor (Samsung
Electronics, Seoul, South Korea). The Present Study This is an important question, as the majority of results rests on self-reports by the
experimental subjects being susceptible to many external factors. Previous studies exclusively focused on
the self-reported emotional effects of cleanliness and provided mixed results. While Liljenquist et al.27
found no effect of a clean-scented room on positive and negative affect, Zhong and Liljenquist1, as well
as Reuven et al.2, found reduced negative emotions by hand washing after recalling an unethical deed of
the past. According to the clean slate effect of washing, the level of participants’ arousal should also be
reset to a more neutral state. To summarize, the present study addresses the three questions described above: (1) we used a
pre-post control group design to investigate whether hand washing would cushion increasing stereotyp-
ing in moral judgments, as suggested by Smith et al.22. Accordingly, we hypothesized that the difference
in morality ratings for moral versus immoral scenes increases over time. Thereby, hand washing may
induce a generalized “clean slate effect”14,15 for immoral as well as moral judgments that counteract this
tendency or, alternatively, washing may elicit even more extreme judgments due to the impression of an
enhanced moral self-image18 (Hypothesis 1, Change in Morality Ratings). (2) We applied eye-tracking
methodology to clarify whether the sampling and processing of information is actually influenced by Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 3 www.nature.com/scientificreports/ hand washing, thus providing an objective measurement. This allows testing of the hypothesis that the
impact of hand washing is limited to the formation of moral judgments and does not touch early pro-
cesses of information gathering (Hypothesis 2, Information Sampling). (3) We tracked pupil size before
and after hand washing as an objective measurement for subjects’ emotional arousal28. If the clean slate
effect of hand washing also affects physiological arousal, pupil diameter should decrease after hand wash-
ing (Hypothesis 3, Physiological Effects). Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 Results
H
h Hypothesis 1: Change in Morality Ratings. We initially calculated an IMAGE SET (moral vs. immoral scenes) x TIME POINT (before vs. after the break) x WASHING CONDITION (washing vs. no washing during the break) mixed-measures ANOVA (Greenhouse-Geisser applied). As expected,
we found a significant effect of the image set on the morality ratings, F(1, 38) = 703.67, p < 0.001,
ηp
2 = 0.95, with much higher ratings for the moral images (M = 7.38, SD = 0.84) than for the immoral
images (M = 2.24, SD = 0.66) (see Fig. 1). Moreover, we found an interaction between the image set
and time point, F(1, 38) = 4.93, p = 0.032, ηp
2 = 0.12, a marginally significant interaction between image
set and washing condition, F(1, 38) = 2.78, p = 0.104, ηp
2 = 0.07, as well as a three-way interaction, F(1,
38) = 4.21, p = 0.047, ηp
2 = 0.10. No further effects occurred, all F(1, 38) ≤ 0.60, p ≥ 0.444, ηp
2 ≤ 0.02. Because we were primarily interested in the potential within-subject changes between the first and sec-
ond block of the experiment, we subsequently investigated whether the morality ratings changed within
the washing group and the no washing group. In fact, the no washing group showed more extreme morality
ratings in block 2 (t2) than in block 1 (t1) in both directions. The ratings for the moral scenes increased
from the first to the second block, t(19) = − 2.11, p = 0.049, d = 0.47, while the ratings for the immoral
scenes decreased, t(19) = 2.21, p = 0.040, d = 0.49. In contrast, in the washing group, neither the morality
ratings of the moral scenes changed nor the morality ratings for the immoral images, both t(19) ≤ 0.57,
p ≥ 0.578, d ≤ 0.13.f p≥
≤
To make this interaction more visible, we computed the difference scores between t2 (block 2) and
t1 (block 1), which served as a dependent variable in a 2 × 2 (IMAGE SET x WASHING CONDITION)
ANOVA. As shown in Fig. Methods
All h The subjects were told that they had to perform a sensorimotor coor-
dination task in order to maintain concentration and attention for the second part of the eye-tracking
session. The subjects played Jenga for two minutes, but half of them previously washed their hands, alleg-
edly due to hygienic reasons (washing group), whereas the control group (no washing group) waited for
an additional two minutes. After the break, the subjects continued with scene observation and evaluation
(block 2). The sequence of scenes was constant across the subjects in favor of the between-subject com-
parison (washing vs. no washing group). Randomized scene sequences usually produce additional noise Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 4 www.nature.com/scientificreports/ in the eye-tracking data, and they do not counterbalance the potential priming effects in the viewing
behavior from one scene to the next (cf.31). However, the order of scenes was pseudo-randomized within
the subjects; that is, three of six successively presented scenes depicted immoral content, while the next
immoral scene was not predictable. p
Moreover, as we were interested in a within-subject effect of hand washing between the first and sec-
ond half of the eye-tracking session, we ensured that both subsets of scenes (18 immoral and 18 moral
scenes before as well as after the break) did not differ in visual saliency. Therefore, each of the 72 scenes
was analyzed by means of a saliency map computation. The standard algorithm34 predicts the fixation
patterns by a decomposition of pictures into several physical features (e.g., contrast and orientation). Additionally, we applied a more recent graph-based visual saliency algorithm (GBVS) to predict the
fixations with an even higher reliability35. Both algorithms revealed no difference between the scenes of
the first and second eye-tracking part, both t(35) ≤ 0.10, p ≥ 0.924.i i
y
g p
p
Finally, in order to check whether the image ratings depended on the specific image sequence in the
eye-tracking session, a third group (comparison group) of 20 subjects with a mean age of 20.75 (SD = 2.65)
observed and rated all images in a randomized order (without eye-tracking and an intermediate break). Eye Movement Parameters. In order to appropriately describe the viewing behavior of study par-
ticipants, we selected parameters that are both very sensitive to the emotional and motivational states of
the observer as well as a suitable indicator for global viewing activity26,31. Methods
All h In accordance with previous
studies on human vision, we therefore focused on the mean length of saccades as an indicator for the
visual step size that participants use to scan the scenes. The detection of a saccade was based on the
following three default measures: amplitude of at least 0.1° visual angle, with a velocity of at least 30°/s,
and with an acceleration of at least 8000°/s2. The minimal saccade velocity was 25°/s after saccade onset. All these values had to be sustained for at least 4 ms for a valid saccade. In contrast, fixations were
defined as periods without saccades36. We also investigated the mean duration of fixations because it is a
sensitive indicator for information processing depth. It is believed that longer fixation durations indicate
a deeper and more extensive processing of visual stimuli37,38. The eye-tracker detected and automatically
parameterized fixations and saccades during each fixation. The first fixation of each trial was excluded
from the analysis because its localization was determined by the preceding fixation spot used for drift
correction. Finally, we applied an entropy measurement to quantify the extent of the spatial exploration
of the scenes. Entropy has the advantage of measuring the spread of fixation distributions without the
necessity to make assumptions about scene-specific geometrical arrangements. Higher entropy values
indicate a more extensive fixation distribution; that is, a more spatially extended exploration of a scene. Thereby, the absolute values in entropy are negligible, as they depend on image resolution and the size of
the Gaussian kernel that we used to convolve the fixation distribution map (leading to a fixation density
map that is used for entropy calculation). This procedure is described in detail elsewhere39. Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. The effect of hand washing on morality ratings. The left panel depicts absolute values, the right
panel depicts the change in morality ratings from block 1 to block 2 by means of difference scores (t2 minus
t1). Vertical lines indicate the standard error of the mean. Figure 1. The effect of hand washing on morality ratings. The left panel depicts absolute values, the right
panel depicts the change in morality ratings from block 1 to block 2 by means of difference scores (t2 minus
t1). Vertical lines indicate the standard error of the mean. the image sequence was randomized across subjects. In this group, the difference scores for the moral
and immoral scenes (mean of last 36 images vs. first 36 images) also differed significantly, t(19) = 2.38,
p = 0.028, d = 0.53, with increased morality ratings for moral scenes (+ 0.158), but slightly decreased
ratings for immoral scenes (− 0.053). g
(
)
To conclude, on a scale ranging from very immoral to very moral, we found much higher morality
ratings for the moral scenes than for the immoral scenes, thus validating our stimulus set. Even more
importantly, we found more stereotyped morality judgments over time (block 2 vs. block 1), but this
effect disappeared when participants washed their hands during the break. Thus, the results support
the notion of a clean slate effect. Hand washing apparently reset the dynamic in participants’ morality
judgments. Hypothesis 2: Information Sampling. With respect to the mean duration of fixations, we initially
calculated a 2 × 2 × 2 (IMAGE SET x TIME POINT x WASHING CONDITION) ANOVA. As shown
in Fig. 2, we found a significant main effect of the image set, F(1, 38) = 103.60, p < 0.001, ηp
2 = 0.73,
with longer fixations on the immoral versus moral scenes. We also found a main effect of the time
point, F(1, 38) = 4.56, p = 0.039, ηp
2 = 0.11, with longer fixations in block 2 versus block 1. The image
set and time point also significantly interacted, F(1, 38) = 15.08, p < 0.001, ηp
2 = 0.28, whereby the effect
of the time point (i.e., longer fixation durations in block 2 vs. www.nature.com/scientificreports/ block 1) was only present on moral
scenes, t(39) = 4.36, p < 0.001, d = 0.69, but not on immoral scenes, t(39) = − 0.38, p = 0.70, d = 0.06. The
ANOVA did not show further significant effects, all F(1, 38) ≤ 0.47, p ≥ 0.495, ηp
2 ≤ 0.01, except a just
significant interaction between image set and washing condition, F(1, 38) = 4.00, p = 0.053, ηp
2 = 0.10,
with a higher difference between immoral and moral scenes in the no washing group versus washing
group. However, and importantly, this slight interaction has to be interpreted with caution. Although
the three-way interaction did not reach statistical significance, it is not apparent how much of the group
difference is due to between-subject differences that already existed from the outset; that is, before the
cleanliness treatment was applied to the washing group. Thus, to clarify whether hand washing actu-
ally affected participants’ fixation durations, we again calculated the difference scores between the two
blocks (t2 minus t1) that represent the temporal change and thus correct for the group differences that
might have already existed before the cleanliness treatment. We used the difference scores as a depend-
ent variable in a 2 × 2 (IMAGE SET × WASHING CONDITION) ANOVA. We found no effect of the
washing condition and no interaction, both F(1, 38) ≤ 0.47, p ≥ 0.495, ηp
2 ≤ 0.01, but the difference in
the fixation duration between block 1 and block 2 differed between the moral and immoral scenes, F(1,
38) = 15.08, p < 0.001, ηp
2 = 0.28, thus reflecting the image set by time point interaction reported above. As shown by Fig. 2, in both experimental groups, the fixation durations on moral scenes were longer after
the break, both t(19) = 2.76, p ≤ 0.012, d ≥ 0.62, while there was no difference between the two groups,
t(38) = 0.99, p = 0.330, d = 0.31. Regarding the immoral scenes, the difference scores neither differed
from zero within the two groups, both |t|(19) ≤ 0.39, p ≥ 0.700, d ≤ 0.09, nor between them, t(19) = 0.11,
p = 0.912, d = 0.04. Consequently, hand washing did not significantly affect the duration of fixations. Results
H
h 1, we again found no main effect of the washing condition, F(1, 38) = 0.03,
p = 0.866, ηp
2 < 0.01, but the difference scores revealed a significant main effect of the image set, F(1,
38) = 4.93, p = 0.032, ηp
2 = 0.12, with a negative difference score for the moral scenes, but a positive dif-
ference score for the immoral scenes. This main effect reflects the image set by the time point interaction
found in the initial three-way ANOVA. Moreover, the image set interacted with the washing condition,
F(1, 38) = 4.21, p = 0.047, ηp
2 = 0.10, thus reflecting the three-way interaction previously found. p
ηpl
g
y
p
y
Finally, in order to verify that the tendency toward more extreme morality ratings in the no washing
group was not an artefact of the constant sequence of scenes, we tested the comparison group, in which Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 5 www.nature.com/scientificreports/ I
h
d h l
h
f
d
h
d
h ifi
In the next step, we investigated the length of saccades that participants made to scan the scenes. The 2 × 2 × 2 (IMAGE SET x TIME POINT x WASHING CONDITION) ANOVA showed a main 6 Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 www.nature.com/scientificreports/ Figure 2. The mean duration of fixations. The left panel depicts absolute values, the right panel depicts the
change in fixation duration from block 1 to block 2 by means of difference scores (t2 minus t1). Vertical lines
indicate the standard error of the mean. Figure 2. The mean duration of fixations. The left panel depicts absolute values, the right panel depicts the
change in fixation duration from block 1 to block 2 by means of difference scores (t2 minus t1). Vertical lines
indicate the standard error of the mean. Figure 3. The mean length of saccades (left panel) and the mean entropy quantifying the extent of spatial
exploration of scenes (right panel). Vertical lines indicate the standard error of the mean. Figure 3. The mean length of saccades (left panel) and the mean entropy quantifying the extent of spatial
exploration of scenes (right panel). Vertical lines indicate the standard error of the mean. effect of the time point, F(1, 38) = 23.71, p < 0.001, ηp
2 = 0.38, with shorter saccades in block 2 versus
1 (see Fig. 3). Moreover, a main effect of the image set occurred, F(1, 38) = 31.42, p < 0.001, ηp
2 = 0.45,
with longer saccades on immoral versus moral scenes. No further effects were found, F(1, 38) ≤ 2.05,
p ≥ 0.160, ηp
2 ≤ 0.05. Accordingly, when using difference scores (t2 minus t1) in a 2 × 2 (IMAGE SET x
WASHING CONDITION) ANOVA, no effect remained, all F(1, 38) ≤ 2.05, all p ≥ 0.160, all ηp
2 ≤ 0.05,
because calculating the difference scores naturally eliminated the main effects of the time point and
image set. ll
d h
f h
l
l
f
fi d b
Th Finally, we investigated the extent of the spatial exploration of images quantified by entropy. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. The effect of hand washing on pupil size. The left panel depicts absolute values, the right panel
depicts the change in pupil size from block 1 to block 2 by means of difference scores (t2 minus t1). Vertical
lines indicate the standard error of the mean. Figure 4. The effect of hand washing on pupil size. The left panel depicts absolute values, the right panel
depicts the change in pupil size from block 1 to block 2 by means of difference scores (t2 minus t1). Vertical
lines indicate the standard error of the mean. To sum up, the viewing behavior changed between block 1 and block 2 in terms of longer fixations
on the moral (but not immoral) scenes, which were paralleled by a less spatially extensive explora-
tion (entropy). Moreover, saccades were longer before versus after the break, and they were longer on
immoral versus moral scenes. However, and in contrast to the morality judgments, hand washing did
not influence eye movement parameters. Consequently, the impact of hand washing did not touch early
processes of information sampling from the environment. Hypothesis 3: Physiological Effects. We initially calculated a 2 × 2 × 2 (IMAGE SET × TIME
POINT × WASHING CONDITION) ANOVA with pupil size as the dependent variable. As shown
by Fig. 4, we found a significant main effect of the image set, F(1, 38) = 196.51, p < 0.001, ηp
2 = 0.84,
with larger pupil sizes when observing immoral versus moral scenes. We also found a main effect of
the time point, F(1, 38) = 53.91, p < 0.001, ηp
2 = 0.59, with larger pupil sizes in block 1 versus block
2. Moreover, we found a marginally significant main effect of the washing condition, F(1, 38) = 2.89,
p = 0.097, ηp
2 = 0.07, with larger pupil sizes in the washing versus no washing group. Also, we found
an interaction between the time point and washing condition, F(1, 38) = 4.61, p = 0.038, ηp
2 = 0.11. The
three-way interaction was marginally significant, F(1, 38) = 2.78, p = 0.104, ηp
2 = 0.07. No interaction
between image set and washing condition, as well as between image set and time point, occurred, both
F(1, 38) ≤ 0.22, p ≥ 0.643, ηp
2 ≤ 0.01. www.nature.com/scientificreports/ p
ηp
In order to further scrutinize the interactions with regard to the impact of the washing treatment, we
again computed the difference scores (t2 minus t1) serving as the dependent variable in a 2 × 2 (IMAGE
SET × WASHING CONDITION) ANOVA. This analysis revealed no main effect of the image set, F(1,
38) = 0.22, p = 0.643, ηp
2 = 0.01, but a marginally significant interaction, F(1, 38) = 2.78, p = 0.104,
ηp
2 = 0.07, that reflected the three-way interaction reported above. Finally, the washing condition showed
a significant main effect, F(1, 38) = 4.61, p = 0.038, ηp
2 = 0.11, that reflected the washing condition by
time point interaction reported above. As Fig. 4 shows, in both conditions (washing and no washing),
the difference in pupil size was significantly negative (one-sample t-tests against zero: all p < 0.001), thus
indicating a smaller pupil size after the break. However, the decrease in pupil size was significantly larger
when subjects had washed their hands between block 1 and block 2. Consequently, hand washing also
affected the physiological level. Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 www.nature.com/scientificreports/ The
2 × 2 × 2 (IMAGE SET × TIME POINT × WASHING CONDITION) ANOVA again showed a main effect
of the time point, F(1, 38) = 9.13, p = 0.004, ηp
2 = 0.19, a main effect of the image set, F(1, 38) = 79.62,
p < 0.001, ηp
2 = 0.68, and an interaction between these two factors, F(1, 38) = 20.65, p < 0.001, ηp
2 = 0.35. As depicted in Fig. 3, the subjects explored moral scenes more spatially extensively in block 1 versus
block 2, t(39) = 5.06, p < 0.001, d = 0.80, while the immoral scenes were equally extensively explored
before versus after the break, t(39) = − 0.302, p = 0.765, d = 0.05. The ANOVA did not show further
significant effects, all F(1, 38) ≤ 1.29, all p ≥ 0.264, all ηp
2 ≤ 0.03. 7 Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 7 Discussion Hand washing is a daily ritual whose fundamental goal is to protect us against physical and biological
contamination and, hence, foster physiological health. However, empirical evidence showed that hand
washing also affects moral judgments. Unmoral deeds enhance the desire for cleansing products in terms
of a Macbeth effect1,3,4, and the impact of washing goes beyond judgment formation, as it induces a kind
of clean slate effect in several domains14–17. The present study addresses three critical questions focused
on the original domain of moral judgments.f First, we found that hand washing between the two blocks induced a clean slate effect in the form of
eliminating the tendency for more extreme moral judgments over time. This finding supports previous
studies that reported clean slate effects regarding cognitive dissonance14, good and bad luck15, failure
experience16, and product evaluation17. In each case, physical cleansing washed away traces of the past
by resetting previous experiences. The present within-subject design revealed such a general clean slate 8 Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 www.nature.com/scientificreports/ effect in the domain of moral judgments, thus generalizing to the judgment of other people’s bad and
good deeds (Hypothesis 1). Second, we found more extreme moral judgments over time for moral and immoral scenes as long
as no hand washing took place in the meantime. This finding corresponds to the results of Smith et
al.22, who found that increased familiarity of information fosters stereotyped judgments. Smith et al. explained this by changes in information processing. According to their findings, increasing familiar-
ity of information leads to less systematic or analytic processing, thus resulting in more stereotyped
judgments. Indeed, we found changes in viewing activity as a signature of image processing from the
first to the second block. This effect was, however, mainly limited to the moral scenes: fixation duration
increased and the amount of spatial exploration decreased on the moral images over time while no
change occurred on the immoral scenes. Moreover, saccades were shorter in block 2 versus 1, and they
were longer on immoral versus moral scenes. This result pattern matches studies showing that images of
negative valence are observed more actively31. Beyond this, we found no hint that hand washing affects
eye-tracking parameters. This result is evidence against an effect of physical cleansing on information
sampling from the environment. Discussion Instead, the effect of hand washing seems to affect the selection and/or
weighting of information stored in memory (e.g., social norms).h Third, the present study provides objective evidence for an impact of physical cleansing on the phys-
iological level. While previous studies limited the focus to self-reports, we analyzed pupil size as an
indicator for emotional arousal28. We found that pupil size decreased from the first to the second meas-
urement block, while hand washing in between the measurement blocks additionally decreased pupil size
(Hypothesis 3). Presumably, the subjects successively habituated to the (im)moral scenes indicated by
a downregulation of arousal. The immoral scenes are especially arousing, as shown by the norm values
of the IAPS manual30. Hand washing seems to facilitate emotional downregulation, as also suggested by
Zhong and Liljenquist1, as well as Reuven et al.2, who found attenuated reports of negative emotions
when subjects washed their hands after they had recalled an unethical deed of the past. Consequently, the
clean slate effect is more than a cognitive reset, and hand washing actually affects the physiological level.ii f
g
g
yf
p y
g
Given the present findings, future studies in the field of embodied cognition research should fathom
the psychophysiological effects of phenomena that have been primarily investigated on the behavioral
level so far – such as physical cleansing. A bulk of studies has already shown that the effects of bodily
sensations are not limited to cognitive processes but they also affect emotional states (cf.10,40). The effect
of hand washing on pupil size and the unaffected eye movement behavior indicate a fruitful avenue for
follow-up studies. Uncovering the psychophysiological mechanisms behind embodiment phenomena not
only increases our understanding of the cognitive consequences of bodily sensations such as morality
judgments. It also helps to exploit the potential for therapeutic and other applied purposes. References 2. Reuven, O., Liberman, N. & Dar, R. The effect of physical cleaning on threatened morality in individuals with obsessive
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We gratefully acknowledge financial support by the European Commission (H2020-FETPROACT-2014,
SEP-210141273, ID: 641321 socSMCs; FP7-ICT-270212 eSMCs) and the European Research Council
(ERC-2010-AdG #269716 – MULTISENSE) (PK). g
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Science 19, 1219–1222 (2008). Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 9 Additional Informationi Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Kaspar, K. et al. Hand Washing Induces a Clean Slate Effect in Moral
udgments: A Pupillometry and Eye-Tracking Study. Sci. Rep. 5, 10471; doi: 10.1038/srep10471 (2015). How to cite this article: Kaspar, K. et al. Hand Washing Induces a Clean Slate Effect in Moral
Judgments: A Pupillometry and Eye-Tracking Study. Sci. Rep. 5, 10471; doi: 10.1038/srep10471 (201 This work is licensed under a Creative Commons Attribution 4.0 International License. The
images or other third party material in this article are included in the article’s Creative Com-
mons license, unless indicated otherwise in the credit line; if the material is not included under the
Creative Commons license, users will need to obtain permission from the license holder to reproduce
the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports | 5:10471 | DOI: 10.1038/srep10471 10
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https://openalex.org/W4220710542
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https://lirias.kuleuven.be/bitstream/20.500.12942/694156/2/Cerebrospinal%20fluid%20tau%20levels%20are%20associated%20with%20abnormal%20neuronal%20plasticity%20markers%20in%20Alzheimers%20disease.pdf
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English
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Cerebrospinal fluid tau levels are associated with abnormal neuronal plasticity markers in Alzheimer’s disease
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Molecular neurodegeneration
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cc-by
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Abstract Background: Increased total tau (t-tau) in cerebrospinal fluid (CSF) is a key characteristic of Alzheimer’s disease (AD)
and is considered to result from neurodegeneration. T-tau levels, however, can be increased in very early disease
stages, when neurodegeneration is limited, and can be normal in advanced disease stages. This suggests that t-tau
levels may be driven by other mechanisms as well. Because tau pathophysiology is emerging as treatment target for
AD, we aimed to clarify molecular processes associated with CSF t-tau levels. Methods: We performed a proteomic, genomic, and imaging study in 1380 individuals with AD, in the preclinical,
prodromal, and mild dementia stage, and 380 controls from the Alzheimer’s Disease Neuroimaging Initiative and
EMIF-AD Multimodality Biomarker Discovery study. Results: We found that, relative to controls, AD individuals with increased t-tau had increased CSF concentrations
of over 400 proteins enriched for neuronal plasticity processes. In contrast, AD individuals with normal t-tau had
decreased levels of these plasticity proteins and showed increased concentrations of proteins indicative of blood–
brain barrier and blood-CSF barrier dysfunction, relative to controls. The distinct proteomic profiles were already
present in the preclinical AD stage and persisted in prodromal and dementia stages implying that they reflect disease
traits rather than disease states. Dysregulated plasticity proteins were associated with SUZ12 and REST signaling,
suggesting aberrant gene repression. GWAS analyses contrasting AD individuals with and without increased t-tau
highlighted several genes involved in the regulation of gene expression. Targeted analyses of SNP rs9877502 in GMNC,
associated with t-tau levels previously, correlated in individuals with AD with CSF concentrations of 591 plasticity
associated proteins. The number of APOE-e4 alleles, however, was not associated with the concentration of plasticity
related proteins. *Correspondence: pj.visser@amsterdamumc.nl;
pj.visser@maastrichtuniversity.nl
1 Alzheimer Center Amsterdam, Department of Neurology, Amsterdam
Neuroscience, Vrije Universiteit Amsterdam, PO Box 7057 1007, MB,
Amsterdam, The Netherlands
Full list of author information is available at the end of the article *Correspondence: pj.visser@amsterdamumc.nl;
pj.visser@maastrichtuniversity.nl
1 Alzheimer Center Amsterdam, Department of Neurology, Amsterdam
Neuroscience, Vrije Universiteit Amsterdam, PO Box 7057 1007, MB,
Amsterdam, The Netherlands
Full list of author information is available at the end of the article © The Author(s) 2022. Visser et al. Molecular Neurodegeneration (2022) 17:27
https://doi.org/10.1186/s13024-022-00521-3 Open Access Cerebrospinal fluid tau levels are associated
with abnormal neuronal plasticity markers
in Alzheimer’s disease Pieter Jelle Visser1,2,3* , Lianne M. Reus1, Johan Gobom4,5, Iris Jansen6, Ellen Dicks1, Sven J. van der Lee1,7,
Magda Tsolaki8, Frans R. J. Verhey2, Julius Popp9,10, Pablo Martinez‑Lage11, Rik Vandenberghe12,13,
Alberto Lleó14, José Luís Molinuevo15,16, Sebastiaan Engelborghs17,18, Yvonne Freund‑Levi3,19, Lutz Froelich20,
Kristel Sleegers21,22, Valerija Dobricic23, Simon Lovestone24, Johannes Streffer17,25, Stephanie J. B. Vos2,
Isabelle Bos2, ADNI, August B. Smit26, Kaj Blennow4,5, Philip Scheltens1, Charlotte E. Teunissen27,
Lars Bertram22,28, Henrik Zetterberg4,5,29,30 and Betty M. Tijms1 CSF analysis CSF samples in EMIF-AD MBD were collected according
to the BIOMARKAPD protocol, [12, 18] and in ADNI as
described elsewhere [19]. Abstract Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 16 Visser et al. Molecular Neurodegeneration (2022) 17:27 Conclusions: CSF t-tau levels in AD are associated with altered levels of proteins involved in neuronal plasticity and
blood–brain and blood-CSF barrier dysfunction. Future trials may need to stratify on CSF t-tau status, as AD individuals
with increased t-tau and normal t-tau are likely to respond differently to treatment, given their opposite CSF prot‑
eomic profiles. Keywords: Alzheimer’s disease, Molecular mechanisms, Biomarker discovery, Heterogeneity, Neuronal plasticity,
Cerebrospinal fluid proteomics Conclusions: CSF t-tau levels in AD are associated with altered levels of proteins involved in neuronal plasticity and
blood–brain and blood-CSF barrier dysfunction. Future trials may need to stratify on CSF t-tau status, as AD individuals
with increased t-tau and normal t-tau are likely to respond differently to treatment, given their opposite CSF prot‑
eomic profiles. Keywords: Alzheimer’s disease, Molecular mechanisms, Biomarker discovery, Heterogeneity, Neuronal plasticity,
Cerebrospinal fluid proteomics Clinical assessment Global cognition was assessed by the Mini-Mental State
Examination (MMSE) [14] and ADAS-Cog 11-item ver-
sion (ADNI) [15]. As a measure for memory function we
used the delayed recall of the logical memory subscale
II of the Wechsler Memory Scale (ADNI), [16] or center
specific verbal word learning tasks (EMIF-AD MBD)
[12]. We selected the Clinical Dementia Rating (CDR)
scale sum of boxes score as a measure of disease severity
[17]. Group definition and staging We selected individuals with AD pathology defined as
abnormally decreased CSF amyloid beta 1–42 (Aβ1-42),
and we subdivided this group into those with abnor-
mally increased total tau (t-tau) and those with normal
t-tau. Based on cognitive performance, AD individuals
were classified in 3 clinical stages as preclinical AD (nor-
mal cognition), prodromal AD (MCI) and mild AD-type
dementia according to study specific criteria [12, 13]. The
control group consisted of individuals with normal cog-
nition, and normal CSF Aβ1-42 and t-tau levels. Backgroundh collaboration under the supervision of Principle Inves-
tigator Michael W. Weiner, MD. The primary goal of
ADNI is to study whether serial biological markers, and
clinical and neuropsychological measures can be com-
bined to measure progression of MCI and early AD and
has enrolled 2850 individuals, see www.adni-info.org. The EMIF-AD MBD study aimed to identify markers for
diagnosis and prognosis of predementia AD. It combined
existing clinical data, samples and scans of 1218 individu-
als with normal cognition, MCI or mild dementia from
prospective cohort studies [12]. g
The amyloid cascade hypothesis poses that Alzheimer’s
disease (AD) starts with amyloid beta (Aβ) aggregation
followed by tau pathology [1]. Increased total tau (t-tau)
levels in cerebrospinal fluid (CSF) are supposed to be
caused by axonal loss [2]. Still, 25% of the AD individuals
with mild cognitive impairment (MCI) or dementia have
normal t-tau levels despite axonal loss [3, 4]. Moreover,
CSF t-tau is abnormally increased in 50% of preclinical
AD individuals, when neurodegeneration is limited [5]. Furthermore, our study in cognitively normal monozy-
gotic twins discordant for amyloid aggregation, suggested
that CSF t-tau levels may rise even before amyloid aggre-
gation reaches abnormal levels [6]. An alternative expla-
nation for the increase in t-tau in AD may be increased
gene expression. A SILK study found that elevated CSF
t-tau levels in AD were associated with increased pro-
duction rather than from spilling of intracellular tau by
dying neurons [7]. A study with induced pluripotent stem
cells (iPSC) showed that AD individuals had increased
tau expression in neuronal progenitor cells compared
to controls [8]. Increased t-tau levels may also result of
increased neuronal activity, [9] or increased neuronal
plasticity as t-tau has an high expression in brain regions
with plastic potential [10]. As the drivers of increased
tau levels in AD remain largely unknown, and because
tau-related mechanisms are emerging as new treatment
targets for AD, we aimed to clarify molecular processes
associated with CSF tau dysregulation. To this end, we
performed a proteomic, genomic and imaging study
including 1380 individuals in the AD continuum, [11]
defined by abnormal CSF Aβ1-42, spanning the clinical
spectrum from preclinical AD to mild dementia, and 380
controls with normal cognition and normal CSF Aβ1-42
and t-tau from the Alzheimer’s Disease Neuroimaging
Initiative (ADNI) and the European Medical Information
Framework for Alzheimer’s disease (EMIF-AD) Multi-
modality Biomarker Discovery (MBD) study [2]. Study participants CSF Aβ1-42 and t-tau were measured locally with
INNOTEST ELISA or INNOBIA AlzBio3 (Fujirebio,
Ghent, Belgium). Cut-offs for Aβ1-42 and t-tau were We selected 961 individuals from ADNI (adni.loni. usc.edu) and 799 individuals from the EMIF-AD MBD
study [12]. ADNI started in 2003 as a public–private Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 3 of 16 cohort-specific in EMIF-AD MBD [12]. Aβ1-42 cut-offs
were determined for each cohort using Gaussian mix-
ture modelling [20]. We measured neurogranin, neuro-
filament-light, YKL-40, Aβ38 and Aβ40 by ELISA [2]. We
performed untargeted mass spectrometry using tan-
dem mass tag (TMT) with 10 + 1 plexing as previously
described, using high-pH reverse phase HPLC for peptide
prefractionation [20, 21]. Peptides were mapped to 2535
proteins using the software ProteomeDiscoverer v.2.2. (Thermo Scientific), using Mascot (MatrixScience) for
protein identification (precursor Dm tolerance 15 ppm,
fragment tolerance 0.05 Da, max missed cleavage sites
2), searching the human subset of the UniProtKB Swis-
sProt database (www.uniprot.org). Percolator (MatrixSci-
ence) was used for scoring peptide specific matches, and
a strict 1% false discovery rate (FDR) was set as threshold
for identification. For reporter ion quantification the fol-
lowing settings were used: Integration tolerance 20 ppm;
Integration Method Most Confident Centroid; normalize
on the reference protein average. The median (IQR) CV
for these proteins was 5.6 (3.8, 8.0). cohort-specific in EMIF-AD MBD [12]. Aβ1-42 cut-offs
were determined for each cohort using Gaussian mix-
ture modelling [20]. We measured neurogranin, neuro-
filament-light, YKL-40, Aβ38 and Aβ40 by ELISA [2]. We
performed untargeted mass spectrometry using tan-
dem mass tag (TMT) with 10 + 1 plexing as previously
described, using high-pH reverse phase HPLC for peptide
prefractionation [20, 21]. Peptides were mapped to 2535
proteins using the software ProteomeDiscoverer v.2.2. (Thermo Scientific), using Mascot (MatrixScience) for
protein identification (precursor Dm tolerance 15 ppm,
fragment tolerance 0.05 Da, max missed cleavage sites
2), searching the human subset of the UniProtKB Swis-
sProt database (www.uniprot.org). Percolator (MatrixSci-
ence) was used for scoring peptide specific matches, and
a strict 1% false discovery rate (FDR) was set as threshold
for identification. For reporter ion quantification the fol-
lowing settings were used: Integration tolerance 20 ppm;
Integration Method Most Confident Centroid; normalize
on the reference protein average. The median (IQR) CV
for these proteins was 5.6 (3.8, 8.0). MBD sample). For related proteins that had identical
values due to fragment a-specificity (e.g. Proteomic process scores (PPS) Proteins and protein fragments (ADNI) values were
standardized according the mean and standard deviation
values of the control group and expressed as z-scores,
with a score of 0 indicating the average concentration
of the control group, z-scores > 0 higher concentrations,
and z-scores < 0 lower concentrations than controls. For
ADNI, we averaged peptides that mapped to the same
protein into a composite protein score when they cor-
related with r > 0.5, and included them as single peptides
otherwise [20]. When the same protein was measured by
different platforms in ADNI, values were averaged if they
correlated with r > 0.5 and otherwise we included them
as separate proteins. From the EMIF-AD MBD TMT
mass spectrometry analysis proteins were included when
observed in at least 92 individuals (30% of EMIF-AD For illustration purposes we combined proteins from
selected GO-BPs, and proteins associated with BBB dys-
function and BACE1 substrates into a PPS by averaging
z-scores of individual proteins belonging to the process
that differed between AD individuals with normal or
increased t-tau in cross-sectional analysis in the total
group. ADNI Aβ1-42 and t-tau were measured on the xMAP platform
(Luminex Corp, Austin, TX). Cut-offs for Aβ1-42 and t-tau
were used as published [19]. Changes in t-tau levels over
time were assessed in ADNI using longitudinal samples
measured within the same batch. YKL-40, neurogranin,
APP beta, neurogranin, neurofilament light, alpha synu-
clein, HBB, CFH, sTREM2, VILIP-1, and BACE1 were
measured by ELISA or related assays, 190 analytes were
analyzed by the Human DiscoveryMAP panel (MAP-
RBM) and 225 proteins were analyzed by targeted mass
spectroscopy [22, 23]. We used the quality checked and
finalised ‘Normalized Intensity’ data as described in
https://adni.loni.usc.edu/wp-content/uploads/2012/
01/2011Dec28-Biomarkers-Consortium-Data-Primer-
FINAL1.pdf Study participants ACTA1 and
ACTA2) we randomly selected one protein for analysis. Other annotations Proteins associated with high choroid plexus expression
were defined based on the Allen Brain Atlas, [32] through
Harmonizome [29] and ABAEnrichment analysis [33]. Blood–Brain barrier (BBB) associated proteins were
selected from reference [34–36]; BACE1 substrates were
selected from reference [37, 38]; Alpha secretase sub-
strates were selected from reference [39, 40]; and gamma
secretase substrates were selected from reference [41]. Predominant cell‑type of protein production Based on the BRAIN RNASeq database (http://www.
brainrnaseq.org), [31] we labelled proteins as being pre-
dominantly produced by a cell type when levels were
higher than 40% of older individuals produced across cell
types. Proteomic annotation
Enrichment analysis We included proteins for enrichment analyses that were
increased or decreased at p < 0.05 relative to the contrast
tested. Biological process enrichment analyses We performed
pathway enrichment analyses for Gene Ontology biologi-
cal processes (GO-BP) using the online Panther applica-
tion and SynGo [24–27]. Transcription factor enrichment analyses We per-
formed transcription factor enrichment analysis using
ChEA through Harmonizome through Enrichr [28–30]. Genotype imputation and quality control Data processing and quality control was performed using
GenomeStudio Software (v2.0.04, Illumina, Inc.), as
described previously [44]. To identify ethnic outliers, a
principal component analysis (PCA) of ancestry was per-
formed based on 1000 Genomes clustering, phase 3 using
PLINK [44]. Individuals of non-European descent and
family relations up to second degree were excluded. After
filtering, the ADNI genotype data included 747 individu-
als and EMIF-AD MBD 931 individuals. SNPs were locally imputed using Minimac 3 to the
Haplotype Reference Consortium reference panel [46]. To account for population structure, principal com-
ponents (PC1-PC20) were computed on a subset of
relatively uncorrelated (r2 < 0.2) single nucleotide poly-
morphisms (SNPs). For ADNI subjects, Apolipopro-
tein E (APOE) ε4 genotype was assessed with two SNPs;
rs429358 the ‘ε4-allele’ and rs7412 the ‘ε2 allele’. APOE
ε4 genotypes in EMIF-AD MBD were generated as
described elsewhere [44]. Genomic assessment EMIF-AD MBD samples were genotyped using the Illu-
mina Global Screening array (Illumina, Incl) and 936
samples passed post-experiment QC [44]. ADNI samples
were genotyped using the Illumina 2.5-M or Illumina
OmniQuad array [45]. Statistics Our main outcomes are the biological process and tran-
scription enrichment analyses for which we report FDR
corrected p-values. For other analysis we report uncor-
rected p-values. Test statistics can be found in the sup-
plemental tables. Group comparisons between AD
subgroups and controls were performed using ANOVA
correcting for age and gender. Change in cognition and
imaging markers were assessed with linear mixed models
including as main terms group, time, and the interaction
group by time, and correcting for age and gender, and
additionally for level of education for cognitive markers. We investigated within the AD individuals with normal
t-tau and AD individuals with increased t-tau whether
protein concentrations increased or decreased with
increasing disease severity ranging from preclinical AD,
prodromal AD, to mild AD-dementia with linear models. A protein was considered to decrease or increase with
disease severity if the linear trend was statistically signifi-
cant or if there was a difference in concentration between
preclinical AD and prodromal AD, preclinical AD and
mild AD-dementia, or between prodromal AD and
mild AD-dementia and the trend analysis supported the
change with severity. Associations between GRS scores Polygenic and other genetic risk score analysis Polygenic and other genetic risk score analysis
Polygenic risk scores (PGRS) for AD were computed for
each subject using PRSice (V2.3) [48]. PGRS were cal-
culated by adding the sum of each allele weighted by
the strength of its association with AD risk as calculated
previously [49]. AD cases were defined as patients clini-
cally diagnosed with AD-type dementia or individuals
with a parental history of AD (i.e., AD-by-proxy) [49]. Clumping was performed prior to calculating PGRS, to
remove SNPs that are in linkage disequilibrium (r2 > 0.1)
within a slicing 1 M bp window. After clumping, PGRS
were computed using various SNP inclusion thresholds. In order to explore how specific genetic alterations were
associated with CSF proteomic profiles, we calculated for
a selection of genesets based on MAGMA analysis (see
results), gene-specific risk scores (GRS) including only
SNPs located on the respective genes with clumping as
described above and computation of GRS using various
SNP inclusion thresholds. We used weights from one
cohort to generate GRS in the other cohort. In order to
reduce the number of tests, GRS scores were selected for
SNP inclusion threshold that best differentiated within
AD between abnormally increased and normal t-tau. Neuroimaging analysis We studied neuroimaging data only from ADNI, because
this study collected longitudinal scans. As a measure of
brain atrophy, we took cortical thickness data from 34
cortical areas from the longitudinal processing pipeline in
Freesurfer version 4.3 for 1.5 T T1-weighted MRI scans,
and v5.1 for 3 T T1-weighted MRI scans (http://adni. Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 4 of 16 of the meta-analysis summary statistics. We considered
for further analysis gene scores based on at least 6 SNPs
and geneset scores with information on at least 6 genes. loni.usc.edu). As a measure of amyloid accumulation, we
used region-specific SUVR values for florbetapir bind-
ing assessed by PET imaging in 34 cortical areas as par-
cellated by Freesurfer v4.5.0 [42]. As a measure of brain
metabolism we analysed fluorodeoxyglucose (FDG)-PET
scans, [43] and determined average glucose metabolism
for brain areas standardized to the average uptake in the
vermis and brain stem according to the Desikan-Killiany
atlas
(http://adni.loni.usc.edu/methods/pet-analysis-
method/pet-analysis/). loni.usc.edu). As a measure of amyloid accumulation, we
used region-specific SUVR values for florbetapir bind-
ing assessed by PET imaging in 34 cortical areas as par-
cellated by Freesurfer v4.5.0 [42]. As a measure of brain
metabolism we analysed fluorodeoxyglucose (FDG)-PET
scans, [43] and determined average glucose metabolism
for brain areas standardized to the average uptake in the
vermis and brain stem according to the Desikan-Killiany
atlas
(http://adni.loni.usc.edu/methods/pet-analysis-
method/pet-analysis/). GWAS and post‑GWAS analyses Concentrations are expressed as z-score relative to the control group (CN) that had normal cognition, normal Aβ1-42 and normal t-tau. Proteins are
sorted according to change relative to control group. Shown are proteins that differed between individuals with AD with increased t-tau or AD
individuals with normal tau from controls. Venn diagram shows number of proteins that differed Fig. 1 Protein concentrations relative to control group in individuals with AD according to t-tau status. Protein concentrations of individuals
with abnormal Aβ1-42 and increased t-tau (a + t + , red) and abnormal Aβ1-42 and normal t-tau (a + t-, blue) in ADNI A and EMIF-AD MBD B. Concentrations are expressed as z-score relative to the control group (CN) that had normal cognition, normal Aβ1-42 and normal t-tau. Proteins are
sorted according to change relative to control group. Shown are proteins that differed between individuals with AD with increased t-tau or AD
individuals with normal tau from controls. Venn diagram shows number of proteins that differed and CSF protein levels were tested with linear models
adjusted for age and sex. decreased levels of 2 proteins in ADNI (1% of proteins
measured) and of 43 proteins in EMIF-AD MBD (3%
of proteins measured) (Fig. 1, Data S2). AD individuals
with normal CSF t-tau, showed an opposite pattern with
increased levels of only 7 proteins in ADNI (3% of pro-
teins measured) and of 60 proteins in EMIF-AD MBD
(4% of proteins measured) and decreases in 107 proteins
in ADNI (43% of proteins measured) and of 411 proteins
in EMIF-AD MBD (28% of proteins measured). Around
50% of the proteins with increased levels in individuals
with increased t-tau had decreased levels in individu-
als with normal t-tau. This indicates that t-tau groups
show disruptions in the same molecular processes, but in
opposite directions. GWAS and post‑GWAS analyses Genome-wide association study (GWAS) analyses on
increased CSF t-tau in subjects with abnormal Aβ1-42
were performed separately in ADNI (abnormal Aβ1-42
and increased t-tau n = 246, abnormal Aβ1-42 and nor-
mal t-tau n = 238) and EMIF-AD MBD (abnormal Aβ1-42
and increased t-tau n = 294, abnormal Aβ1-42 and nor-
mal t-tau n = 155) using PLINK software (V1.90). We
used a logistic regression model including PC1-PC3 and
sex. Genome-wide significance was defined as p ≤ 5e-08. Meta-analysis on ADNI and EMIF-AD MBD GWAS
summary statistics was performed using METAL [47]. MAGMA software was used to compute gene scores and
geneset scores for biological pathways based on p-values Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 5 of 16 Fig. 1 Protein concentrations relative to control group in individuals with AD according to t-tau status. Protein concentrations of individuals
with abnormal Aβ1-42 and increased t-tau (a + t + , red) and abnormal Aβ1-42 and normal t-tau (a + t-, blue) in ADNI A and EMIF-AD MBD B. Concentrations are expressed as z-score relative to the control group (CN) that had normal cognition, normal Aβ1-42 and normal t-tau. Proteins are
sorted according to change relative to control group. Shown are proteins that differed between individuals with AD with increased t-tau or AD
individuals with normal tau from controls. Venn diagram shows number of proteins that differed Fig. 1 Protein concentrations relative to control group in individuals with AD according to t-tau status. Protein concentrations of individuals
with abnormal Aβ1-42 and increased t-tau (a + t + , red) and abnormal Aβ1-42 and normal t-tau (a + t-, blue) in ADNI A and EMIF-AD MBD B. Concentrations are expressed as z-score relative to the control group (CN) that had normal cognition, normal Aβ1-42 and normal t-tau. Proteins are
sorted according to change relative to control group. Shown are proteins that differed between individuals with AD with increased t-tau or AD
individuals with normal tau from controls. Venn diagram shows number of proteins that differed Fig. 1 Protein concentrations relative to control group in individuals with AD according to t-tau status. Protein concentrations of individuals
with abnormal Aβ1-42 and increased t-tau (a + t + , red) and abnormal Aβ1-42 and normal t-tau (a + t-, blue) in ADNI A and EMIF-AD MBD B. Results Of the individuals with AD, 788 (57%) had increased CSF
t-tau levels and 592 (43%) normal t-tau levels. Abnormal
and normal t-tau groups showed generally similar demo-
graphics and baseline cognitive performance (Data S1a). We first determined stability of t-tau levels over time
in 499 ADNI participants who had repeated CSF sam-
pling up to 7 years. CSF t-tau increased in the prodromal
AD stage at similar rates for both t-tau groups, and this
increase did not differ from the increase in t-tau in the
control group (Figure S1a). At last follow-up, 20% of the
AD individuals with normal t-tau at baseline developed
abnormally increased t-tau at follow-up and these indi-
viduals had t-tau levels just below the cut-point at base-
line (Figure S1b). Taken together, these findings suggest
that CSF t-tau levels reflect a trait rather than a stage. Enrichment analyses with GO-BP for CSF proteins
increased in the AD group with abnormal t-tau rela-
tive to controls, showed in both ADNI and EMIF-AD
MBD involvement of plasticity-related processes such
as nervous system development, axonogenesis, syn-
apse assembly, myelination, gliogenesis, angiogenesis,
mitogen-activated protein kinase (MAPK) signaling,
cell-cycle, gene expression and glycolysis (Fig. 2 selec-
tion of representative processes; Figure S2 shows
synaptic processes; Data S3a, S3b show all enriched
processes). Abnormality in each of these processes has
been reported before in separate studies, [8, 50–55]
and our findings now suggest that they are part of com-
mon plasticity response. We also observed involvement l
We then studied CSF proteome profiles for t-tau sub-
groups in 559 individuals with proteomic data (Data
S1b). This included targeted proteomics of 248 proteins
in ADNI and targeted and untargeted proteomics of
1458 proteins in EMIF-AD MBD (Data S2). Compared
to controls, AD individuals with increased CSF t-tau had
increased levels of 130 proteins in ADNI (52% of ADNI
proteins measured) and of 477 proteins in EMIF-AD
MBD (33% of EMIF-AD MBD proteins measured) and Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 6 of 16 Fig. 2 Association of selected GO biological processes and transcription factors with CSF proteins, MAGMA geneset score, and cell type. Results A,B GO-BP
and SUZ12 and REST transcription factors enriched for proteins in AD individuals with increased t-tau (a + t +) or normal t-tau (a + t-) with increased
(red) or decreased (blue) concentrations relative to controls in the total sample (A) or in the preclinical AD stage (B). P-values of all GO-BPs are listed
in Data S3a. C P-value of association of GO-BP with abnormal t-tau in GWAS-based MAGMA genset analysis. P-values of all GO-BPs are listed in Data
S4d. D Proportion of proteins with cell-type specific expression. A: ADNI cohort, E: EMIF-AD MBD cohort; A + t + : abnormal Aβ1-42 and increased
t-tau; A + t-: abnormal Aβ1-42 and normal t-tau; CN A + t + : abnormal Aβ1-42 and increased t-tau in preclinical AD stage; CN A + t-: abnormal Aβ1-42
and normal t-tau in preclinical AD stage. Oligo = oligodendrocyte; endothelial = endothelial cell. (protein enrichment) and Data S4d (MAGMA
analysis) Fig. 2 Association of selected GO biological processes and transcription factors with CSF proteins, MAGMA geneset score, and cell type. A,B GO-BP
and SUZ12 and REST transcription factors enriched for proteins in AD individuals with increased t-tau (a + t +) or normal t-tau (a + t-) with increased
(red) or decreased (blue) concentrations relative to controls in the total sample (A) or in the preclinical AD stage (B). P-values of all GO-BPs are listed
in Data S3a. C P-value of association of GO-BP with abnormal t-tau in GWAS-based MAGMA genset analysis. P-values of all GO-BPs are listed in Data
S4d. D Proportion of proteins with cell-type specific expression. A: ADNI cohort, E: EMIF-AD MBD cohort; A + t + : abnormal Aβ1-42 and increased
t-tau; A + t-: abnormal Aβ1-42 and normal t-tau; CN A + t + : abnormal Aβ1-42 and increased t-tau in preclinical AD stage; CN A + t-: abnormal Aβ1-42
and normal t-tau in preclinical AD stage. Oligo = oligodendrocyte; endothelial = endothelial cell. (protein enrichment) and Data S4d (MAGMA
analysis) through histone acetylation [58, 59]. Previous studies
showed evidence of REST/SUZ12 de-repression in AD
brain tissue, iPSC neurons from individuals with sporadic
AD, and tangle bearing AD neurons [8, 60, 61]. Results Analysis of cell-type specific proteins revealed that
proteins of all major brain cell types differed in con-
centration between AD individuals with increased and
normal t-tau and that plasticity proteins were typically
neuron-specific (Fig. 2, Data S2). i
We then investigated whether the opposite protein pat-
terns could already be detected in the preclinical stage
of AD. We found that relative to controls, preclinical
AD individuals with increased t-tau had higher levels of
plasticity proteins while preclinical AD individuals with
normal t-tau showed lower plasticity protein levels. Pre-
clinical AD individuals with normal t-tau also showed
higher concentrations of proteins associated with bar-
rier dysfunction (Table 1, Fig. 2b, Fig. 3, Data S2 column
BK-BN, Data S3a for all enriched GO-BP). These findings
indicate that both aberrant neuronal plasticity processes
and barrier dysfunction are very early events in AD. i
We continued analysis by comparing CSF protein con-
centrations between AD individuals with increased t-tau
and normal t-tau directly. AD individuals with increased
t-tau showed, relative to those with normal t-tau, an
increase in the concentration of 167 proteins in ADNI
(67% of ADNI proteins measured) and of 768 proteins in
EMIF-AD MBD (53% of EMIF-AD MBD proteins meas-
ured) and a decrease in the concentration of 2 proteins
in ADNI (1% of proteins measured) and of 136 proteins
in EMIF-AD MBD (9% of proteins measured). These We next investigated how protein concentrations
changed with increasing disease severity stages. In AD
individuals with increased t-tau levels, the concentra-
tion of 59 (ADNI) to 129 proteins (EMIF-AD MBD)
decreased with increasing disease severity and the con-
centration of 2 (ADNI) to 34 proteins (EMIF-AD MBD) Table 1 Top 20 proteins that differed in preclinical AD from the control group according to CSF t-tau status
T-tau Total tau, CN Control group with normal cognition, normal Aβ1-42 and normal t-tau
a Protein associated with Blood Brain Barrier dysfunction
b Proteins with high expression in choroid plexus. Results Interest-
ingly, of the proteins with increased gene expression in
these previous studies that were measured in our CSF
study, the majority showed increased CSF concentrations
in AD individuals with increased t-tau: 4 of 4 (CALB1,
NRXN3, SCN3B, SNAP25) proteins from reference [60];
56 of 67 (84%) proteins from reference [8]; and 200 of 238
(84%) proteins from reference [61] (annotated in data S2,
column DF-DG). of cytokine signaling, leukocyte activation, oxidative
stress, mitochondrial dysfunction, and apoptosis. Amy-
loid production was increased, reflected by increased
levels of amyloid precursor protein (APP), Aβ1-40, and
Aβ1-38, and increased levels and activity of BACE1, and
increased concentrations in substrates of the main
secretases involved in Aβ metabolism (i.e., BACE1, alpha
and gamma secretase; annotated in Data S2, column
DA-DC). Increased Aβ production is known to set off the
amyloid cascade in autosomal dominant AD, [56] and our
data suggest that increased amyloid production may also
play a role in sporadic AD [57]. To identify potential driv-
ers of increased protein levels we performed CHeA tran-
scription factor-binding site enrichment analysis, which
indicated SUZ12 (p-FDR corrected = 1.62E-11) and
REST (p-FDR corrected = 1.04E-9) as most significantly
enriched. SUZ12 and REST repress gene transcription AD individuals with normal t-tau showed, relative to
the control group, decreased levels of proteins associ-
ated with neuronal plasticity and regulation of MAPK
signaling, with concomitant lower levels of APP, Aβ1-40
and Aβ1-38, and secretase substrates (Fig. 2, Figure S2; Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 7 of 16 Visser et al. Molecular Neurodegeneration (2022) 17:27 Data S3a, S3b for all enriched processes). Proteins with
decreased concentrations in the AD group with normal
t-tau, also converged on SUZ12 and REST transcription
factors, suggesting that increased gene repression activ-
ity is driving this CSF proteomic profile. Furthermore,
the AD group with normal t-tau showed 67 proteins with
increased levels relative to controls. Twenty of these pro-
teins were associated with blood–brain barrier (BBB)
dysfunction (annotated in Data S2, column DD) and 25
proteins had a high expression in choroid plexus (ABAE-
nrichment minimum pFWER = 0.004), suggesting blood-
CSF barrier (BCSFB) dysfunction (annotated in Data S2,
column DE). Increased proteins were mainly seen in the
EMIF-AD MBD cohort, as ADNI proteomics was tar-
geted for brain specific proteins. proteins were enriched for the same processes that dif-
fered for each group relative to controls (Figure S3, Data
S3a). Results Full list of proteins is provided in Data S2, column BK-BN
Contrast
Cohort
Number of
proteins that
differ
Top 20 proteins with largest effect size
Preclinical AD with increased t-tau
Proteins with increased concentration relative to CN
EMIF-AD MBD
262
YWHAH, LAMP5, PCSK5, CHI3L1, SMOC1, ADCYAP1, SPP1,
GDA, CRYM, TAGLN3, PLXNA2, PCDH8, HPRT1, CPD, GAP43,
ENPP5, CAMK2A, PKM, MELTF, NAXE
ADNI
93
NRGN, PKM, VSNL1, GOT1, ALDOA, SPP1, GOT2, HGF, NEO1,
NCSTN, MOG, APLP2, SOD1, BACE1, APP, CHI3L1, FABP3,
ENO2, NCAM2, SPON1
Proteins with decreased concentration relative to CN
EMIF-AD MBD
19
S100A6, ADAMDEC1, GPLD1, ANXA5, KNG1, SERPINA4,
HSPA1A, IGHV4-30–2, CPB2, IGHV4-30–4, FLNA, IGHV3-30,
PRAP1, IGLV3-10, HSPA6, ABI3BP, IGHV2-70D, HSPA7, PON1
ADNI
2
GOLM1, LEP
Preclinical AD with normal t-tau
Proteins with increased concentration relative to CN
EMIF-AD MBD
36
SLC39A12b, ADIPOQa, ANGPTL7, LGI1b, CD9b, KRT24, IFI30a,
TTRb, FOLR1b, NCMAP, SLC5A5b, SELPLG, ENPP2b, F5b, CTSA,
KRT12, KRT9, SIAEb, COL15A1, KRT10
ADNI
3
EDN1, APOE-e4, FGF4b
Proteins with decreased concentration relative to CN
EMIF-AD MBD
102
HS6ST1, CDH9, ASTN1, PLXNA1, APP, PMP2, STMN3, CDH8,
PRKAR1A, GABARAPL2, GALNT6, PITHD1, GALNT1, CPM,
MMP17, FAM19A2, CD99, FURIN, TAGLN, CDH12
ADNI
24
abeta1-40, abeta1-38, ADGRL1, CADM3, NEO1, NPTX1,
MCAM, CHGB, PCSK1, NEGR1, L1CAM, UBB, PTPRN, CAC‑
NA2D1, TIMP1, PAM, BTD, VEGFA, DAG1, NBL1 Table 1 Top 20 proteins that differed in preclinical AD from the control group according to CSF t-tau status Number of
proteins that
differ Preclinical AD with increased t-tau Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 8 of 16 CN
MCI
Dem
-4
-3
-2
-1
0
1
2
3
4
PPS
Nervous system development
CN
MCI
Dem
-4
-3
-2
-1
0
1
2
3
4
PPS
Gene expression
CN
MCI
Dem
-4
-3
-2
-1
0
1
2
3
4
PPS
Cytokine-mediated signalling
CN
MCI
Dem
-4
-3
-2
-1
0
1
2
3
4
PPS
Leukocyte activation
CN
MCI
Dem
-4
-3
-2
-1
0
1
2
3
4
PPS
Angiogenesis
CN
MCI
Dem
-4
-3
-2
-1
0
1
2
3
4
PPS
Myelination
CN
MCI
Dem
-4
-3
-2
-1
0
1
2
3
4
PPS
Blood-brain barrier
CN
MCI
Dem
-4
-3
-2
-1
0
1
2
3
4
PPS
BACE1 substrates
Fig. 3 Association of protein process score (PPS) with disease severity. Data are based on cross-sectional analysis and combines data from ADNI
and EMIF-AD MBD. Results In grey PPS of control group with normal cognition and normal CSF Aβ1-42 and t-tau; In red PPS of AD individuals with increased
t-tau; In blue PPS of AD individuals with normal t-tau. CN = cognitively normal; MCI = mild cognitive impairment; Dem = dementia CN
MCI
Dem
-4
-3
-2
-1
0
1
2
3
4
PPS
Nervous system development nt
CN
MCI
Dem
-4
-3
-2
-1
0
1
2
3
4
PPS
Gene expression CN
MCI
Dem
-4
-3
-2
-1
0
1
2
3
4
PPS
Cytokine-mediated signallin ng
CN
MCI
Dem
-4
-3
-2
-1
0
1
2
3
4
PPS
Leukocyte activation Cytokine-mediated signalling PPS PPS PPS CN
MCI
Dem
-4
-3
-2
-1
0
1
2
3
4
PPS
Angiogenesis CN
MCI
Dem
-4
-3
-2
-1
0
1
2
3
4
PPS
BACE1 substrates CN
MCI
Dem
-4
-3
-2
-1
0
1
2
3
4
PPS
Myelination CN
MCI
Dem
-4
-3
-2
-1
0
1
2
3
4
PPS
Blood-brain barrier Myelination PPS PPS PPS PPS Fig. 3 Association of protein process score (PPS) with disease severity. Data are based on cross-sectional analysis and combines data from ADNI
and EMIF-AD MBD. In grey PPS of control group with normal cognition and normal CSF Aβ1-42 and t-tau; In red PPS of AD individuals with increased
t-tau; In blue PPS of AD individuals with normal t-tau. CN = cognitively normal; MCI = mild cognitive impairment; Dem = dementia Presence of the APOE ε4 allele, the major genetic AD
risk factor, was more common in AD individuals with
increased t-tau compared to AD individuals with nor-
mal t-tau (66% vs 53% p < 0.001) and was in both AD
groups more common than in controls (17%, p < 0.001). Compared to controls, AD individuals with increased
t-tau and with normal t-tau had both higher AD PGRS
across SNP inclusion thresholds (Fig. 4a, Data S4a)
[49]. PGRS did not differ between AD individuals with
increased t-tau and normal t-tau after stratification for
clinical stage and correction for APOE ε4 carriership
and age (Data S4a), suggesting that these groups have
a similar AD genetic risk architecture. Next, we per-
formed an exploratory GWAS within AD individuals to
identify other potential genetic markers associated with
increased t-tau. We found tentative associations with
SNPs in the APBB2 gene (nominal p-value 1.63–2.92E-
06, Data S4b). Results The top 3 genes associated with increased
t-tau from gene-based analyses included, in addition to
APBB2, TBC1D10B (nominal p = 2.9E-04), a Rab GTPA
activating protein involved in MAPK signaling, and
LRP3 (nominal p = 3.36E-04), a low-density lipoprotein
receptor protein (Data S4c). The top 3 GO-BP gene-sets
associated with increased t-tau from MAGMA analysis
were positive regulation of cellular process (self-con-
tained p = 1.07E-04), protein K29-linked ubiquitination
(self-contained p = 1.52E-04) and negative regulation
of actin nucleation (self-contained p = 1.57E-04, Data
S4d). Twenty-eight percent of GO-BPs enriched for increased. Proteins of which the concentration decreased
with disease severity were enriched for neuronal plastic-
ity and proteins of which the concentration increased
were enriched for mitochondrial outer membrane per-
meabilization and complement activation (Fig. 3, Data
S2, S3a (all enriched GO-BP), Figure S3). In AD individu-
als with normal t-tau, the concentration of 48 (ADNI) to
265 (EMIF-AD MBD) proteins decreased with increas-
ing disease severity and the concentration of 10 proteins
(ADNI) to 54 (EMIF-AD MBD) increased with disease
severity (Fig. 3, Data S2, S3a (all enriched GO-BP), Fig-
ure S3). Proteins of which the concentration decreased
with disease severity were enriched for neuronal plas-
ticity and proteins of which the concentration increased
proteins included BBB related proteins (n = 32) and pro-
teins with increased expression in the choroid plexus
(n = 14). Because the concentration of plasticity pro-
teins decreased in both AD individuals with increased
t-tau and normal t-tau, differences in plasticity proteins
between the groups that were present in the preclinical
AD stage persisted in the prodromal and dementia stage
(Fig. 3). The decrease in concentration of neuronal plas-
ticity proteins with disease severity aligns with the obser-
vation of decreased gene expression of neuronal plasticity
proteins with increasing disease severity in post-mortem
AD studies [57, 62]. Next, we investigated whether genetic factors were
related to increased t-tau in AD individuals in the com-
bined ADNI and EMIF-AD MBD cohorts (n = 1067). Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 9 of 16 Fig. 4 Genetic analysis. A PGRS across SNP thresholds SNP weights were based on [49]. Full data (including data after correction for APOE ε4
genotype and demographics, and separate for cohort and clinical stage) are shown in Supplemental Data S4a. Results a + t + : abnormal Aβ1-42 and
increased t-tau (red); a + t-: abnormal Aβ1-42 and normal t-tau (blue). B Number of proteins associated with genetic risk score (GRS) and processes
associated with these proteins. Left site shows number of proteins with a positive correlation with CSF t-tau GWAS-based GRS (coloured outer
ring) and right site indicates to which GO-biological processes (GO-BP) these proteins below (GO-BP CSF, grey outer ring). Shown are the 10 GRS
with the highest number of positive correlations with CSF proteins. The size of the arrows visualizes the number of correlations between GRS and
protein concentration. For example, the GO-BP gene expression GRS showed a positive correlation with 256 proteins and these proteins were
predominantly associated with nervous system development, gene expression and cell adhesion. Arrows to CSF GO-BP are shown in case the GRS
correlated with at least 8 proteins from the CSF GO-BP. Supporting data in Data S5a. ECM = Extracellular matrix Fig. 4 Genetic analysis. A PGRS across SNP thresholds SNP weights were based on [49]. Full data (including data after correction for APOE ε4
genotype and demographics, and separate for cohort and clinical stage) are shown in Supplemental Data S4a. a + t + : abnormal Aβ1-42 and
increased t-tau (red); a + t-: abnormal Aβ1-42 and normal t-tau (blue). B Number of proteins associated with genetic risk score (GRS) and processes
associated with these proteins. Left site shows number of proteins with a positive correlation with CSF t-tau GWAS-based GRS (coloured outer
ring) and right site indicates to which GO-biological processes (GO-BP) these proteins below (GO-BP CSF, grey outer ring). Shown are the 10 GRS
with the highest number of positive correlations with CSF proteins. The size of the arrows visualizes the number of correlations between GRS and
protein concentration. For example, the GO-BP gene expression GRS showed a positive correlation with 256 proteins and these proteins were
predominantly associated with nervous system development, gene expression and cell adhesion. Arrows to CSF GO-BP are shown in case the GRS
correlated with at least 8 proteins from the CSF GO-BP. Supporting data in Data S5a. ECM = Extracellular matrix lowest self-contained p-value. For each of the GRS, we
used weights from one cohort to generate a GRS in the
other cohort. We then correlated the individual GRS of
each gene or GO-BP with individual protein concen-
trations (Data S5a). Results A positive correlation means that
a higher GRS is associated with a higher protein con-
centration. The largest number of positive correlations
of GRS with CSF protein concentrations were observed
for the GO-BP gene expression GRS (288 proteins),
the GO-BP regulation of MAPK cascade GRS (146
proteins), and the GO-BP histone H3 acetylation GRS
(127 proteins, Fig. 4b, left coloured part, Data S5a). The proteins that showed a positive correlation with
these GRS were typically associated with nervous sys-
tem development, gene expression, and cell adhesion CSF proteins that differed in concentration between
AD t-tau groups, differed also in GO-BP gene-set score
between the t-tau groups (Fig. 2; Data S3a). This sug-
gests that interindividual differences in CSF t-tau
concentrations may have partly a genetic basis. To
investigate whether genetic markers influenced CSF
protein concentrations, we correlated subject level GRS
with subject level CSF protein concentrations in 188
individuals with abnormal Aβ1-42. We used the GRS of
the top 3 genes, the GRS of the top 3 GO-BP and the
GRS of 9 GO-BPs that differed between abnormal and
normal t-tau groups in both CSF and MAGMA geneset
analysis in Fig. 2. Given the association of CSF profiles
with REST and SUZ12 we also selected the GRS of 2
GO-BPs associated with histone acetylation with the CSF proteins that differed in concentration between
AD t-tau groups, differed also in GO-BP gene-set score
between the t-tau groups (Fig. 2; Data S3a). This sug-
gests that interindividual differences in CSF t-tau
concentrations may have partly a genetic basis. To
investigate whether genetic markers influenced CSF
protein concentrations, we correlated subject level GRS
with subject level CSF protein concentrations in 188
individuals with abnormal Aβ1-42. We used the GRS of
the top 3 genes, the GRS of the top 3 GO-BP and the
GRS of 9 GO-BPs that differed between abnormal and
normal t-tau groups in both CSF and MAGMA geneset
analysis in Fig. 2. Given the association of CSF profiles
with REST and SUZ12 we also selected the GRS of 2
GO-BPs associated with histone acetylation with the Visser et al. Molecular Neurodegeneration (2022) 17:27 Visser et al. Molecular Neurodegeneration (2022) 17:27 Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 10 of 16 Page 10 of 16 Visser et al. Molecular Neurodegeneration (2022) 17:27 (Fig. 4b, right grey part). Results These findings support the
notion that the increase in plasticity-related processes
in CSF has in part a genetic background. Moreover,
we tested the association of SNP rs9877502 in GMNC,
previously associated with CSF t-tau levels without
stratification for amyloid aggregation, with tau status
[44, 63, 64]. Consistent with previous data, the A-allele
frequency at this site was more common in AD indi-
viduals with increased t-tau compared to those with
normal t-tau (nominal p-value = 0.02). We then asso-
ciated this SNP with CSF protein levels using additive
models. 591 of 1705 proteins (35%) showed an increase
in CSF level with increasing number of rs9877502
A-alleles (Data S5b). These proteins were enriched
for neuronal plasticity related processes (Data S5c),
and SUZ12 (p-FDR corrected = 5.27e-24) and REST
(p-FDR corrected = 6.34e-18) signaling. 105 proteins
(6%) showed a negative correlation with the number
of rs9877502 A-alleles, including 36 proteins associ-
ated with BBB dysfunction and 36 immunoglobulins. In a smilar way, we associated the number of APOE-e4
alleles with CSF protein levels in individuals with AD. Forty-five proteins showed an increase in CSF levels
with increasing number of APOE-e4 alleles (Data S5b)
and these proteins were enriched for GO-BP learning
or memory (p-FDR corrected = 0.046). The concentra-
tion of 36 proteins showed a negative correlation with
the number of APOE-e4 alleles (Data S5b) and were not
enriched for any GO-BP. Results from our molecular process and transcription
factor enrichment analysis suggested that the increased
levels of neuroplasticity proteins in the AD group with
increased t-tau could result from an increase in mitogenic
MAPK signaling, or from a reduction in gene repression
by REST and SUZ12. The de-repression of gene expres-
sion by REST/SUZ12 in AD has previously been reported
in post-mortem AD studies and in iPSC neurons from
individuals with sporadic AD [8, 60, 61]. Our finding
that a subset of proteins coded by genes with increased
expression in these previous studies had increased CSF
concentrations in AD individuals with increased t-tau as
well, further supports the role of REST/SUZ12 de-repres-
sion in AD. Both APP and tau are regulated by REST or
SUZ12, and this may explain why the increases in levels
of Aβ production markers and tau occur in tandem [6]. Results The increased concentration of neuronal plasticity pro-
teins might reflect a compensatory mechanism triggered
by tau-induced synaptic dysfunction such as impairment
of presynaptic vesicle release, trafficking of glutamatergic
receptors, and maturation of dendritic spines [65]. AD
neuropathological studies suggested that such a plastic-
ity response is likely pathological and results in aberrant
synaptic sprouting (both at axons and at dendrites), dis-
organised capillairies and an abnormal cortical myelin
architecture [51, 52, 66]. Increased expression of APP
and tau may initiate a vicious cycle. APP and Aβ peptides
can activate MAPK signaling, [53, 67, 68] which may fur-
ther increase APP production[69, 70]. The APP intracel-
lular domain (AICD) stimulates gene transcription, [71]
and may increase expression of APP and BACE1 [72]. Abnormal tau may increase gene expression through
depletion of H3K9me2, altered spatial open chromatin
organization, or altered H3K9 acetylation [73, 74].h Finally, we studied the effect of t-tau on disease pro-
gression using longitudinal ADNI data. Relative to controls, AD groups with increased t-tau
and normal t-tau showed both faster memory decline and
PET-amyloid accumulation in the preclinical stage and
showed faster decline in all cognitive and imaging meas-
ures in the prodromal and dementia stage. Compared to
AD individuals with normal t-tau, those with increased
t-tau declined faster on cognitive tests and in glucose
metabolism in the prodromal AD stage and showed a
faster decline in cortical thickness in the prodromal and
dementia stage (Fig. 5, Data S6). g
y
[
]
The group of AD individuals with normal t-tau was
characterized by decreased concentrations of plasticity
related proteins and increased concentrations of BBB
and BCSFB permeability related proteins. The increase
in barrier permeability may be caused by accumula-
tion of Aβ in vessel walls and toxic effects of Aβ on cells
that constitute these barriers [75, 76]. This can loosen
tight junctions and increase paracellular transport [75,
77–79]. Impaired BBB and BCSFB function may lead
to hypoplasticity through impairments in their physi-
ological role in glucose transport, capillary perfusion,
and neurogenesis, [79] and possibly also by increasing
REST signaling, which can be triggered by ischemia
[80]. The increase in concentrations of CSF proteins
that are produced in the choroid plexus suggests
BCSFB dysfunction. The choroid plexus is involved in
the clearance of Aβ, [81] and the increase in concentra-
tion of proteins expressed in the choroid plexus could
be a response to Aβ induced inflammation [82–84]. Discussion Our main finding is that AD individuals with
increased CSF t-tau levels showed increased concen-
trations of proteins associated with neuronal plas-
ticity, while AD individuals with normal t-tau levels
showed decreased concentrations of plasticity pro-
teins. These opposite CSF proteomic profiles were
already observed in the preclinical stage of AD, and
persisted in prodromal and mild dementia stages,
indicating that they reflect different disease traits
rather than different disease states. Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 11 of 16 Fig. 5 Longitudinal cognitive and imaging changes according to diagnostic group and clinical stage Upper images: change in cognitive markers. A MMSE, B CDR sum of boxes, C Logical memory, and D ADAS-Cog. Change of controls are shown in grey, of AD individuals with increased t-tau
in red (a + t +), and of AD individuals with normal t-tau (a + t-) in blue. 1. Slope p < 0.05 different from control; 2. Slope p < 0.05 different from 0; 3. Slope p < 0.05 different between AD individuals with increased t-tau (a + t +) and AD individuals with normal t-tau (a + t-). Lower images: Annual
rate of change in imaging markers. E amyloid aggregation, F cortical thickness and G FDG-PET glucose metabolism. PET amyloid data are not
shown for AD individuals with dementia with normal t-tau because of low number of participants (n = 6). Data are shown in Data S6. Data are from
ADNI only. CN = normal cognition; MCI = mild cognitive impairment; a + t + = abnormal Aβ1-42 and increased t-tau; a + t- = abnormal Aβ1-42 and
normal t-tau Fig. 5 Longitudinal cognitive and imaging changes according to diagnostic group and clinical stage Upper images: change in cognitive markers. A MMSE, B CDR sum of boxes, C Logical memory, and D ADAS-Cog. Change of controls are shown in grey, of AD individuals with increased t-tau
in red (a + t +), and of AD individuals with normal t-tau (a + t-) in blue. 1. Slope p < 0.05 different from control; 2. Slope p < 0.05 different from 0; 3. Slope p < 0.05 different between AD individuals with increased t-tau (a + t +) and AD individuals with normal t-tau (a + t-). Lower images: Annual
rate of change in imaging markers. E amyloid aggregation, F cortical thickness and G FDG-PET glucose metabolism. Discussion The
genetic pattern associated with t-tau levels in individuals
with AD is thus different from the genetic processes asso-
ciated with AD-type dementia, which include immunity,
lipid metabolism, and intracellular trafficking [49]. This
indicates that within the AD genetic risk architecture
other genetic processes could influence AD phenotypes. levels in this and other datasets) [44, 63, 64] with levels of
696 CSF proteins in AD individuals. GMNC is involved
in neuronal plasticity and regulation of gene expres-
sion [89, 90]. We also found that GRS associated with
t-tau were involved in neuronal plasticity and that these
GRS showed an association with the concentration of
plasticity-related proteins. On the contrary, the number
of APOEe4 alleles was not associated with the concen-
tration of proteins involved in neuronal plasticity. The
genetic pattern associated with t-tau levels in individuals
with AD is thus different from the genetic processes asso-
ciated with AD-type dementia, which include immunity,
lipid metabolism, and intracellular trafficking [49]. This
indicates that within the AD genetic risk architecture
other genetic processes could influence AD phenotypes. study showed that increased CSF t-tau levels were associ-
ated with long-term depression (LTD) after transcranial
magnetic stimulation in the motor cortex [96]. As this
study was performed in demented individuals, it is possi-
ble that LTD reflects synaptic failure. Future studies that
combine electrophysiological measures with proteomic
data in human beings are required to study this question
in more detail. We further found that AD individuals with increased
t-tau also showed increased cytokine signaling and leu-
kocyte activation. This activation could result from sol-
uble tau oligomers, [97] although it is also possible that
both increased t-tau levels and microglia activation result
from amyloid pathology. A strength of the study is the large sample size available
for proteomic analysis and the use of two independent
cohorts. Even though these cohorts performed proteom-
ics in a different way (targeted proteomics in ADNI, non-
targeted proteomics in EMIF-AD MBD) and the overlap
in proteins measured in both studies was limited, enrich-
ment analysis showed very similar results between the
cohorts (Fig. 2), which supports the robustness of our
findings. The consistency across cohorts was also pre-
sent on the protein level. Discussion Of the 167 proteins measured
in both cohorts, only 7 proteins (5%) showed a difference
between the cohorts on both major outcomes (AD with
normal t-tau vs control and AD with increased t-tau ver-
sus controls, Data S2). A limitation is that CSF proteom-
ics was performed cross-sectionally and the differences
between disease stages need to be confirmed in longitu-
dinal studies. In addition, the association between change
in CSF protein concentrations and biological processes
needs to be further addressed in functional studies. The
sample size for genetic analysis was small and larger sam-
ples are needed to confirm these results. l
As we found a wide range of processes deregulated,
it is possible that this may reflect an aspecific response
to brain pathology. This seems, however, unlikely as the
same set of proteins that was increased in AD individu-
als with increased t-tau was decreased in AD individuals
with normal t-tau suggesting that these processes have a
common denominator. In addition, we found that these
proteins were enriched for SUZ12 and REST transcrip-
tion factors, which are master regulators of neuronal
plasticity. Moreover, abnormalities in nervous system
development related processes, myelination, angiogen-
esis, MAPK signaling, cell-cycle, gene expression and
glycolysis have all been reported in separate AD post-
mortem, animal or cell studies [8, 50–55]. Still, some
dysregulated processes may only be indirectly involved
in plasticity. For example, increased levels of proteins
associated with glycolysis could be a response to the high
energy demand resulting from increased neuronal activ-
ity [91]. It is unlikely that the increased levels of plastic-
ity proteins in AD individuals with increased t-tau simply
reflect cell death as levels of plasticity-associated proteins
were highest in the preclinical AD stage, when neurode-
generation is limited, and decreased with increasing dis-
ease severity. Discussion PET amyloid data are not
shown for AD individuals with dementia with normal t-tau because of low number of participants (n = 6). Data are shown in Data S6. Data are from
ADNI only. CN = normal cognition; MCI = mild cognitive impairment; a + t + = abnormal Aβ1-42 and increased t-tau; a + t- = abnormal Aβ1-42 and
normal t-tau Our GWAS and GRS analyses suggested that levels of
tau and neuroplasticity proteins partly depend on genes
involved in gene expression, although no genome-wide
significant signals were identified. APBB2 showed the
strongest association with CSF t-tau in these analyses. While markers in APBB2 do not show strong evidence
for association with AD risk in GWAS comparing con-
trols with individuals with AD-dementia [49], the gene
represents an interesting functional candidate as APBB2
binds AICD. Alternative splicing of APBB2 increased
the Aβ42/ Aβ40 ratio [87], and overexpression increased
Aβ1-40, APP, and AICD levels and changed gene expres-
sion [68, 87, 88]. In addition, we found associations of
rs9877502 in GMNC (previously associated with t-tau AD individuals with normal t-tau may have cerebral
amyloid angiopathy (CAA), a condition in which Aβ is
deposited in cerebral and leptomeningeal blood vessels
[85]. CAA is common in individuals with AD and cor-
relates with Aβ plaque deposition and neurofibrillary
tangles [85]. CAA has been associated with normal lev-
els of t-tau levels and a decrease in Aβ40 levels, which
was also observed in our AD individuals with normal
t-tau [86]. Although t-tau levels were within normal
limits, this group showed a typical AD genetic pattern
and a progressive disease course with increases in cog-
nitive and AD biomarkers abnormalities, which sup-
ports the view that this is a subtype of AD, and not a
different disease entity. Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 12 of 16 Visser et al. Molecular Neurodegeneration Page 12 of 16 levels in this and other datasets) [44, 63, 64] with levels of
696 CSF proteins in AD individuals. GMNC is involved
in neuronal plasticity and regulation of gene expres-
sion [89, 90]. We also found that GRS associated with
t-tau were involved in neuronal plasticity and that these
GRS showed an association with the concentration of
plasticity-related proteins. On the contrary, the number
of APOEe4 alleles was not associated with the concen-
tration of proteins involved in neuronal plasticity. Funding g
This work has been supported by ZonMW Memorabel grant programme
#73305056 (BMT), #733050512 (SL) and #733050824 (BMT and PJV), the
Swedish Research Council (#2018–02532, HZ), the European Research
Council (#681712, HZ) and Swedish State Support for Clinical Research
(#ALFGBG-720931, HZ), the Alzheimerfonden (Grant no. AF-930934) and
Dtiftelsen Gamla tjänarinnor (JG), and the Innovative Medicines Initiative Joint
Undertaking under EMIF grant agreement #115372 (PJV, HZ, SV, IB). HZ is a
Wallenberg Academy Scholar. The Leuven cohort was funded by Stichting
Alzheimer Onderzoek (#11020, #15005, #13007) and the Vlaamse Impulsfi‑
nanciering voor Netwerken voor Dementie-onderzoek (IWT #135043). The
Lausanne cohort was supported by the Swiss National Research Foundation
SNF (#320030_141179). Data was used for this project of which collection and
sharing was funded by the Alzheimer’s Disease Neuroimaging Initiative (ADNI)
(National Institutes of Health Grant U01 AG024904) and DOD ADNI (Depart‑
ment of Defense award number W81XWH-12–2-0012). ADNI is funded by
the National Institute on Aging, the National Institute of Biomedical Imaging
and Bioengineering, and through generous contributions from the following:
AbbVie, Alzheimer’s Association; Alzheimer’s Drug Discovery Foundation;
Araclon Biotech; BioClinica, Inc.; Biogen; Bristol-Myers Squibb Company; Cer‑
eSpir, Inc.; Cogstate; Eisai Inc.; Elan Pharmaceuticals, Inc.; Eli Lilly and Company;
EuroImmun; F. Hoffmann-La Roche Ltd and its affiliated company Genentech,
Inc.; Fujirebio; GE Healthcare; IXICO Ltd.; Janssen Alzheimer Immunotherapy
Research & Development, LLC.; Johnson & Johnson Pharmaceutical Research
& Development LLC.; Lumosity; Lundbeck; Merck & Co., Inc.; Meso Scale
Diagnostics, LLC.; NeuroRx Research; Neurotrack Technologies; Novartis
Pharmaceuticals Corporation; Pfizer Inc.; Piramal Imaging; Servier; Takeda Phar‑
maceutical Company; and Transition Therapeutics. The Canadian Institutes of
Health Research is providing funds to support ADNI clinical sites in Canada. Private sector contributions are facilitated by the Foundation for the National
Institutes of Health (www.fnih.org). The grantee organization is the Northern
California Institute for Research and Education, and the study is coordinated
by the Alzheimer’s Therapeutic Research Institute at the University of Southern
California. ADNI data are disseminated by the Laboratory for Neuro Imaging at
the University of Southern California. Supplementary Information The online version contains supplementary material available at https://doi.
org/10.1186/s13024-022-00521-3. The online version contains supplementary material available at https://doi.
org/10.1186/s13024-022-00521-3. Additional file 1: Data S1. Participant characteristics. S1a: Characteristics
of individuals with CSF Aβ1-42 and tau measurements available; S1b:
Characteristics of individuals with CSF proteomic data. Data S2. Protein
annotation and statistics of group comparisons of protein levels. Data
S3a. Full list of GO biological processes associated with proteins that
differ according to group and clinical stage. Data S3b. SynGO enriched
synaptic cellular components and biological processes that differ accord‑
ing to group. Data S4a. Estimated marginal means of AD GWAS-based
polygenic risk scores in controls, AD individuals with increased t-tau and
AD individuals with normal t-tau. Data S4b. Top 1000 SNPS from GWAS
on AD individuals with increased t-tau and normal t-tau in pooled ADNI
and EMIF-AD MBD cohorts. Data S4c. Difference in MAGMA gene score
between AD individuals with increased t-tau and normal t-tau based
on t-tau GWAS in pooled ADNI and EMIF-AD MBD cohorts. Data S4d. Difference in GO biological process MAGMA geneset score between AD
individuals with increased t-tau and normal t-tau based on t-tau GWAS in
pooled ADNI and EMIF-AD MBD cohorts. Data S5a. Correlation between
genetic risk score and CSF protein level in individuals with abnormal Aβ1-
42. Data S5b. Association of the number of GMNC rs9877502-A risk alleles
and number of APOE-e4 alleles with CSF protein concentrations in a linear
model in individuals with AD. Data S5c. GO-BP processes enriched for
proteins that have a positive or negative association with the number of
rs9877502-A risk alleles in an additive model. Data S6. Annual change in
imaging measures. Acknowledgements We thank Drs. Andre Franke and Michael Wittig as well as Mrs. Tanja Wesse and
Sanaz Sedghpour Sabet for their help with the genome-wide genotyping experi‑
ments. Further, we thank Dr. Fabian Kilpert and Shengjun Hong for his help with
the processing and management of the genome-wide genotyping data. Data used in preparation of this article were obtained from the Alzheimer’s
Disease Neuroimaging Initiative (ADNI) database (adni.loni.usc.edu). As such, the
investigators within the ADNI contributed to the design and implementation of
ADNI and/or provided data but did not participate in analysis or writing of this
report. A complete listing of ADNI investigators can be found at: http://adni.loni.
usc.edu/wpcontent/uploads/how_to_apply/ADNI_Acknowledgement_List.pdf Conclusions CSF t-tau levels in AD are associated with altered levels
of proteins involved in neuronal plasticity and blood–
brain and blood-CSF barrier dysfunction. The asso-
ciation of increased CSF t-tau with neuronal plasticity
protein levels provides support for the role of tau in
neuronal plasticity and gene expression and implies that
increased CSF t-tau levels may not simply reflect axonal
loss [8, 73, 74]. It will be of critical importance to stratify
future trials on t-tau status, as individuals with increased
t-tau and normal t-tau are likely to respond differently
to treatment, given their opposite proteomic profiles. The dosing of amyloid and tau antibodies may need to
be tailored to t-tau levels, because of differences in BBB/
BCSFB integrity between those with increased and nor-
mal t-tau. Treatments with drugs that target amyloid
production, hyperexcitation, or hyperplasticity, such The observation that increased t-tau levels are associ-
ated with an increase in concentration of plasticity pro-
teins, aligns with observations that tau is associated with
increased neuronal excitability in neurophysiological
studies [92]. In an AD mice model it was shown that tau
could be an enabler of neural network dysfunction caused
by amyloid pathology as tau reduction reduced amyloid-
related overexcitation [92, 93]. Tau may also have direct
effects on excitability as overexpression of human tau
in mice models increased delta/theta power and reduc-
tion of tau expression counteracted these changes [94]. Another study showed that nuclear translocation of tau
led to increased expression of VgluT1, which is involved
in glutamatergic synaptic transmission [95]. A clinical Visser et al. Molecular Neurodegeneration (2022) 17:27 Page 13 of 16 as bace inhibitors, histone modifiers and anti-epileptic
drugs, could only be effective in AD individuals with
increased t-tau in the predementia stage, when the CSF
concentration of amyloid production markers and plas-
ticity related proteins are highest. Retrospective analysis
of previous trial data according to CSF t-tau status may
lead to a better understanding why effects in these trials
were absent or minimal and whether AD individuals with
increased t-tau and normal t-tau have a different treat-
ment response. Future studies on the mechanisms that
lead to t-tau associated proteomic profiles, such as REST
and SUZ12 signaling, will help to clarify AD pathophysi-
ology and may eventually lead to novel drug targets. Conclusions REST transcription factors associated with proteins that differed between
AD individuals with increased t-tau and normal t-tau and proteins that
changed with disease severity. REST transcription factors associated with proteins that differed between
AD individuals with increased t-tau and normal t-tau and proteins that
changed with disease severity. Abbreviations
AD Al h i
’ d AD: Alzheimer’s disease; ADNI: Alzheimer’s Disease Neuroimaging Initiative;
AICD: APP intracellular domain; APOE: Apolipoprotein E; APP: Amyloid precur‑
sor protein; Aβ: Amyloid beta; BBB: Blood–Brain barrier; BCSFB: Blood-CSF
barrier; CAA: Cerebral amyloid angiopathy; CDR: Clinical Dementia Rating; CN:
Cognitively normal; CSF: Cerebrospinal fluid; ECM: Extracellular matrix; EMIF-
AD: European Medical Information Framework for Alzheimers disease; FDG:
Fluorodeoxyglucose; FDR: False discovery rate; GO-BP: Gene Ontology biologi‑
cal processes; GRS: Gene-specific risk scores; GWAS: Genome-wide associa‑
tion study; iPSC: Induced pluripotent stem cells; LD: Linkage disequilibrium;
LTD: Long-term depression; MAPK: Mitogen-activated protein kinase; MBD:
Multimodality Biomarker Discovery; MCI: Mild cognitive impairment; MMSE:
Mini-Mental State Examination; PC1: Principal component; PCA: Principal
component analysis; PET: Positron Emission Tomography; PGRS: Polygenic risk
scores; PPS: Proteomic process scores; SNP: Single nucleotide polymorphisms;
SUVR: Standardized uptake value; T-tau: Total tau; TMT: Tandem mass tag. Authors’ contributions Conceptualization: PJV, BMT. Methodology proteomics EMIF-AD: JG, HZ. Formal analyses: BMT, PJV, LR, JG, VD, SH, ED. Resources: JG, LR, IJ, ED, SH, SL,
VD, MT, FRJV, JP, PMLA, RV, AL, JLM, SE, YFL, LF, KS, LB, SL, JS, SV, IB, DP, GS, KB, PS,
CET, HZ, PJV. Data curation: SV, IB. Writing—Original Draft: BMT, PJV. Writing—
Review & Editing: all authors. Supervision: PJV, BMT. Funding acquisition: PJV,
SL, HZ, JB, KB, LB, KS. All authors read and approved the final manuscript. Author details 6. Konijnenberg E, Tomassen J, den Braber A, Ten Kate M, Yaqub M, Mulder
SD, Nivard MG, Vanderstichele H, Lammertsma AA, Teunissen CE, et al. Onset of Preclinical Alzheimer Disease in Monozygotic Twins. Ann Neurol. 2021;89:987–1000. 1 Alzheimer Center Amsterdam, Department of Neurology, Amsterdam
Neuroscience, Vrije Universiteit Amsterdam, PO Box 7057 1007, MB, Amster‑
dam, The Netherlands. 2 Alzheimer Center Limburg, School for Mental Health
and Neuroscience, Maastricht University, PO Box 616, 6200, MD, Maastricht,
The Netherlands. 3 Department of Neurobiology, Care Sciences and Society,
Division of Neurogeriatrics, Karolinska Institutet, Stockholm, Sweden. 4 Clini‑
cal Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal,
Sweden. 5 Department of Psychiatry and Neurochemistry, Institute of Neu‑
roscience and Physiology, Sahlgrenska Academy at the University of Goth‑
enburg, Mölndal, Sweden. 6 Department of Complex Trait Genetics, Center
for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU
University, Amsterdam, the Netherlands. 7 Section Genomics of Neurodegen‑
erative Diseases and Aging, Department of Clinical Genetics, Vrije Universiteit
Amsterdam, Amsterdam UMC, Amsterdam, the Netherlands. 8 1St Department
of Neurology, AHEPA University Hospital, Thessaloniki, Makedonia, Greece. 9 Old
Age Psychiatry, University Hospital Lausanne, Lausanne, Switzerland. 10 Depart‑
ment of Geriatric Psychiatry, University Hospital of Psychiatry and University
of Zürich, Zürich, Switzerland. 11 Fundación CITA-Alzhéimer Fundazioa, San
Sebastian, Spain. 12 Neurology Service, University Hospitals Leuven, Leuven,
Belgium. 13 Laboratory for Cognitive Neurology, Department of Neurosciences,
KU Leuven, Leuven, Belgium. 14 IIB-Sant Pau, Hospital de La Santa Creu I Sant
Pau, Universitat Autonoma de Barcelona, Barcelona, Spain. 15 Barcelonaβeta
Brain Research Center (BBRC), Barcelona, Spain. 16 Alzheimer’s Disease Unit
and Other Cognitive Disorders Unit, Hospital Clinic de Barcelona, Barcelona,
Spain. 17 Reference Center for Biological Markers of Dementia (BIODEM),
Institute Born‑Bunge, University of Antwerp, Antwerp, Belgium. 18 Depart‑
ment of Neurology, UZ Brussel and Center for Neurosciences, Vrije Universiteit
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ences, Örebro University, Örebro, Sweden. 20 Department of Geriatric Psychiatry,
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Germany. 21 Complex Genetics Group, VIB Center for Molecular Neurology,
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Centers (AUMC), Amsterdam Neuroscience, Netherlands. 28 Center for Lifespan
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via the PRIDE partner repository with the dataset identifier https://doi.org/
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from the authors. Datasharing may be restricted by consent given by research
participants within each contributing cohort and by European GDPR regula‑
tions, which currently exclude data sharing with a number of non-European
countries. Statistical data can be found in the supplementary information files. via the PRIDE partner repository with the dataset identifier https://doi.org/
10.6019/PXD019910. Access to other EMIF-AD MBD data can be requested
from the authors. Datasharing may be restricted by consent given by research
participants within each contributing cohort and by European GDPR regula‑
tions, which currently exclude data sharing with a number of non-European
countries. Statistical data can be found in the supplementary information files. Additional file 2: Figure S1. Longitudinal change in CSF total tau. Figure
S2. Enrichment of synaptic processes in individuals with AD according to
t-tau status. Figure S3. Enriched GO biological processes and SUZ12 and Additional file 2: Figure S1. Longitudinal change in CSF total tau. Figure
S2. Enrichment of synaptic processes in individuals with AD according to
t-tau status. Figure S3. Enriched GO biological processes and SUZ12 and Page 14 of 16 Visser et al. Molecular Neurodegeneration (2022) 17:27 Visser et al. Molecular Neurodegeneration Competing interests
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Identification and tissue expression profile of genes from three chemoreceptor families in an urban pest, Periplaneta americana
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Scientific reports
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Identification and tissue expression
profile of genes from three
chemoreceptor families in an urban
pest, Periplaneta americana
Yan Chen1,*, Ming He2,*, Zhao-Qun Li3, Ya-Nan Zhang4 & Peng He2 received: 01 February 2016
accepted: 19 May 2016
Published: 09 June 2016 Periplaneta americana is a notorious urban pest prevalent in human habitats; very little is known
about its chemosensory mechanism. Employing the advanced next-generation sequencing technique,
in the present study, we conducted transcriptome sequencing and analysis of the antennae of the
adult males and females as well as their mouthparts using an Illumina platform. This resulted in the
discovery of a huge number of the members of all major known chemosensory receptor families in
P. americana, including 96 odorant receptors (ORs), 53 ionotropic receptors (IRs), and 33 gustatory
receptors (GRs). Tissue expression profiles showed most of them mainly expressed in antennae and
phylogenetic analysis demonstrated the expansion in the clade distinguishing them from other
functionally well-known Lepidoptera species. A high percentage of chemosensory receptor genes (ORs
in particular) showing female antenna bias in mRNA expression was observed. Our results provide
a basis for further investigations on how P. americana coordinates its chemosensory receptor genes
in chemical communication with environments, and for development of novel pest management
approaches. Insects communicate with their environment through chemosensation to help them accomplish a large number
of essential physiological processes, such as mate-finding, host location, and alarming their conspecifics. Insects
employ three major groups of chemosensory receptors, namely odorant receptors (ORs), variant ionotropic
receptors (IRs), and gustatory receptors (GRs)1–3. Insect ORs and GRs possess seven transmembrane domains;
compared to mammalian G-protein coupled receptors, orientation of these domains is reverse, with an intracel-
lular N-terminus and an extracellular C-terminus4. Analysis of available insect genome sequences indicates that
the OR family has undergone rapid evolution in a species-specific manner. A highly variable number of insect
ORs have been identified in different insect species; for example, 62 ORs occur in Drosophila melanogaster5,
79 in Anopheles gambiae6, 170 in Apis mellifera7, 259 in Tribolium castaneum8, and 66 in Danaus plexippus9. In
D. melanogaster antenna, only one OR gene is expressed in most dendritic membranes of the olfactory sensory
neurons (OSNs)10. OR co-receptor (ORco), being one of the most conserved OR genes among various species,
pre-dates other ORs that appear only in winged insects11. ORco interacts with other ligand-specific ORs to form
an ORx-ORco complex, which functions as a ligand-gated cation channel12. Pheromone receptors (PRs), one of
the sub-classes of ORs, are specifically activated by sex pheromone components and have been widely studied in
Lepidoptera13,14. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 01 February 2016
accepted: 19 May 2016
Published: 09 June 2016 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Tissues
♂Antennae
♀Antennae
Mouthparts
Total size
6.67 Gb
6.29 Gb
6.35 Gb
GC content
36.80%
36.82%
41.15%
Number of transcripts
304,023
Total unigene count
248,192
Genes with homologues in NR
40,294
Total transcript nucleotides
210,868,904
Total unigene nucleotides
143,462,349
N50 transcript length
1155 nt
N50 unigene length
795 nt
Longest unigene length
32,380 nt
Median unigene length
327 nt
Table 1. Summary of P. americana transcriptome assembly. Table 1. Summary of P. americana transcriptome assembly. GRs, the other members of the chemosensory superfamily, are likely to have the same ancestor as ORs and also
have a seven transmembrane structure15. Insect GRs mainly respond to non-volatile ligands with the exception
of CO2. Based on the known substances to which they respond, GRs were grouped with sugar receptors16, CO2
receptors17, and bitter or other receptor18. Although GRs do not have a common coreceptor such as ORco, they
are also supposed to form heteromeric complexes, without fixed combinations19. IR, a new family of olfactory
receptors, has the same ancestor as the mammalian ionotropic glutamate receptors (iGluRs)20. IRs are involved
in insect odorant reception, as was first shown by the combined misexpression experiments and their subcel-
lular localization in olfactory organs in D. melanogaster20. Further research showed that IRs differed from ORs
in responding to carboxylic acids and amines21. IRs can be classified into two distinct subfamilies with different
ancestors, namely, the conserved “antennal IRs” and the species-specific “divergent IRs.” The antennal IRs may
represent the original olfactory receptor family of insects. IR8a and IR25a, belonging to antennal IRs, are two
IR co-receptors22; they usually exhibit higher expression than other IRs23,24. Besides the olfactory function, IRs
also play a key role in gustation. In D. melanogaster, IR52c and IR52d are co-expressed in the taste sensilla of the
foreleg of males and regulate sexual behaviour, whereas IR76b is involved in salt tasting25. Cockroaches are a big group of ancient insects of the order Blattodea. Of the approximately 4,600 cockroach spe-
cies, about 30 are associated with human habitats. American cockroach, Periplaneta americana, one of the notori-
ous urban pests is common and difficult to control. It can tolerate a wide range of environments, from Arctic cold to
tropical heat. Periplanone-A and periplanone-B are two major sex-pheromone components emitted from the female
P. americana, which trigger strong electroantennogram responses and behavioural response of conspecific male
individuals26. www.nature.com/scientificreports/ To avoid toxic baits, cockroaches rapidly evolved their gustatory system responsible for glucose
aversion27. However, little is known about the chemosensory mechanism of these essential chemicals (such as
sex pheromone components, periplanone-A and periplanone-B, and glucose) in this species. Only few che-
mosensory receptor gene fragments have been identified in another cockroach species, Blattella germanica28. Therefore, in the present study, we sequenced and analyzed three transcriptomes of the adults of P. americana
using next-generation sequencing, and identified 190 putative chemosensory receptor genes. These included 96
ORs, 61 iGluRs/IRs, and 33 GRs. To understand their potential functions, we conducted gene ontology (GO)
annotation and scanned the tissue-specific expression of these sequences, as well. We also examined their phy-
logenetic relationships with some other insect species. Identification and tissue expression
profile of genes from three
chemoreceptor families in an urban
pest, Periplaneta americana
Yan Chen1,*, Ming He2,*, Zhao-Qun Li3, Ya-Nan Zhang4 & Peng He2 Not much is known about the recognition mechanism in other insect species with sex phero-
mones of different chemical structure. 1Key Lab of Optimal Utilization of Natural Medicine Resources, Department of Pharmacology of Chinese Material
Medica, Guizhou Medical University, Huaxi District, Guiyang, Guizhou 550025, People’s Republic of China. 2State Key
Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide
and Agricultural Bioengineering, Ministry of Education, Guizhou University, Huaxi District, Guiyang 550025, People’s
Republic of China. 3Key Laboratory of Tea Biology and Resource Utilization, Ministry of Agriculture, Tea Research
Institute, Chinese Academy of Agricultural Science, Hangzhou, 310008, People’s Republic of China. 4College of
Life Sciences, Huaibei Normal University, Huaibei, 235000, People’s Republic of China. *These authors contributed
equally to this work. Correspondence and requests for materials should be addressed to Y.-N.Z. (email: ynzhang_
insect@163.com) or P.H. (email: phe1@gzu.edu.cn) 1 Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 Results
S In the molecular function category, the genes expressed in the three organs were mostly enriched for
binding (e.g., nucleotide, ion, and odorant binding) and catalytic activity (e.g., hydrolase and oxidoreductase). In
the biological process category, most common were the cellular and metabolic processes. In the cellular compo-
nent terms, the most abundant were cell and cell part (Fig. 1B). Identification of P. americana OR/IR/GR genes. The unigenes related to candidate olfactory receptors
(ORs/IRs/GRs) were identified based on the keyword searches of the BLASTx and Pfam family annotations. To
avoid missing any P. americana OR/IR/GR genes, the predicted unigene protein sequences were also analyzed
using PSI-BLASTp with OR/IR/GR sequences from B. germanica30 and Z. nevadensis31. In all, we identified 190
unigenes belonging to the chemosensory receptor family in the transcriptome of male antennae, female antennae,
and mouthparts of P. americana. These included 96 ORs (including one ORco gene), 61 IRs/iGluRs (53 IRs and 8
iGluRs), and 33 GRs, all of which shared high identities with other insect OR (~34–88%), IR (~33–95%), and GR
(~29–84%) genes after re-BLASTx identification. Information on the OR, IR, and GR genes including the unigene
references, lengths, and the best BLASTx hits are listed in Supplementary Table S2. Tissue expression profile of P. americana OR/IR/GR genes. Specific tissue expression pattern usually
indicates specific gene functions. Thus, quantitative polymerase chain reaction (qPCR) was conducted to investi-
gate the tissue expression profile of the candidate P. americana OR/IR/GR genes (Figs 2–4). For OR and IR genes,
we selected four tissues, namely the male antennae, female antennae, maxillary palps (Mp), and legs (Le). The
results indicated that these OR or IR-encoding genes were expressed exclusively in the antennae (especially in
female antennae) of the olfactory organ. Based on statistical analysis, 79 of the 85 P. americana (Pame) ORs and
43 of the 46 PameIRs showed antenna-biased expression pattern (11 PameORs and 4 PameIRs were not amplified
in all the tested tissues despite attempts using multiple primer pairs). Of these, PameORco had the highest expres-
sion among all the ORs. Only 4 PameORs and one PameIRs were dominantly expressed in the male antennae, 53
PameORs and 30 PameIRs were female antennae-biased, and 22 PameORs and 11 PameIRs were equal expressed
in the male and female antennae (Figs 2 and 3). Results
S Sequencing and assembly of transcriptome. Three transcriptomes from male antennae (mA), female
antennae (fA), and mouthparts (Mo) were sequenced using the Illumina HiSeq™ 4000 platform (Illumina,
Tianjin, China) and assembled with Trinity (version: r20140413p1)29. About 45.91 (mA), 43.45 (fA), and 43.17
(Mo) million reads were obtained for each transcriptome. After filtering, 44.45 (mA), 41.92 (fA), and 42.34 (Mo)
million clean reads were generated, which comprised of 6.67 (mA), 6.29 (fA), and 6.35 (Mo) gigabases (Gb), with
a longest unigene length of 32,380 nt and a median length of 327 nt after combined assembly of these three tran-
scriptome. Finally, these reads were assembled into 304,023 transcripts and 248,192 unigenes, with N50 lengths
of 1,155 and 795 nt, respectively (Table 1). In addition, the unigenes with a sequence length >500 nt accounted for
29.14% of the transcriptome assembly (Supplementary Fig. S1). The transcriptome raw reads have been deposited
with the NCBI SRA database (accession number: SRR3089536, SRR3089537, and SRR3089538). Homology analysis and gene ontology annotation. BLASTx homology searches of all the 248,192
unigenes showed that 40, 294 (16.23%) had homologous genes in the non-redundant (NR) protein database with
a cut-off E-value of 10−5. The best match percentage (35.32%) was with Zootermopsis nevadensis sequences, fol-
lowed by sequences from Acyrthosiphon pisum (5.30%), Stegodyphus mimosaum (4.10%), T. castaneum (3.10%),
and Diaphorina citri (2.60%) (Fig. 1A). p
g
Gene ontology (GO) annotations for all the unigenes were obtained using the Blast2GO pipeline, according to
the BLASTx search against NR. The GO annotations were used to classify the transcripts into functional groups
according to specific GO categories. Among the 248,192 unigenes, 38,307 (15.43%) could be assigned to various Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 2 cientificreports/
Figure 1. Annotation summaries for P. americana unigenes. (A) Species distribution of unigenes with the
best hit annotation terms in the non-redundant (NR) database. (B) Gene ontology (GO) classifications of
P. americana unigenes. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Annotation summaries for P. americana unigenes. (A) Species distribution of unigenes with the
best hit annotation terms in the non-redundant (NR) database. (B) Gene ontology (GO) classifications of
P. americana unigenes. Figure 1. Annotation summaries for P. americana unigenes. (A) Species distribution of unigenes with the
best hit annotation terms in the non-redundant (NR) database. (B) Gene ontology (GO) classifications of
P. americana unigenes. GO terms. Results
S Due to their predominant expression in male antennae (the
Male antennae/Female antennae mRNA expression level >10), two other highly expressing PameORs, PameOR1,
and PameOR2, with expression levels in the male antennae being 16.8- and 36.9-fold higher than in the female
antennae, respectively (Fig. 2A–C) were observed, suggesting the two ORs may participate in female sex phero-
mones recognition. Seven PameORs, PameOR17, 23, 33, 40, 43, 90, and 92, three PameIRs, PameIR23, 24, and 25
(Fig. 3A–C) demonstrated female-specific expression (Female antennae/Male antennae >10). We also found 2
PameORs (PameOR63 and 67) and 4 PameIRs (PameIR1, 10, 16 and 17) to be dominantly expressed in the major Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 3 cientificreports/
Figure 2. Tissue expression profile of PameOR genes based on relative mRNA quantity. (A) Heat map
illustrating the Log10 transformation of mRNA expression levels of the PameORs in different tissues. (B) Relative
mRNA expression level of all the PameOR genes; the level of PameOR1 expression in the male antennae was set
as 1. (C) Relative mRNA expression level of each PameOR gene in a represented stack; the level of PameOR in
the male antennae was set as 1. mA, male antennae, fA, female Mp, maxillary palps, Le, Legs. www.nature.com/scientificreports/ Figure 2. Tissue expression profile of PameOR genes based on relative mRNA quantity. (A) Heat map
illustrating the Log10 transformation of mRNA expression levels of the PameORs in different tissues. (B) Relative
mRNA expression level of all the PameOR genes; the level of PameOR1 expression in the male antennae was set
as 1. (C) Relative mRNA expression level of each PameOR gene in a represented stack; the level of PameOR in
the male antennae was set as 1. mA, male antennae, fA, female Mp, maxillary palps, Le, Legs. chemosensory organs not only in the antennae but also in the maxillary palps. However, 4 PameORs (PameOR17,
74, 75, and 81), 4 PameIRs (PameIR21, 31, 35 and 42), and all 6 iGluRs (PameiGluR1~PameiGluR6) were not
observed to have an obvious chemosensory organ-biased expression. Five tissues were selected for investigation of PameGR genes: antennae mixed from both the sexes, maxillary
palps, labial palps (Lp), mouthparts (Mor, without maxillary palps and labial palps), and legs. The results showed
that 21 of the 32 PameGRs expressed majorly in the maxillary palps/labial palps (PameGR27 was not detected in
all the tested tissues; Fig. Results
S 4A–C); this included dominant expression of PameGR20, 26, and 28 in the maxillary
palps and labial palp-biased expression of PameGR19. y
g
p
Five tissues were selected for investigation of PameGR genes: antennae mixed from both the sexes, maxillary
palps, labial palps (Lp), mouthparts (Mor, without maxillary palps and labial palps), and legs. The results showed
that 21 of the 32 PameGRs expressed majorly in the maxillary palps/labial palps (PameGR27 was not detected in
all the tested tissues; Fig. 4A–C); this included dominant expression of PameGR20, 26, and 28 in the maxillary
palps and labial palp-biased expression of PameGR19. Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 4 www.nature.com/scientificreports/ Phylogenetic analysis
To further unco er the functional role of P americana OR/IR/GR genes ph loge
Figure 3. Tissue expression profile of PameIR genes based on relative mRNA quantity. (A) Heat map
illustrating the Log10 transformation of mRNA expression levels of the PameIRs in different tissues. (B) Relative
mRNA expression levels among all the PameIR genes; the expression level of PameIR1 in the male antennae was
set as 1. (C) Relative mRNA expression level of each PameIR gene in a represented stack; the relative expression
of PameIR in the male antennae was set as 1. mA, male antennae, fA, female Mp, maxillary palps, Le, Legs. Figure 3. Tissue expression profile of PameIR genes based on relative mRNA quantity. (A) Heat map
illustrating the Log10 transformation of mRNA expression levels of the PameIRs in different tissues. (B) Relative
mRNA expression levels among all the PameIR genes; the expression level of PameIR1 in the male antennae was
set as 1. (C) Relative mRNA expression level of each PameIR gene in a represented stack; the relative expression
of PameIR in the male antennae was set as 1. mA, male antennae, fA, female Mp, maxillary palps, Le, Legs. Phylogenetic analysis. To further uncover the functional role of P. americana OR/IR/GR genes, phyloge-
netic trees were constructed using sequences of typical OR/IR/GR genes from other insect species for which
the whole genome or transcriptome data were available. Eighty-five PameORs were observed to be distributed
in five major groups (Group A, B, C, D, and E) with other insect species ORs (Fig. 5). Bombyx mori PR and A. pisum OR groups were found to be independent groups without any orthologues of PameORs. Results
S Group C was
the ORco group, which is the most conventional OR among various species; only one ORco gene, PameORco,
was grouped in the same sub-group with another cockroach, B. germanica, BgerORco gene. PameOR55 was the
only PameOR in Group A, which was grouped with 6 BmorORs and 23 DmelORs. PameOR75 and PameOR90
belonged to Group D with 29 BmorORs, 3 DmelORs, and 4 ZnevORs. Group B was a Blattaria-specific group
with 62 PameORs, 26 ZnevORs, and 2 BgerORs. Group E was also a Blattaria-specific group that included 19
PameORs, 30 ZnevORs, and 3 BgerORs. Phylogenetic analysis. To further uncover the functional role of P. americana OR/IR/GR genes, phyloge-
netic trees were constructed using sequences of typical OR/IR/GR genes from other insect species for which
the whole genome or transcriptome data were available. Eighty-five PameORs were observed to be distributed
in five major groups (Group A, B, C, D, and E) with other insect species ORs (Fig. 5). Bombyx mori PR and A. pisum OR groups were found to be independent groups without any orthologues of PameORs. Group C was
the ORco group, which is the most conventional OR among various species; only one ORco gene, PameORco,
was grouped in the same sub-group with another cockroach, B. germanica, BgerORco gene. PameOR55 was the
only PameOR in Group A, which was grouped with 6 BmorORs and 23 DmelORs. PameOR75 and PameOR90
belonged to Group D with 29 BmorORs, 3 DmelORs, and 4 ZnevORs. Group B was a Blattaria-specific group
with 62 PameORs, 26 ZnevORs, and 2 BgerORs. Group E was also a Blattaria-specific group that included 19
PameORs, 30 ZnevORs, and 3 BgerORs. Four major iGluR groups were the antennal IR, divergent IR, NMDA iGluR, and non-NMDA iGluR groups. IR8a and IR25a were co-receptors present in the IR group. Two PameIRs, PameIR8 and PameIR25, were observed
to be distributed in these two co-receptors groups, respectively. Six PameIRs, PameIR26, 27, 31, 38, 51, and 53,
were distributed in the IR75 and IR64a groups. PameIR1 and PameIR10 are the orthologous genes of antennal Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 5 www.nature.com/scientificreports/ ntificreports/
Figure 4. Tissue expression profile of PameGR genes based on relative mRNA quantity. (A) Heat map
illustrating the Log10 transformation of mRNA expression levels of the PameGRs in different tissues. Results
S (B) Relative
mRNA expression level of all the PameGR genes; the level of expression of PameGR1 in the antennae was set
as 1. (C) Relative mRNA expression level of each PameGR gene in a represented stack; the level of expression
of PameGR in the antennae was set as 1. An, antennae, Mp, maxillary palps, Lp, labial palps Mor, mouthparts
without Mp and Lp, Le, Legs. Figure 4. Tissue expression profile of PameGR genes based on relative mRNA quantity. (A) Heat map
illustrating the Log10 transformation of mRNA expression levels of the PameGRs in different tissues. (B) Relative
mRNA expression level of all the PameGR genes; the level of expression of PameGR1 in the antennae was set
as 1. (C) Relative mRNA expression level of each PameGR gene in a represented stack; the level of expression
of PameGR in the antennae was set as 1. An, antennae, Mp, maxillary palps, Lp, labial palps Mor, mouthparts
without Mp and Lp, Le, Legs. IR21a and IR68a groups, respectively. PameIR6, 15, 44, and 48 were four genes that were grouped with the anten-
nal IR41a. However, no orthologous genes of IR87a, IR7, IR41a, IR93, and IR76b were found in P. americana. Two
major divergent IR groups of Blattaria were found in the IR tree, namely the A and B group. Group A contained
26 PameIRs and 49 ZnevIRs. Group B contained 11 PameIRs and 48 ZnevIRs (Fig. 6). p
( g
)
In the GR phylogenetic tree, 16 PameGRs and 3 PameGRs were distributed in the sugar and CO2 receptors,
respectively (Fig. 7). No orthologues of fructose receptors were noticed. The other 12 PameGRs were grouped in
another clade “bitter/other receptors” without functionally identified orthologues. Discussion In the present study, we determined the repertoire of chemosensory receptor families (ORs, IRs, and GRs) in a
notorious urban pest, P. americana. These receptor genes have potential significance as targets for developing
new pest control strategies, as well as for elucidating the molecular mechanisms that underlie insect-host inter-
actions. Transcriptomes (19.31 Gb in total) of three chemosensory organs, male antennae, female antennae, and
mouthparts, were sequenced; this was higher than the transcriptomes processed in most of the other studies32–34. After extensive sequencing and assembly using the Trinity software, we identified 96 ORs, 61 IRs/iGluRs (53 IRs Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 6 www.nature.com/scientificreports/ Figure 5. Phylogenetic tree of P. americana PameORs and other typical insect ORs. PameORs are
highlighted in red. Species abbreviations: Apis, A. pisum; Pame, P. americana; Bger, B. germanica; Znev,
Zootermopsis nevadensis; Dmel, D. melanogaster; Tdom, Lygus lineolaris; Bmor, B. mori. Figure 5. Phylogenetic tree of P. americana PameORs and other typical insect ORs. PameORs are
highlighted in red. Species abbreviations: Apis, A. pisum; Pame, P. americana; Bger, B. germanica; Znev,
Zootermopsis nevadensis; Dmel, D. melanogaster; Tdom, Lygus lineolaris; Bmor, B. mori. and 8 iGluRs), and 33 GRs in this species. The number of ORs was much higher than in the two other Blattaria
species, Z. nevadensis (60 ORs, genome data)31 and B. germanica (5 ORs, antennal transcriptome data)30. It was
also higher than the 62 ORs found in D. melanogaster15 and the 79 ORs in A. gambiae6,35, but was much lower
than the number of ORs reported in T. castaneum (259 ORs)8, A. mellifera (170 ORs)7, and L. moratoria (142
ORs)36. The number of IRs was lower than the numbers found in Z. nevadensis (136 IRs)31 and higher than those
found in B. germanica (5 IRs, antennal transcriptome data)30, but it was much higher than the 14 IRs found in A. gossypii24 and Sogatella furcifera23, 57 in D. melanogaster20,22,37, and 22 in A. gambiae38. The number of GRs was
much lower than that in Z. nevadensis (75 GRs)31, D. melanogaster (68 GRs)15, Anopheles gambiae (72 GRs)6, and
B. mori (65 GRs)39, but was higher than in A. mellifera (13 GRs)7. Unlike other ancient insects, which either lack
ORco (Lepismachilis y-signata) or possess three ORco genes (Thermobia domestica)11, only one ORco gene was
found in P. americana, which has implies a fully developed ORx/ORco-based olfactory system. Discussion This result is same
as that observed in another cockroach, B. germanica, where also only one ORco gene, BgerOR1, an orthologue of
PameORco, was present in the antennal transcriptome30. IR8a and IR25a are also coreceptors in the IR system20. PameIR8 and PameIR25 were found clustered in the IR8a and IR25a group with members from other insect
species, which are candidate IR coreceptors in P. americana and the orthologous genes, ZnevIR8, ZnevIR25, and
BgerIR4 for the IR coreceptors from two other Blattaria species. These findings suggest that the adaptation of
distinct species to their hosts has led to the diversification of ORs, IRs, and GRs during their evolution. and 8 iGluRs), and 33 GRs in this species. The number of ORs was much higher than in the two other Blattaria
species, Z. nevadensis (60 ORs, genome data)31 and B. germanica (5 ORs, antennal transcriptome data)30. It was
also higher than the 62 ORs found in D. melanogaster15 and the 79 ORs in A. gambiae6,35, but was much lower
than the number of ORs reported in T. castaneum (259 ORs)8, A. mellifera (170 ORs)7, and L. moratoria (142
ORs)36. The number of IRs was lower than the numbers found in Z. nevadensis (136 IRs)31 and higher than those
found in B. germanica (5 IRs, antennal transcriptome data)30, but it was much higher than the 14 IRs found in A. gossypii24 and Sogatella furcifera23, 57 in D. melanogaster20,22,37, and 22 in A. gambiae38. The number of GRs was
much lower than that in Z. nevadensis (75 GRs)31, D. melanogaster (68 GRs)15, Anopheles gambiae (72 GRs)6, and
B. mori (65 GRs)39, but was higher than in A. mellifera (13 GRs)7. Unlike other ancient insects, which either lack
ORco (Lepismachilis y-signata) or possess three ORco genes (Thermobia domestica)11, only one ORco gene was
found in P. americana, which has implies a fully developed ORx/ORco-based olfactory system. This result is same
as that observed in another cockroach, B. germanica, where also only one ORco gene, BgerOR1, an orthologue of
PameORco, was present in the antennal transcriptome30. IR8a and IR25a are also coreceptors in the IR system20. PameIR8 and PameIR25 were found clustered in the IR8a and IR25a group with members from other insect
species, which are candidate IR coreceptors in P. americana and the orthologous genes, ZnevIR8, ZnevIR25, and
BgerIR4 for the IR coreceptors from two other Blattaria species. Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 Discussion Two PameORs (PameOR63 and PameOR67)
and four PameIRs (PameIR1, 10, 16, and 17) were expressed both in the antennae and maxillary palps, indicating
that they might play key roles not only in olfaction but also in gustation, as previously reported in mosquito46
and fly47. Moreover, 4 PameORs (17, 74, 75, and 81) and 4 PameIRs (21, 31, 35 and 42, and six iGluRs (iGluR1–
6) exhibited non-chemosensory organ-biased expression. All of them expressed in the legs to a certain extent,
which was consistent with some previous studies36,47,48. However, we could not ignore their olfactory function; as
reported earlier, IR expression in the legs affected the mating behaviour of fly47.i roles of OR/IR in olfaction, most Locusta migratoria OR/IR genes displayed olfactory tissue specific expression. However, our results showed that Pame OR/IR genes not only exhibited antenna-biased expression patterns, but
also antennae/maxillary palps-biased and other expression patterns. Two PameORs (PameOR63 and PameOR67)
and four PameIRs (PameIR1, 10, 16, and 17) were expressed both in the antennae and maxillary palps, indicating
that they might play key roles not only in olfaction but also in gustation, as previously reported in mosquito46
and fly47. Moreover, 4 PameORs (17, 74, 75, and 81) and 4 PameIRs (21, 31, 35 and 42, and six iGluRs (iGluR1–
6) exhibited non-chemosensory organ-biased expression. All of them expressed in the legs to a certain extent,
which was consistent with some previous studies36,47,48. However, we could not ignore their olfactory function; as
reported earlier, IR expression in the legs affected the mating behaviour of fly47.i fl
PameGRs expressed mainly in the gustatory organs Mp and Lp, which was consistent with the findings in D. melanogaster49. Insect GRs have been classed in four groups: CO2-, fructose-, non-fructose sugar- receptors, and
bitter/other receptors50. Based on the analysis of phylogenetic tree, we could assign a functional group to most of
the PameGRs. The CO2 receptors are relatively conserved among the insect species51. Three putative CO2 receptors,
PameGR4, 13, and 28 were identified in P. americana. However, only PameGR28 displayed maxillary palps-biased
expression, indicating that it had a greater possibility to function as a CO2 receptor than the other two putative
receptors. They exhibited apparent orthologous relationships with the Z. nevadensis GR genes, PameGR4/ZnevGR16
and PameGR13/PameGR28/ZnevGR7. The presence of three CO2 receptors was consistent with the number of
these receptors in most other insect species, except D. Discussion These findings suggest that the adaptation of
distinct species to their hosts has led to the diversification of ORs, IRs, and GRs during their evolution. i
In some lepidopteran species, the expressions of ORs in male antennae are mostly higher than or equal to that
in female antennae; for example, in Spodoptera litura, 17 of the 25 ORs are male antenna-biased and only 2 of
25 ORs show female-biased expression40. In contrast, in B. mori, only 10/47 of the ORs (female/male >2) show
a female-biased expression41. In P. americana, a lot of PameORs (53 out of 85) and PameIRs (30 out of 57) have
a female antennae-biased expression; this was in contrast to the findings in M. sexta (7/70, RNAseq data, male/
female >10)42 and B. mori (2/68, RT-PCR data, female specific)43. In another study on B. germanica, the authors
selected two ORs, BgerOR1 and BgerOR2, and one IR, BgerIR5, to investigate their tissue expression pattern by
qPCR. All these three genes displayed female antenna-biased expression30. However, this female antenna-biased
expression pattern is expected, as in some hymenopteran species, such as the two parasitoid wasps, Chouioia
cunea44 and Microplitis mediator45, ORs and IRs have been reported to play a crucial role in female individuals for
finding suitable host to lay eggs. However, cockroaches, which are the early ancestors, lack the internal oviposi-
tors. Thus, we hypothesize that the female adults would find more food sources, using food volatiles as cues for
the olfactory receptors, to meet the energy requirements of pregnant or egg-carrying females. Consistent with the Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 7 www.nature.com/scientificreports/ Figure 6. Phylogenetic tree of P. americana PameIRs and other typical insect iGluRs and IRs. PameIRs are
highlighted in red. Bger, B. germanica; Znev, Zootermopsis nevadensis; Dmel, D. melanogaster; Bmor, B. mori. Figure 6. Phylogenetic tree of P. americana PameIRs and other typical insect iGluRs and IRs. PameIRs are
highlighted in red. Bger, B. germanica; Znev, Zootermopsis nevadensis; Dmel, D. melanogaster; Bmor, B. mori. roles of OR/IR in olfaction, most Locusta migratoria OR/IR genes displayed olfactory tissue specific expression. However, our results showed that Pame OR/IR genes not only exhibited antenna-biased expression patterns, but
also antennae/maxillary palps-biased and other expression patterns. Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 Discussion We
hypothesize that some other GRs, not present in this group, might play a role in detecting fructose. are several lineage-specific expansions of Blattaria species; the P. americana and Z. nevadensis bitter/other receptors
are common with bitter receptors of other insect species39,42,54. Eleven orthologous gene pairs (ZnevGR16/PameGR4,
ZnevGR7/PameGR13/PameGR28, ZnevGR14/PameGR19, ZnevGR15/PameGR10, ZnevGR18/PameGR11,
ZnevGR10/PameGR21, ZnevGR8/PameGR30, ZnevGR12/PameGR3, ZnevGR34/PameGR20, ZnevGR47/PameGR22,
and ZnevGR69/PameGR2/PameGR15) were observed in these two species indicating that the bitter/other receptors
are conserved. BmorGR9 in B. mori55 and DmelGR43a in D. melanogaster were defined as fructose receptors16. It has
recently been reported that DmelGR43a can respond to a range of sugars19. However, we did not find any fructose
receptor orthologue in P. americana, or in the genome of other known species, Z. nevadensis and A. pisum. We
hypothesize that some other GRs, not present in this group, might play a role in detecting fructose. yp
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g
p
g
p y
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Adults of female P. americana release two major active sex pheromone components, periplanone-A and
periplanone-B, to solicit the male adults26. Thus, like in the lepidopterans, male antenna predominate expression
is the standard to find PR13,56. PameOR1 and PameOR2, the higher expressing PameORs in the same clade, are
proposed to be the candidate PRs, with their expression levels in the male antennae being much higher than in the
female antennae and feebly detected in other tissues. Certainly, the putative PR function needs further elucidation
by in vivo and in vitro studies. Besides ORs, IRs, such as IR52c and IR52d of D. melanogaster, are also involved in
the mating behaviour47. In P. americana, we observed only one IR, PameIR2, to possess male-antennae dominant
expression. This IR belonged to the divergent IR of the Blattaria-specific group A with no orthologous genes,
and far separated from DmelIR52c and Dmel52d. However, whether the two major sex pheromone components
activate the two respective PRs or PameIR2 needs to be investigated further.i p
g
In conclusion, based on the analyses of transcriptomic data, we identified a large number totally 190 new
chemosensory receptors in the urban pest, P. americana; these included 96 ORs, 61 iGluRs/IRs, and 33 GRs,
which is much number than many other insect species. Our results provide a valuable resource for investigating
and elucidating the mechanisms of olfaction and gustation in P. americana. Discussion melanogaster, which only has two receptors, DmelGR21a and
DmelGR63a51. The sugar receptors (SRs) were first reported in D. melanogaster. Eight genes, DmelGR5a, 64a-f, and
61, which are active for a number of non-fructose sugars in vivo by coding for a single GR or for heterodimers19. Fourteen putative SRs were identified in P. americana, which were present in same group in the phylogenetic tree as
the DmelSRs. The number of SRs is variable in different insect species due to the distinct host and food they have; two
have been reported in A. mellifera7, five in M. sexta42, B. mori39, and Z. nevadensis31, eight in D. melanogaster19 and A. gambiae6, 10 in D. plexippus42, and 16 in T. castaneum52. Eleven of the 14 SRs in P. americana showed Mp/Lp-biased
expression pattern, including equal expression of 10 SRs in Mp and Lp. One SR was predominantly expressed in Mp,
indicating the Mp/Lp are both important organs to sense sugars. Consistent with this observation, the sugar-binding
sites are present in contact with chemosensory hairs on the maxillary palps of P. americana53. Four pairs of ort-
hologues were observed, namely PameGR7/PameGR9/PameGR29/BmorGR4, PameGR25/ZnevGR6/ZnevGR5,
PameGR12/PameGR23/ZnevGR1, and PameGR6/ZnevGR2. However, the functional information about these orthol-
ogous genes is not available. The other 15 PameGRs remain clustered with the predicted bitter/other receptors. There Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 8 www.nature.com/scientificreports/ Figure 7. Phylogenetic tree of P. americana PameGRs and other typical insect GRs. PameGRs are
highlighted in red. Bger, B. germanica; Znev, Zootermopsis nevadensis; Dmel, D. melanogaster; Bmor, B. mori,
Agam, A. gambiae. Figure 7. Phylogenetic tree of P. americana PameGRs and other typical insect GRs. PameGRs are
highlighted in red. Bger, B. germanica; Znev, Zootermopsis nevadensis; Dmel, D. melanogaster; Bmor, B. mori,
Agam, A. gambiae. are several lineage-specific expansions of Blattaria species; the P. americana and Z. nevadensis bitter/other receptors
are common with bitter receptors of other insect species39,42,54. Eleven orthologous gene pairs (ZnevGR16/PameGR4,
ZnevGR7/PameGR13/PameGR28, ZnevGR14/PameGR19, ZnevGR15/PameGR10, ZnevGR18/PameGR11,
ZnevGR10/PameGR21, ZnevGR8/PameGR30, ZnevGR12/PameGR3, ZnevGR34/PameGR20, ZnevGR47/PameGR22,
and ZnevGR69/PameGR2/PameGR15) were observed in these two species indicating that the bitter/other receptors
are conserved. BmorGR9 in B. mori55 and DmelGR43a in D. melanogaster were defined as fructose receptors16. It has
recently been reported that DmelGR43a can respond to a range of sugars19. However, we did not find any fructose
receptor orthologue in P. americana, or in the genome of other known species, Z. nevadensis and A. pisum. Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 Methods Insect samples. P. americana were purchased from an insect rearing factory in Anhui province, China. We
collected 200 antennae and mouthparts each, from the adults of both the sexes (male/female = 1:1) for tran-
scriptome sequencing. We dissected various tissues (male/female = 1:1, approximately 100 antennae of both the
genders, 80 maxillary palps, 80 labial palps, and 80 mouthparts without maxillary palps and labial palps, and 100
legs for each replicate experiment) collected from the adults under a microscope in three replicates for each tissue
type. The tissue samples were stored in liquid nitrogen at −80 °C until further use. Insect samples. P. americana were purchased from an insect rearing factory in Anhui province, China. We
collected 200 antennae and mouthparts each, from the adults of both the sexes (male/female = 1:1) for tran-
scriptome sequencing. We dissected various tissues (male/female = 1:1, approximately 100 antennae of both the
genders, 80 maxillary palps, 80 labial palps, and 80 mouthparts without maxillary palps and labial palps, and 100
legs for each replicate experiment) collected from the adults under a microscope in three replicates for each tissue
type. The tissue samples were stored in liquid nitrogen at −80 °C until further use. cDNA library construction and Illumina sequencing. Total RNA was extracted using TRIzol reagent
(Invitrogen Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA degradation and contamina-
tion was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer spectrophotometer
(IMPLEN, CA, USA). RNA concentration was measured using Qubit RNA Assay Kit in Qubit 2.0 Fluorometer
(Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent
Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The cDNA library construction and Illumina
sequencing of the samples were performed by Novogene Bioinformatics Technology Co. Ltd., Beijing, China. A total amount of 3 μg RNA was used as input material for the RNA sample preparations. Sequencing libraries
were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s
recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was puri-
fied from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using diva-
lent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5×). First strand
cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Methods Second
strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining over-
hangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3′ ends of DNA
fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to
select cDNA fragments of preferentially 150–200 bp length, the library fragments were purified with AMPure XP
system (Beckman Coulter, Beverly, USA). Thereafter, 3 μL USER Enzyme (NEB, USA) was added to size-selected,
adaptor-ligated cDNA and incubated at 37 °C for 15 min followed by 5 min at 95 °C before PCR. PCR was per-
formed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. Finally, the
PCR products were purified (AMPure XP system) and the library quality was assessed on an Agilent Bioanalyzer
2100 system. De novo assembly of short reads and gene annotation. Raw data (raw reads) in the fastq format were
first processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing the
reads containing adapter, reads containing ploy-N, and low quality reads from the raw data. Simultaneously, Q20,
Q30, GC-content, and sequence duplication level of the clean data were calculated. All the downstream analyses
were based on clean data with high quality. De novo transcriptome assembly was performed using the short reads
assembly program Trinity (version: r20140413p1) with min_kmer_cov set to 2 by default and all other parameters
were set to default values. The overlap settings used for the assembly were 30 bp and 80% similarity, and all the
other parameters were set to their default values. Unigenes >150 bp were aligned by BLASTx with protein databases, including Nr, Swiss-Prot, KEGG, and
COG (e-value <10−5), to identify proteins with high sequence identity and to assign putative functional anno-
tations. Next, we used the Blast2GO program (version: b2g4pipe_v2.5, e-value = 1.0E-6) (https://www.blast2go. com/) to obtain GO annotations of the unigenes and we obtained the GO functional classifications using WEGO
software (http://wego.genomics.org.cn/cgi-bin/wego/index.pl). Phylogenetic analysis. Amino acid sequences of the selected ORs, iGluRs/IRs, and GRs were aligned with
the MAFFT (E-INS-I parameter)57. Thereafter, PhyML 3.1 with LG substitution model was used to construct a
maximum likelihood phylogenetic tree using Bayesian analysis. The OR dataset comprised ORs in the available
databases from: A. pisum58,59, B. germanica30, D. melanogaster15, B. mori41, Z. nevadensis31, and T. domestica11. The
IR dataset comprised IRs from B. germanica30, B. Discussion As a crucial first step towards under-
standing of their functions, we also conducted a comprehensive examination of the tissue expression patterns
and phylogenetic tree of these olfactory receptor genes, which demonstrated that most of these OR, IR, and GR
genes were expressed in the chemosensory organs and most OR and IR genes are female antenna-biased expres-
sion indicating their key role in female physiological behaviours. Our findings provide the foundation for future
research into the olfactory and gustatory system of P. americana, such as the calling behaviour. It could also help
in identifying a large number of potential target genes for controlling this notorious pest. Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 9 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ were calculated according to the 2−ΔΔCq method. The relative fold-changes in the different tissues were calculated
and normalized based on the transcript levels in the male antennae. were calculated according to the 2−ΔΔCq method. The relative fold-changes in the different tissues were calculated
and normalized based on the transcript levels in the male antennae. Statistical analysis. Data (mean ± SE) from various samples were subjected to one-way nested analysis of
variance (ANOVA) followed by a least significant difference test (LSD) for mean comparison using SPSS Statistics
17.0 (IBM, Chicago, IL, USA). Statistical analysis. Data (mean ± SE) from various samples were subjected to one-way nested analysis of
variance (ANOVA) followed by a least significant difference test (LSD) for mean comparison using SPSS Statistics
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nogaster37. The GR dataset comprised GRs from D. melanogaster15, A. gambiae6, Z. nevadensis31, and A. mellifera7. Finally, the trees were viewed and group edited with FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/). RNA extraction and cDNA synthesis. Total RNA was extracted using EasyPure RNA Kit (TransGen
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Biotech, Beijing, China). The mRNA levels were quantified using ADP-ribosylation factor (the cDNA sequence
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cDNA sequence was identified from this study) as the reference genes. Means and standard errors were obtained
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9. Smadja, C. M. et al. Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. How to cite this article: Chen, Y. et al. Identification and tissue expression profile of genes from three
chemoreceptor families in an urban pest, Periplaneta americana. Sci. Rep. 6, 27495; doi: 10.1038/srep27495
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chemoreceptor families in an urban pest, Periplaneta americana. Sci. Rep. 6, 27495; doi: 10.1038/srep27495
(2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports | 6:27495 | DOI: 10.1038/srep27495 12
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Ecoeficiencia: clave de la responsabilidad ambiental empresarial en el sector textil
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Ecoeficiencia: clave de la responsabilidad ambiental
empresarial en el sector textil
Eco-efficiency: The Key to Corporate Environmental
Responsibility in the Textile Industry
Remedios Pitre-Redondo
Universidad de la Guajira, Riohacha, Colombia.
Santander De la Ossa-Guerra
Universidad de Sucre, Sincelejo, Colombia.
Hugo Hernández-Palma
Universidad del Atlántico, Barranquilla, Colombia.
Resumen
Objetivo: Este estudio tuvo como finalidad identificar si las pequeñas y medianas empresas textiles (PYMES),
ubicadas en el Departamento del Atlántico, Colombia, realizan prácticas de Ecoeficiencia para el control de
residuos contaminantes. Método: se utilizó una investigación de tipo descriptivo, con un enfoque cuantitativo y
un diseño de campo no experimental, en la que se aplicó una encuesta cerrada, dirigida a una muestra de 53
empresas. Resultados y discusión: Entre los principales hallazgos se encontró que el 56% de las PYMES
participantes no realizan acciones para reducir el volumen o la peligrosidad de sus residuos, ya que no los
consideran importantes ni rentables. Por lo tanto, estos procesos no son ecoeficientes y el compromiso con la
preservación del medio ambiente y la sostenibilidad de las PYMES en estudio es insuficiente. Conclusiones:
Dado que el sector textil es uno de los recursos con menor índice de reutilización del mundo, es fundamental que
este vínculo con los procesos de gestión sostenible y la ecoeficiencia sea claro y constante, apuntando a una
internalización de los procesos de responsabilidad social, apoyándose en el Estado, las familias, las personas y las
leyes para su constante evolución y desarrollo.
Palabras clave: Desarrollo sostenible, Prácticas ambientales, Responsabilidad social, Residuos sólidos.
Clasificación JEL: Q01, Q50, Q56.
Autor de Correspondencia
hugoghernandezp@mail.uniatlant
ico.edu.co
Recibido: 03-03-2020
Aceptado: 08-07-2020
Publicado: 30-11-2020
Copyright © 2020
Desarrollo Gerencial
Abstract
Objective: This study aimed to identify whether small and medium sized textile companies (SMEs), located in
the Department of Atlántico, Colombia, currently carry out Eco-efficiency practices for the control of waste,
contaminating residues, among other aspects. Method: A descriptive type of research was used, with a
quantitative approach and a non-experimental field design, in which a closed survey was applied, directed at a
sample of 53 companies. Results and discussion: Among the main findings, it was found that 56% of the
participating SMEs do not take actions to reduce the volume or hazardousness of their waste, since they do not
consider it important or profitable. Therefore, these processes are not eco-efficient and the commitment to
environmental preservation and sustainability of the SMEs under study is insufficient. Conclusions: Given that
the textile sector is one of the resources with the lowest rate of reuse in the world, it is fundamental that this
link with the processes of sustainable management and eco-efficiency be clear and constant, aiming at an
internalization of the processes of social responsibility, relying on the State, families, people and laws for their
constant evolution and development.
Keywords: Sustainable development, Social responsibility, Environmental practices, Solid waste.
JEL classification: Q01, Q50, Q56.
Como citar este artículo (APA):
Pitre-Redondo, R., De la Ossa-Guerra, S., & Hernández-Palma, H. (2020). Ecoeficiencia: clave de la responsabilidad ambiental
empresarial en el sector textil. Desarrollo Gerencial, 12(2), 1-20. https://doi.org/10.17081/dege.12.2.3870
1
Remedios Pitre Redondo, Santander De la Ossa Guerra, Hugo Hernández Palma
Introducción
Con el paso de los años, el ser humano ha pasado de ser una especie nómada el cual se dedicaba a la
casa y a los cultivos estacionarios a convertirse hoy en día en seres sedentarios; caracterizados por el
establecimiento de comunidades y el desarrollo de industrias a gran escala para lograr suplir las
necesidades de sus iguales. A partir de este punto, es innegable reconocer el actual impacto que ha
tenido esta tendencia y estilo de vida en el medio ambiente; pues el cambio climático con el pasar de
cada segundo está teniendo mayores connotaciones y trayendo consecuencias que podrían llegar a ser
irreversibles si no se toman las acciones necesarias a tiempo. Basta con revisar los números de emisiones
de CO2 (Dióxido de carbono), que al sol de hoy han alcanzado los números más altos en los últimos
800.000 años, según reportes de Oceanografía Scripps (2019) y al año 2007 según reportes de
Intergovernmental Panel on Climate Change (2007), el 19% de esta cifra corresponde a la
responsabilidad del sector industrial.
En tal sentido, la sociedad actual está tomando conciencia acerca del impacto que tiene la industria
sobre el deterioro del medio ambiente; según Suárez y Molina (2014), el desarrollo industrial si bien
induce una fuerte reactivación socioeconómica, contribuye a la generación de diversas problemáticas
ambientales como es el caso de la contaminación, el desequilibrio en los ecosistemas y la extinción de
fauna y flora y, así mimo, da paso a la génesis de problemáticas sociales, entre ellas y quizás la más
relevante, el detrimento de la salud pública principalmente, a partir de la contaminación atmosférica
(Ballester, et. al., 2003; Vargas, 2005) y de la contaminación en las cuencas hídricas (Maldonado, 2009)
Este hecho viene como resultado de un extenso proceso histórico por el cual ha pasado la sociedad
para poco a poco ir reconociendo el cambio climático como una problemática real, la cual es resultado de
la acción indiscriminada del hombre y de sus industrias que por mucho tiempo se desarrollaron obviando
las necesidades y el impacto hacia los ecosistemas.
Lo anterior, ha sido el punto de partida para la creación de leyes proteccionistas que impulsan
estrategias direccionadas a la evaluación, prevención y control de los factores desligados de la actividad
industrial, que influyen en la aparición de problemas ambientales (Torres, Barreto y Rincón, 2015). Pero
así mismo, ha motivado a las empresas que desean ser competitivas a desarrollar sistemas de gestión que
les ayuden a reducir su huella ambiental, por medio del mejoramiento de sus procesos, la adquisición de
tecnológicas y prácticas de producción limpias, el posicionamiento de políticas de optimización de recursos
e incluso, el eco-diseño de sus productos; todo ello con la finalidad de hacer un uso efectivo de sus
recursos, reducir costes y eco-posicionarse en el mercado.
Desarrollo Gerencial 12(2):1-20, Julio-Diciembre, 2020. DOI: https://doi.org/10.17081/dege.12.2.3870
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Remedios Pitre Redondo, Santander De la Ossa Guerra, Hugo Hernández Palma
El eco-posicionamiento, según la Fundación Fórum Ambiental (2014) se ha popularizado entre las
empresas por cuestiones como la calidad, el ahorro, el mercado, la imagen, las oportunidades de negocio
y, las consideraciones de tipo ético y social.
De acuerdo con Eryuruk (2012) las empresas de hoy deben rediseñar sus estructuras existentes, para
convertirse en ambientalmente responsables, de este modo puedan aumentar su credibilidad en el medio
pero, así mismo, producir más, con menos recursos, lo que evita el desperdicio de las materias primas,
disminuye las emisiones y reduce los daños al medio ambiente (Mansour et. al., 2006).
Uno de los sectores industriales con mayor grado de contaminación ambiental en Colombia, es el
textil; de acuerdo con el informe de Colombia Productiva (2012) la mayoría de las empresas de este
sector no cuentan con políticas de sostenibilidad ni de Responsabilidad Social Empresarial RES ya que el
67% de estas empresas no elabora ningún tipo de informe de sostenibilidad, el 25% no tiene gestión de
aguas residuales, el 58% no cuenta con un sistema de control de emisiones atmosféricas y solo el 4,4%
de las empresas recuperan o reutilizan residuos de su producción. Estas cifras son alarmantes debido a
que la producción textil en Colombia sigue en aumento, mientras que la mayoría de estas empresas
adolecen de iniciativas para reducir su impacto medioambiental, siendo evidentes factores de riesgo
desligados de si operación, como es el caso de los residuos químicos altamente contaminantes.
En atención a lo anterior, surge el concepto de Ecoeficiencia, una práctica que intenta sostener el valor
ecológico de las empresas al tiempo que potencia el crecimiento económico del mercado; así pues, las
empresas que implementan prácticas de Ecoeficiencia consiguen aumentar su competitividad,
contribuyendo, al tiempo, al desarrollo sostenible del territorio, puesto que, añaden mayor valor a sus
productos con un menor consumo de recursos naturales y disminuyen la contaminación, lo que ayuda a
mitigar los riesgos ambientales que pueden llegar a afectar a sus stakeholders internos y externos.
En este sentido, se destaca que las prácticas relacionadas con la preservación del ambiente por parte
de las organizaciones vienen como resultados de lo que se llama la Responsabilidad Social Empresarial
RSE, lo cual es un propósito importante para toda organización, así como para los grupos de interés, pues
permite lograr sus objetivos de mejor manera y tener un impacto positivo en el entorno y es una serie de
obligaciones y compromisos tanto legales, como éticos, nacionales e internacionales que adquieren las
empresas con los llamados grupos de interés, derivados del impacto generado por la existencia, actividad,
además las operaciones de las organizaciones, las cuales son producidas en el contexto social, laboral,
medioambiental, así como también al relacionado a los derechos humanos, en tal aspecto, este tiene un
efecto significativo en el proceso de gestión de las empresas, tanto a lo relacionado a sus diversas
actividades productivas y comerciales, así como en el desarrollo de las relaciones con los mencionados
grupos de interés (Duran, Fuenmayor, Sergio y Hernández, 2016).
Desarrollo Gerencial 12(2):1-20, Julio-Diciembre, 2020. DOI: https://doi.org/10.17081/dege.12.2.3870
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Remedios Pitre Redondo, Santander De la Ossa Guerra, Hugo Hernández Palma
Bajo estas ideas, se aporta que actualmente Colombia es una nación que dentro del marco de la
sostenibilidad se encuentra atrasado con relación a otros países, pues no se toman en cuenta diversos
procesos o regulaciones que permitirían asegurar la prevalencia de los ecosistemas en el país (Cuartas y
Méndez, 2016).
A demás, desde ciertas posturas, se aporta que brindar especial atención a las necesidades del medio
ambiente sobre las necesidades de las organizaciones puede traer como resultados un declive de la
calidad competitiva de dichas empresas frente a los mercados; trayendo como resultado un declive en los
índices económicos y laborales de los países y regiones. Ciertamente, se debe tener en cuenta que hoy en
día se vive una época caracterizada por los mercados altamente competitivos; donde los constantes
avances tecnológicos por resultado de la globalización obligan a las empresas a siempre buscar la
innovación y la calidad como premisa para mantener su estatus competitivo (Parra et al, 2018).
A partir de este planteamiento, surge la disyuntiva de encontrar un equilibrio entre la calidad de los
procesos productivos de manera que se tengan en cuenta las necesidades del medio ambiente a través de
procesos de gestión sostenible; concepto direccionado a la gestión de los recursos encaminado a la
calidad de los procesos cuidando la necesidad de los ecosistemas (Custodio y De Oliveira, 2016)
A partir de lo anterior se considera que la Ecoeficiencia es una alternativa para alcanzar este valor de
posicionamiento dentro de los mercados, tomando en consideración el cambio climático como hecho
catalizador de acciones enfocadas a la sostenibilidad. De esta manera, el presente artículo de
investigación se desarrolló con la finalidad de identificar si las pequeñas y medianas empresas (Pymes)
textiles, ubicadas en el departamento del Atlántico, Colombia en la actualidad llevan a cabo prácticas de
Ecoeficiencia para el control de despilfarros, desechos contaminantes, consumo de energía, entre otros;
aspectos que apuntan al posicionamiento de prácticas ambientalmente responsables. Si bien, según la
Superintendencia de Sociedades (2013), el departamento del Atlántico solo aporta el 3,62% de la
producción textil nacional, se selecciona este segmento geográfico para el estudio, dada la evolución que
este ha tenido en los últimos años en el sector textil, además, porque, a partir de su posición privilegiada
y vocación exportadora, el departamento del Atlántico ha dado paso a dos importantes clústeres: uno de
confecciones y otro diseño textil.
Fundamentación Teórica
Responsabilidad Social Empresarial RSE
La RSE es uno de los elementos más representativos de las organizaciones en la actualidad pues ha
generado una gran nivel de debate sobre su alcance e implicaciones dentro de las diversas industrias
(Raufflet, Lozano, Duque y de la Torre, 2012; Padilla et al., 2017) proponiendo cambios en las prácticas
Desarrollo Gerencial 12(2):1-20, Julio-Diciembre, 2020. DOI: https://doi.org/10.17081/dege.12.2.3870
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Remedios Pitre Redondo, Santander De la Ossa Guerra, Hugo Hernández Palma
empresariales de manera que estas cambien direccionamiento hacia la sociedad, respondiendo a las
exigencias de las comunidades que piden empresas más consientes con el impacto de sus actividades en
la sociedad y en el medio ambiente (Solarte y Duque, 2017).
En este sentido, se aporta que la RSE es una obligación que poseen las empresas con relación al
impacto que estas llegan a generar en su entorno y como estas tienen un importante efecto sobre las
dimensiones social, económica y ambiental (Ramírez et al., 2018). Del mismo modo, esta busca garantizar
que el impacto positivo equitativo, sustentable y que cause el menor impacto posible a las llamadas
partes interesadas. Ciertamente la RSE implica que estas sean conscientes sobre el papel que tienen
dentro un todo más complejo donde las organizaciones, al ser un fenómeno humano se involucren con las
comunidades para aporta a las construcciones de una nueva sociedad (Pérez, Espinoza y Peralta, 2016;
Castro, et al., 2019).
De esta manera, se convierte en un apunte al cambio, al desarrollo y a la calidad de las organizaciones
que a la larga permite crear una percepción más humana de los consumidores, lo cual permitirá que la
organización como marca tenga un reconocimiento positivo y una mayor aceptación.
Asimismo, en el libro verde se hacen mención a dos importantes dimensiones de la RSE (Salazar,
Hidalgo y Manríquez, 2017; Abreo, Pinzón y Chacón, 2018); los cuales se enfocan a lo interno, referido a
prácticas que se llevan a cabo dentro de la organización (Ortiz y Bandeira, 2015) y la dimensión externa,
referida al impacto que las acciones tomadas por la organización tienen en el entorno de esta
(Chumaceiro, Hernández, Yori y Ziritt, 2013).
A partir de aquí, el Centro para la Acción de la Responsabilidad Social Empresarial en Guatemala
[Centra RSE] (2006) citado en De Grazia (2006) propone la pirámide de la responsabilidad social
empresarial como medio de apoyo a las organizaciones que busquen implementarla; priorizando áreas de
acción referidas a esta responsabilidad social empresarial. En esta pirámide se establecen los niveles de
los criterios para diseñar políticas funcionales direccionadas al cambio sostenible y responsable:
Figura 1. Pirámide de la responsabilidad social empresarial
Diseño/ejecucion de
politicas de estado
Proyección
comunidad
Proyección familia
Desarrollo de la persona
Cumplimiento régimen legal
Fuente: Elaboración propia de autores basado en CENTRA RSE (2006).
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Autores como Barros Retamozo y González (2015) aportan que las políticas de estado permiten que las
acciones vayan acorde a los planteamientos legales para responder a las proyecciones comunitarias. Así
mismo generando un involucramiento con las familias, creando un espacio de confianza entre empresapersona para promover el desarrollo de las personas dentro y fuera de esta, siempre cumpliendo con los
estándares legales requeridos, que fungen como la base de las acciones en todo momento. En este
sentido, se reconoce que dentro de la responsabilidad social empresarial existen los llamados grupos de
interés (Stakeholders); los cuales se direccionan a las partes interesadas o afectadas por las actividades
(Freeman, 2010). Entre los grupos de interés se pueden mencionar a los trabajadores o las comunidades
los cuales establecen sus intereses hacia la relación de las organizaciones son las temáticas políticas,
sociales o ambientales. Dentro del presente artículo se realiza un énfasis a la relación entre la comunidad,
las empresas y el medio ambiente al reconocer el impacto que se tiene sobre esta.
Del cambio climático a la Ecoeficiencia
Por muchos años se pensó que el cambio climático era un hecho ficticio sin soporte científico hasta
que en los años 70 la comunidad científica comenzó a preocuparse por los constantes cambios de clima
que sucedían para esa época, comenzando a hacer las primeras predicciones sobre el calentamiento
global (Peterson, Connolley y Fleck, 2008; Deryugina, 2013). Con base a ello, ciertas décadas después los
gobiernos del mundo apoyados en esta comunidad científica llevan a cabo en 1994 la Convención Marco
de las Naciones Unidas sobre el Cambio Climático la cual se encaminó a estabilizar los niveles de
contaminación generados a nivel mundial estableciendo un concepto de cambio climático que lo
comprende desde la actividad humana que altera la composición de la atmosfera mundial (Oscullo y Haro,
2016).
A partir de estos primeros pasos se generan nuevos documentos como el Protocolo De Kioto de la
Convención Marco de las Naciones Unidas sobre el Cambio Climático en 1998 hasta llegar al 2019, donde
no solo existen rigurosas regulaciones sobre los temas ambientales, si no que se han generado modelos y
propuestas que buscan mantener la rentabilidad y competitividad de la industria sin dejar a un lado los
compromisos adquiridos en años anteriores.
Dentro de estos procesos se destaca la Ecoeficiencia (Deryugina, 2013), término que se atribuye a la
relación existente entre el valor de un producto y/o servicio y los impactos medioambientales derivados
de todo el proceso desde su comienzo hasta que este deja de ser utilizado (Durán, Calderón y Montero,
2018). Este proceso de Ecoeficiencia trae como resultado la vinculación de la conciencia ambiental dentro
de las organizaciones, de manera que se logre tener el mayor margen de producción y ganancias, con el
menor consumo de recursos y energía y así reducir el impacto sobre el medio ambiente (Carmelo, 2018).
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En tal sentido y basado en Rincón y Wellens (2011) se presentan los cinco principios de la
Ecoeficiencia:
Figura 2. Principios de la eco-eficiencia
1. Consumo de agua por unidad de valor agregado neto
2. Requerimientos energéticos por unidad de valor agregado neto
3. Contribución al calentamiento global por unidad de valor agregado neto
4. Dependencia de sustancias que deterioran la capa de ozono por unidad
de valor agregado neto
5. Residuos sólidos generados por unidad de valor agregado neto
Fuente: Rincón y Wellens (2011).
Se destaca que al implementar la Ecoeficiencia dentro de una organización implica plantear una
iniciativa de valor pues al promover el desarrollo socioeconómico con un bajo coste de recursos (Vallejo y
Cabrera, 2017), las mismas empresas se convierten en lideres dentro de su sector al ser estas las que
generan una verdadera innovación (Sánchez, 2011). Aquí se plantea un interesante propuesto al afirmar
que el daño ambiental proviene de los procesos para lograr los objetivos y no de los objetivos, por lo que
son estos los que deben ser mejorados y trabajados (Cantú, 2015).
Se aporta que el empleo de la Ecoeficiencia permite a que las organizaciones sean vistas como parte
de un todo en los sistemas naturales (González y Pérez, 2011), por lo que los procesos para su desarrollo
y administración deberían poseer un enfoque sobre los componentes de la industria y la biosfera, la
relación del flujo de la materia con el sistema industrial y considerar la tecnología como medio de
transición para el logro de la no sustentabilidad (Carrillo, 2009).
Actualmente existen diversos enfoques al momento de medir la Ecoeficiencia, pero uno de los más
representativos es el brindado por Desimone y Popoff (2000):
Reducción del derroche de recursos apoyándose en la mejora continua.
Disminución del volumen y toxicidad de los residuos.
Reducción del consumo energético y emisiones contaminantes.
Disminución de los riesgos de incumplimiento de leyes y se mejoran las relaciones de la
comunidad y la empresa.
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En este sentido, se considera que un punto fundamental para lograr la Ecoeficiencia se relaciona con el
empleo de las llamadas Mejores Tecnologías Disponible (MTD), término que se direcciona al uso de los
equipamientos tecnológicos más avanzados y que su uso permite optimizar de la manera más adecuada
los procesos de una organización. Ciertamente se ha reconocido el uso de las MTD para la reducción del
impacto ambiental, ligado al rol ecológico que buscan tomar las nuevas tecnologías en la actualidad (Jiang
et al., 2012)
Método
El presente estudio se desarrolló desde un enfoque cuantitativo, el cual permitió el análisis de
resultados numéricos para su interpretación como procesos humanos. Del mismo modo, se direccionó
desde la investigación descriptiva y exploratoria, para lograr identificar si las pequeñas y medianas
empresas (Pymes) textiles, ubicadas en el departamento del Atlántico cumplen con las claves de la
Ecoeficiencia. El diseño empleado fue de campo, no experimental y transversal pues la investigación
requirió de realizar una medición única en el lugar de origen del fenómeno estudiado, pero sin llevar a
cabo cualquier manipulación de las variables objeto de estudio dentro de la muestra seleccionada.
Este estudio se aplicó a los gerentes, administradores y jefes de producción de las PYMES textiles del
departamento del Atlántico, Colombia. La muestra de la investigación es de tipo no probabilística (Otzen y
Manterola, 2017), para la selección de esta, se consultaron las bases de datos de la Cámara de Comercio
de Barranquilla, en donde se identificaron las pymes textiles ubicadas en el departamento del Atlántico,
legalmente constituidas y, a partir de allí, se seleccionaron de manera aleatoria 53 empresas.
Las pymes elegidas fueron contactadas vía telefónica y, previo consentimiento informado, se les fue
aplicado el instrumento, al cual dieron respuesta de manera efectiva. Para llevar a cabo el mencionado
proceso de recolección de la data, el presente artículo se apoyó en el uso de un cuestionario cerrado con
opciones dicotómicas (Si o No), el cual se configuró a partir de los principios básicos de la Ecoeficiencia,
estructurando de esta manera seis preguntas clave que permiten evidencias el grado de compromiso y
responsabilidad ambiental de las empresas seleccionadas como muestra en este estudio. La distribución
de las preguntas del cuestionario responde de la siguiente manera a las claves de la Ecoeficiencia:
Tabla 1. Distribución del instrumento
Indicador
Reducción del derroche de recursos apoyándose en la mejora continua.
Disminución del volumen y toxicidad de los residuos.
Ítem
Ítem 1 y 5
Ítem 2
Reducción del consumo energético y emisiones contaminantes.
Ítem 3 y 4
Disminución de los riesgos de incumplimiento de leyes y se mejoran las relaciones de la
comunidad y la empresa.
Ítem 6
Fuente: Elaboración Propia de autores (2019)
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Se destaca que este cuestionario se basa en los cinco principios de la Ecoeficiencia que son
enumerados por Rincón y Wellens (2011). Este instrumento fue validado por un grupo de tres expertos en
el área de sostenibilidad empresarial y responsabilidad social corporativa. Del mismo modo, a este
instrumento se le aplico un proceso para determinar su confiabilidad a través del alfa de Cronbach la cual
en este caso fue de 0,67 por lo que se considera como un valor aceptable en estudios de índole social
(Oviedo y Arias, 2005). Se destaca que los datos fueron procesados por medio del programa estadístico
SPSS.
Resultados
En esta sección se estructuran los resultados del cuestionario aplicado a los representantes de las 53
empresas seleccionadas como muestra en esta investigación, los cuales se estructuran a partir de cada
una de las seis preguntas claves que les fueron formuladas durante la etapa de recolección. Una de las
claves de la Ecoeficiencia es el control de los despilfarros de los recursos mediante la mejora continua; de
acuerdo con el estudio el 76% de las pymes textiles participantes efectúa estos procesos de control de
despilfarro; sin embargo, el 24% restante afirma no llevar a cabo tales procesos. (Gráfico 1).
Gráfico 1. Control de despilfarros
¿La empresa controla el despilfarro de los recursos mediante la mejora continua?
24%
76%
Si
No
Cuando las empresas no tienen un control preciso sobre sus desperdicios no solo corren con el riesgo
de que aumenten sus costos de producción, sino de que se disminuya de manera sustancial su
rendimiento y la percepción de valor que poseen sus clientes en torno a los productos y servicios que
ofrece, esto último puede explicarse de la siguiente manera: si los clientes perciben que la empresa no es
consciente de reciclar, y reutilizar sus residuos de producción o de establecer técnicas para disminuir sus
despilfarros, podrían preferir a empresas que presten los mismos servicios o productos pero cuya
producción sea más amigable con la naturaleza, en aras de apoyar a compañías ambientalmente
responsables y así, disminuir la huella ambiental industrial. Por fortuna, la mayoría de las compañías
objeto de estudio, afirman tener un control preciso de sus despilfarros.
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Ahora bien, las empresas textiles utilizan diversos insumos químicos considerados como peligrosos en
su proceso de producción, los cuales de no ser gestionados de la manera adecuada pueden constituirse
como residuos altamente contaminantes para el medio ambiente; de allí la importancia de que las
empresas textiles se esfuercen por disminuir el volumen y cambiar algunos insumos químicos que utilizan
en la manufactura de sus productos por sustitutos con menor nivel de toxicidad. Se preguntó a las pymes
textiles objeto de estudio si en la actualidad se esfuerzan por disminuir el volumen y el nivel de toxicidad
de sus residuos, los resultados dejan en claro que la mayoría de estas entidades, representada por el
56% no llevan a cabo acciones para disminuir el volumen ni la peligrosidad de sus residuos y que, solo el
43% de estas empresas desarrolla este tipo de acciones. (Gráfico 2).
Gráfico 2. Disminución volumen y toxicidad de residuos
¿La empresa se esfuerza por dismuir el volumen y toxicidad de residuos?
44%
56%
Si
No
El hecho de que estas empresas no gestionen de la manera adecuada sus residuos, más aún si estos
son peligrosos (sustancias químicas), se constituye como un riesgo evidente para el medio ambiente y
para los stakeholders externos, principalmente la comunidad. A partir de esta falta de control, los residuos
pueden alterar el equilibrio de los ecosistemas, contaminar las cuencas hídricas y afectar de manera
profunda la salud de los animales y seres humanos. Otro de los cuestionamientos que se expusieron en
el instrumento de investigación se basó en indagar si estas empresas controlaban su consumo de energía
y emisiones contaminantes por medio de prácticas de producción limpia; el 72% de las pymes
participantes dio una respuesta afirmativa; el 28% de las pymes restantes, expresó que no llevaban a
cabo prácticas de producción limpias para el control de dichos aspectos (Gráfico 3).
Gráfico 3. Prácticas de Producción limpia
¿En la empresa se controla el consumo de energía y emisiones
contaminantes por medio de practicas de producción limpia?
28%
72%
Si
No
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Seguidamente se preguntó a las empresas si en la actualidad incorpora MTD para optimizar su
producción; el 72% niega implementar este tipo de procesos; mientras que el 28% restante asegura si
ejecutarlos. (Gráfico 4).
Gráfico 4. MTD para optimizar producción
¿En la actualidad la empresa incorpora mejores tecnologías disponibles para
optimizar su producción?
28%
72%
Si
No
Las MTD, además, contribuyen a disminuir el impacto ambiental a través de la prevención de la
contaminación y el diseño de productos ecológicos, lo que crea valor para las partes de interés y
posiciona a la empresa como ambientalmente responsable.
Otro punto importante, que se estudió a partir del instrumento, se centra en el desarrollo de
estrategias orientadas a reducir costos en el consumo de recursos (energéticos) y materias primas;
muchas empresas no cuentan con el capital económico para incorporar prácticas de producción limpia o
MTD, sin embargo, pueden diseñar e incorporar otro tipo de estrategias que les permita minimizar el
consumo de recursos, más aun si se trata de aquellos categorizados como no renovables como la energía
eléctrica convencional; según los resultados del estudio, el 56% de las pymes participantes afirma que
no desarrolla citadas estrategias; mientras que el 44% restante asegura si ejecuta este tipo de
estrategias. (Gráfico 5)
Gráfico 5. Estrategias reducción consumo
¿En la actualidad la empresa desarrolla estrategias orientadas a reducir costos en el consumo
de recursos/materias primas?
44%
56%
Si
No
Finalmente, se preguntó a las empresas si consideraban que aumentando la seguridad ambiental
podrían generar valor para sus partes de interés y aumentar su credibilidad en el mercado; el 84% estuvo
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de acuerdo con esta afirmación; mientras que el 16% ignora que estas prácticas surtan beneficios para su
operación. (Gráfico 6)
Gráfico 6. Seguridad Ambiental para crear valor
¿Considera que aumentando la seguridad ambiental podrá generar valor para sus
partes de interes y aumental su credibilidad en el mercado?
16%
84%
Si
No
De esta manera, se observa que un gran porcentaje de las organizaciones que forman parte de la
muestra reconocen el gran valor que genera la mejoría de la seguridad ambiental para las partes
interesadas y así mejorar su credibilidad en el mercado.
Discusiones
A partir de los resultados del trabajo de campo, puede afirmarse que la mayoría de las pymes textiles
ubicadas en el departamento del Atlántico no son empresas eco-eficientes; tal aseveración se sustenta en
que, si bien la mayoría de las empresas asegura controlar sus despilfarros, estas no llevan a cabo
procesos que les permitan disminuir el volumen y el grado de toxicidad de los insumos que utilizan en su
producción, así pues, de poco sirve que una empresa regule las pérdidas de materiales, sino controla por
ejemplo, el uso de sus materias primas o la cantidad de desechos peligrosos que dispone en el medio
ambiente.
En consonancia con lo anterior, Rincón (2001) manifiesta que las empresas no pueden ser
competitivas mientras no eliminen sus desperdicios y no trabajen con parámetros de productividad; pero,
así mismo, esta competitividad depende de que las compañías minimicen el tiempo y la cantidad de
recursos que utilizan en su producción; en este orden de ideas, las pymes textiles objeto de estudio,
tienen grandes retos para ser competitivas a nivel nacional e internacional, puesto que, según el estudio,
la mayoría de estas empresas no cuentan con estrategias orientadas a reducir el consumo de recursos
(energéticos) y materias primas, lo cual conlleva a altos costos de producción y, por consiguiente, a
aumentar la huella ambiental de su operación.
El consumo desmedido de energía, de acuerdo con Blanco y Arce (2012) produce grandes impactos
ambientales, ya que, algunas centrales productoras de este recurso no renovable (Al menos en Colombia,
por su plantas de generación convencional), utilizan procesos de combustión de los derivados de petróleo
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para generarlo; así mismo, Duart (2007) manifiesta que el uso de la energía eléctrica en la industria
contribuye al aumento en las emisiones de CO2, situación que tiene graves repercusiones sobre el
calentamiento global y al aumento de las tasas de contaminación atmosférica. De igual modo, el consumo
descontrolado de materias primas no solo genera una sobreexplotación de los recursos naturales, sino
que incrementa la tasa de despilfarros y residuos que pueden contaminar el medio ambiente.
A partir de lo anterior, las empresas textiles deben reconocer la importancia de reducir el consumo de
energía, según Campos et al. (2008) citado en Del Pilar et. al., (2013), “Las empresas industriales pueden
lograr ahorros de energía de hasta un 40%, algunos sin inversión de capital, mediante la aplicación de
métodos de gestión energética” (p. 115); pero así mismo la importancia de reducir al máximo la
utilización de materias primas no renovables y de incursionar en materiales sustitutos que cumplan con la
misma función a un costo ambiental más bajo (Cañizares y Jurado, 2019).
Dentro del cuestionario se trabajó sobre la aplicación de las MTD para reducir el consumo de recursos,
donde se encontró un bajo porcentaje representativo de su aplicación a nivel organizacional. De este
modo, se aporta que las prácticas de producción limpias, más allá de evitar la emisión de residuos y de
minimizar el uso indiscriminado de recursos energéticos, según Van y Herrera (2007) permiten la
optimización y mejoramiento de procesos empresariales, promueven la innovación tecnológica
(ecodiseño), la sostenibilidad e incluyen el concepto de ciclo vida como orientación de la gestión
ambiental empresarial.
De la mano de las prácticas de producción limpia, surgen las MTD o Mejores Tecnologías Disponibles,
las cuales, según Evrard et. al., (2016) permiten la reducción del uso de recursos finitos y, así mismo la
efectividad en el uso de materias primas, para beneficiar a la empresa, el consumidor y la sociedad en
general. Estos procesos de reducción y optimización, de acuerdo con Serna (2010) pueden levarse a cabo
a partir de que las empresas determinen los sistemas y equipos que permitan la disminución de
consumos, costos energéticos y costos ambientales; estos sistemas son reconocidos en el medio como las
Mejores Tecnologías Disponibles (MTD) y su propósito es reducir al máximo los consumos, los despilfarros
y los residuos de la producción.
Si bien estas MTD ofrecen grandes beneficios a las empresas, en el caso de las pymes textiles del
departamento del Atlántico no han sido implementadas a cabalidad, de hecho el 72% de estas empresa
expresa que en la actualidad no incorpora este tipo de tecnología a su operación; las razones no se
especifican en el estudio, sin embargo, se estima que estas empresas no acuden a las MTD porque no
están dispuestas a invertir recursos económicos o, así mismo, porque no ven su realmente utilidad,
dejando de lado los grandes ahorros que estas MTD pueden generarles. Lo mencionado se enlaza con la
pregunta direccionada a saber si se reducían los índices de consumos de materia prima, donde las
respuestas fueron dividas.
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De allí la importancia de que las compañías objeto de estudio, hagan un uso consiente de sus materias
primas, creen una cultura de reducción, reutilización y reciclaje, además, reemplacen los insumos nocivos
para el medio ambiente, como son las tintas con base química, por elementos biodegradables, los cuales
no solo les ayudarán a prevenir enfermedades de su fuerza laboral y la disminución de costes, sino que
permitirán la salvaguarda del medio ambiente y la salud pública.
A su vez, se percibe que las organizaciones perciben el valor de mejorar la seguridad ambiental como
herramienta para su relación con los grupos de interés y su reputación en el mercado. Cuando las
empresas están enajenadas de los beneficios que pueden percibir al ser ambientalmente responsables,
difícilmente se direccionaran a modificar sus prácticas, procesos de producción y tecnologías, ya que esto
supone para ellas más gastos que utilidades; sin embargo, según Pérez y Camacho (2013) cuando las
empresas logran ser reconocidas en el medio como una entidad con alto sentido de responsabilidad social
y ambiental, esta puede mantener e incrementar su rentabilidad, puesto que se apalanca su imagen,
posicionamiento y trascendencia de la marca en el mercado.
Es claro que, la mayoría de las empresas participantes en el estudio saben que, aumentando la
seguridad ambiental, podrían generar valor para sus partes de interés y, de este modo, incrementar su
credibilidad en el mercado; no obstante, son evidentes los grandes obstáculos que estas entidades tienen
que sobrepasar para ser empresas ambientalmente responsables, dado que sus procesos y condiciones
actuales están yendo en contravía de las crecientes exigencias en la legislación ambiental y los
requerimientos de los consumidores “verdes” actuales. A partir de lo anterior, las pymes textiles deben
implementar herramientas de gestión de Ecoeficiencia que les permitan diagnosticar las oportunidades de
mejora ambiental, optimizar el consumo energético y de materias primas, establecer incluso medidas de
ahorro de agua, tales como las auditorías ambientales, los sistemas de gestión medio ambiental ISO
14001 y programas de contabilidad ambiental.
Conclusiones
En primera instancia, se reconoce que la Responsabilidad Social Empresarial es un elemento de cambio
dentro de las dinámicas empresariales de la actualidad; la cual brinda la oportunidad de direccionar a las
organizaciones a convertirse entes más humanos, los cuales reconocen su importante papel dentro de la
sociedad y de las comunidades como elemento promotor del crecimiento y del desarrollo local y regional.
De este modo, la vinculación a la Ecoeficiencia como proceso de cambio se presenta como una
interesante propuesta para alcanzar altos estándares de calidad y de competitividad de las empresas
vinculando estos objetivos con las necesidades del medioambiente, que, si bien en el pasado parecía
ideas contrarias, hoy en día se comprueba que ambas van de la mano a través de una relación de
sostenibilidad e innovación. De esta manera, al ser el sector textil el generador uno de los recursos con
Desarrollo Gerencial 12(2):1-20, Julio-Diciembre, 2020. DOI: https://doi.org/10.17081/dege.12.2.3870
14
Remedios Pitre Redondo, Santander De la Ossa Guerra, Hugo Hernández Palma
menor índice de reutilización en el mundo, se hace fundamental que esta vinculación con procesos de
gestión sostenible y Ecoeficiencia sean claros y constantes, apuntando a una internalización de los
procesos responsabilidad social, apoyándose en el estado, las familias, las personas y las leyes para su
constante evolución y desarrollo.
Puede visualizarse en el mercado actual un compromiso creciente por parte de las empresas por
contribuir a la sostenibilidad ambiental; estos avances además de tener un impacto positivo en la
naturaleza, en la medida que se disminuye la contaminación, también tiene una incidencia en la
mitigación de enfermedades que perjudican a los stakeholders externos y, cuyo foco son los residuos de
la actividad industrial; sin embargo, al efectuar el estudio en las pymes textiles del departamento de
Atlántico, Colombia, tal compromiso por la preservación y sostenibilidad ambiental es insuficiente, en
primer lugar porque sus acciones son superfluas y estas direccionadas a reparar problemáticas
coyunturales.
En segundo lugar, porque se centran más en desarrollar acciones para su propio beneficio que para
beneficio ambiental, por ejemplo, estas empresas reducen la cantidad de despilfarros para ahorrar
recursos y disminuir costos, pero, no reducen el volumen y toxicidad de los insumos que utilizan y que
luego se convierten en residuos potencialmente peligrosos para el medio ambiente.
Los resultados obtenidos permiten observar que si bien las empresas reconocen la importancia de
emplear mecanismos de seguridad ambiental en sus procesos y buscan disminuir el derroche de recursos,
aún deben trabajar de mejor manera la reducción de materia prima, contaminación y recursos energéticos
que se consumen dentro de estas. Al igual en el empleo de las mejores tecnologías disponibles como base
de apoyo para la consecución de estas metas y puntos clave de la Ecoeficiencia.
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https://iris.unipa.it/bitstream/10447/527842/2/Crespi%20et%20al.%20-%202020%20-%20Unraveling%20Moral%20Reasoning%20in%20Amyotrophic%20Lateral%20Sclerosis%20How%20Emotional%20Detachment%20Modifies%20Moral%20Judgment.pdf
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Unraveling Moral Reasoning in Amyotrophic Lateral Sclerosis: How Emotional Detachment Modifies Moral Judgment
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BRIEF RESEARCH REPORT
published: 21 August 2020
doi: 10.3389/fpsyg.2020.02083 Edited by:
Fabio Giovannelli,
University of Florence, Italy Reviewed by:
Francesca Trojsi,
University of Campania Luigi Vanvitelli,
Italy
Mattia Siciliano,
Second University of Naples, Italy *Correspondence:
Chiara Crespi
chiara.crespi@unipv.it;
chiara.crespi@iusspavia.it Specialty section:
This article was submitted to
Neuropsychology,
a section of the journal
Frontiers in Psychology Received: 19 May 2020
Accepted: 28 July 2020
Published: 21 August 2020 Unraveling Moral Reasoning in
Amyotrophic Lateral Sclerosis: How
Emotional Detachment Modifies
Moral Judgment Chiara Crespi1*, Gaia Chiara Santi2, Alessandra Dodich3, Federica Lupo4,
Lucia Catherine Greco5, Tommaso Piccoli4, Christian Lunetta5 and Chiara Cerami2,6 1 Department of Brain and Behavioral Sciences, University of Pavia, Pavia, Italy, 2 Istituto Universitario di Studi Superiori,
Pavia, Italy, 3 CeRiN, Center for Mind/Brain Sciences, University of Trento, Rovereto, Italy, 4 Department of Experimental
Biomedicine and Clinical Neurosciences, University of Palermo, Palermo, Italy, 5 NEuroMuscular Omnicentre, Niguarda Ca’
Granda Hospital, Milan, Italy, 6 IRCCS Mondino Foundation, Pavia, Italy In the last decade, scientific literature provided solid evidence of cognitive deficits
in amyotrophic lateral sclerosis (ALS) patients and their effects on end-life choices. However, moral cognition and judgment are still poorly investigated in this population. Here we aimed at evaluating both socio-cognitive and socio-affective components
of moral reasoning in a sample of 28 ALS patients. Patients underwent clinical and
neuropsychological evaluation including basic cognitive and social cognition measures. Additionally, we administered an experimental task including moral dilemmas, with
instrumental and incidental conditions. Patients’ performances were compared with
a control group [healthy control (HC)], including 36 age-, gender-, and education-
matched healthy subjects. Despite that the judgment pattern was comparable in ALS
and HC, patients resulted less prone to carry out a moral transgression compared to
HC. Additionally, ALS patients displayed higher levels of moral permissibility and lower
emotional arousal, with similar levels of engagement in both instrumental and incidental
conditions. Our findings expanded the current literature about cognitive deficits in ALS,
showing that in judging moral actions, patients may present non-utilitarian choices
and emotion flattening. Such a decision-making profile may have relevant implications
in applying moral principles in real-life situations and for the judgment of end-of-life
treatments and care in clinical settings. In the last decade, scientific literature provided solid evidence of cognitive deficits
in amyotrophic lateral sclerosis (ALS) patients and their effects on end-life choices. However, moral cognition and judgment are still poorly investigated in this population. Here we aimed at evaluating both socio-cognitive and socio-affective components
of moral reasoning in a sample of 28 ALS patients. Patients underwent clinical and
neuropsychological evaluation including basic cognitive and social cognition measures. Additionally, we administered an experimental task including moral dilemmas, with
instrumental and incidental conditions. Patients’ performances were compared with
a control group [healthy control (HC)], including 36 age-, gender-, and education-
matched healthy subjects. Unraveling Moral Reasoning in
Amyotrophic Lateral Sclerosis: How
Emotional Detachment Modifies
Moral Judgment Despite that the judgment pattern was comparable in ALS
and HC, patients resulted less prone to carry out a moral transgression compared to
HC. Additionally, ALS patients displayed higher levels of moral permissibility and lower
emotional arousal, with similar levels of engagement in both instrumental and incidental
conditions. Our findings expanded the current literature about cognitive deficits in ALS,
showing that in judging moral actions, patients may present non-utilitarian choices
and emotion flattening. Such a decision-making profile may have relevant implications
in applying moral principles in real-life situations and for the judgment of end-of-life
treatments and care in clinical settings. In the last decade, scientific literature provided solid evidence of cognitive deficits
in amyotrophic lateral sclerosis (ALS) patients and their effects on end-life choices. However, moral cognition and judgment are still poorly investigated in this population. Here we aimed at evaluating both socio-cognitive and socio-affective components
of moral reasoning in a sample of 28 ALS patients. Patients underwent clinical and
neuropsychological evaluation including basic cognitive and social cognition measures. Keywords: amyotrophic lateral sclerosis, emotion detachment, moral cognition, moral judgment, social cognition Subjects Twenty-eight patients with a diagnosis of either probable
or definite ALS (Brooks et al., 2000) (25 males; mean
age = 57.79 ± 11.72 years; mean education = 11.50 ± 4.78 years;
mean disease duration from the onset = 1.84 ± 1.47 years)
were enrolled (Table 1) for the present study. According to the
clinical presentation, ALS patients were classified as 19 spinal and
nine bulbar onsets. We excluded patients with global cognitive
deficits, as revealed by a mini mental state examination (MMSE)
raw score < 23 (Duchesne et al., 2005; Diniz et al., 2007;
Mitchell, 2017), or comorbid psychiatric disorders potentially
interfering with cognitive functioning, as well as patients with
respiratory disorders (forced vital capacity < 70% of predicted
capacity), severe dysarthria, and communication difficulties
potentially invalidating the administration and/or interpretation
of neuropsychological assessment. Morality is defined as the set of values and habits adopted
by a cultural group in order to orient its social conduct
(Moll et al., 2005). Tasks involving moral dilemmas—i.e.,
situations where an agent cannot fulfill, with his/her choice, all
applicable moral requirements—classically measure the patient’s
ability to evaluate actions made in respect to a set of virtues
(i.e., “moral judgment”). Moral reasoning is defined as the
sum of all mental conscious processes that allows the agent
to achieve moral judgment (Moll et al., 2005). From the
neuroscience perspective, neurocognitive processes underlying
moral reasoning primarily encompass two main dimensions:
the socio-cognitive and the socio-affective components (Bzdok
et al., 2012). In evaluating a moral dilemma, subjects are
often required to define the “moral acceptability/permissibility”
of the leading character’s behavior (e.g., completely morally
unacceptable or completely morally acceptable), referred to the
individual’s ability to evaluate the character’s action as tolerable,
according to the personal moral values and habits, and to evaluate
the experience considering the relative “emotional valence”
(e.g., pleasantness/unpleasantness) and “emotional arousal” (e.g.,
activation/calm). These emotional dimensions represent those
parameters that typically account for most of the variance in the
final moral judgment (Lotto et al., 2014). Thirty-six age-, gender-, and education-matched healthy
controls (HCs; 25 males; mean age = 52.97 ± 14.03 years;
mean education = 9.97 ± 3.17 years) were recruited at local
senior community centers. They underwent a clinical interview,
a neurologic examination, and a brief neuropsychological
assessment in order to test cognitive efficiency. Subjects Medical history
positive for neuropsychiatric disorders, positive neurologic
examination, MMSE raw score <28 (Measso et al., 1993;
Crespi et al., 2014, 2016; Van Patten et al., 2019), as well as
verbal and visuospatial delayed memory performances (i.e., Rey
Auditory Verbal Learning test, Rey Figure Recall task) below
25th percentile according to the Italian normative values, were
considered as exclusion criteria for HC enrollment. A recent paper on moral judgment in ALS and behavioral
variant of frontotemporal dementia (bvFTD) (Semler et al.,
2019) proved comparable poor patient’s attitude in evaluating
a moral situation in both neurodegenerative conditions. In this
study, experimental assessment included the Moral Competence
Test, the Ethics Position Questionnaire, and the Idler Index of
Religiosity to respectively test moral competence, moral position,
and religiosity. Impairment in moral judgment was regardless
of ethic and religiosity scores and did not correlate with basic
cognitive performances (Semler et al., 2019). INTRODUCTION Crespi C, Santi GC, Dodich A,
Lupo F, Greco LC, Piccoli T,
Lunetta C and Cerami C (2020)
Unraveling Moral Reasoning
in Amyotrophic Lateral Sclerosis: How
Emotional Detachment Modifies Moral
Judgment. Front. Psychol. 11:2083. doi: 10.3389/fpsyg.2020.02083 Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative multisystem disorder that
selectively targeted the motor system (Hardiman et al., 2017). A variable range of non-motor
behavioral and cognitive manifestations affects patients, with a subgroup of ALS developing a
frank frontotemporal dementia (FTD) syndrome (Hardiman et al., 2017). Cognitive disorders often
characterize the clinical phenotype, with a major involvement of language and executive abilities August 2020 | Volume 11 | Article 2083 Frontiers in Psychology | www.frontiersin.org Moral Cognition in ALS Crespi et al. (Consonni et al., 2013; Beeldman et al., 2016; Strong et al., 2017). Behavioral changes may occur with predominant apathy and
depression (Lillo et al., 2011). (Consonni et al., 2013; Beeldman et al., 2016; Strong et al., 2017). Behavioral changes may occur with predominant apathy and
depression (Lillo et al., 2011). other
neurodegenerative
patients
(e.g.,
FTD)
investigated
the relationship between moral judgment, basic and social
cognition abilities (Mendez et al., 2005; Mendez and Shapira,
2009; Gleichgerrcht et al., 2011) showing low performance,
compared to control subjects, in a subtask requiring an
immediate
emotionally
driven
moral
judgment
(Mendez
et al., 2005). The role of affective and emotional processes
on moral decision-making in bvFTD is also supported by
results of Gleichgerrcht et al. (2011). p
(
)
In the last decade, scientific literature provided evidence of
social cognition deficits in ALS, particularly, in the domains
of recognition and processing of emotional materials (Lulé
et al., 2005; Papps et al., 2005; Zimmerman et al., 2007;
Palmieri et al., 2010; Crespi et al., 2014), affective decision-
making (Meier et al., 2010; Girardi et al., 2011), and empathy
(Gibbons et al., 2007; Cavallo et al., 2011; Cerami et al., 2014). Notwithstanding such a large literature on cognitive deficits
and socio-emotional disorders in ALS, moral cognition has
been poorly investigated yet. Research studies on the field
mainly investigated ethical implications of end-of-life treatment
decisions, supporting the independence between basic cognitive
and behavioral impairments and patient decisions on the use
of invasive medical devices (Böhm et al., 2016). In this view,
the literature suggests the need for longitudinal assessment of
decision-making skills to early recognize possible alterations and
their effects on end-of-life choices (Khin Khin et al., 2015). INTRODUCTION In this view, our research study was aimed at investigating
how affective and emotional dimensions influence the decision-
making process of ALS patients in a moral dilemmas task. Frontiers in Psychology | www.frontiersin.org Basic Cognitive and Social Assessment
of Amyotrophic Lateral Sclerosis
Patients Basic cognitive functioning in patients was evaluated with the
Italian version of the Edinburgh Cognitive and Behavioral ALS
Screen (ECAS) battery. This is a widely used multi-domain
brief assessment designed to evaluate a range of cognitive
functions typically affected in ALS (i.e., ALS-Specific domains:
executive functions, social cognition, fluency, and language)
(Poletti et al., 2016). In addition to executive (reverse digit span, The influence of affective parameters on moral judgment
processes
in
ALS
patients
is
a
matter
of
debate. No
detailed
information
about
the
socio-affective
dimension
of moral reasoning was available yet. Some studies on August 2020 | Volume 11 | Article 2083 Frontiers in Psychology | www.frontiersin.org 2 Moral Cognition in ALS Crespi et al. TABLE 1 | Demographic, clinical, and neuropsychological features of the sample. (A) Demographic and clinical features
ALS
HC
Statistics
Female/Male ratio
3/25
11/25
χ2(1) = 0.057
Age in years (mean ± SD)
57.79 ± 11.72
52.97 ± 14.03
t(62) = 0.603
Education in years (mean ± SD)
11.50 ± 4.79
9.97 ± 3.17
t(62) = 0.075
Disease duration in years (mean ± SD)
1.84 ± 1.47
–
–
Age at the onset (mean ± SD)
55.15 ± 11.54
–
–
ALS-FRSr (mean ± SD)
29.80 ± 7.18
–
–
(B) Cognitive and behavioral assessment
% out of cutoff score*
% borderline score*
Mini mental state examination (mean ± SD)
26.29 ± 2.44
(6/28) 21.4%
–
ECAS-global score (mean ± SD)
106.54 ± 12.49
(4/28) 14.28%
(1/28) 3.57%
ECAS-specific (mean ± SD)
77.72 ± 11.09
(4/28) 14.28%
(3/28) 10.71%
ECAS-non-specific (mean ± SD)
28.61 ± 3.0
(1/28) 3.57%
(3/28) 10.71%
SET global score (mean ± SD)
12.57 ± 3.78
(5/28) 17.9%
(3/28) 10.7%
SET emotion attribution condition (mean ± SD)
4.50 ± 1.36
(2/28) 7.1%
(5/28) 17.9%
SET intention attribution condition (mean ± SD)
4.24 ± 1.60
(4/28) 14.3%
(6/28) 21.4%
SET causal inference condition (mean ± SD)
3.94 ± 1.60
(5/28) 17.9%
(4/28) 14.3%
Ek-60F global score (mean ± SD)
46.71 ± 6.70
(4/28) 14.3%
(2/28) 7.1%
The table reports, (A) demographic information for patients and controls, as well as clinical characteristics of the patient group. (B) Illustrates scores obtained from
the cognitive and behavioral assessment in patients, reporting mean and standard deviation (SD), the proportion of patients scoring out of the cutoff score and that of
patients obtaining a borderline score. Basic Cognitive and Social Assessment
of Amyotrophic Lateral Sclerosis
Patients ALS, amyotrophic lateral sclerosis; HC, healthy control; ALS-FRSr, ALS functional rating scale revised; ECAS, Edinburgh cognitive
and behavioral ALS screen battery; SET, story-based empathy task; Ek-60F, Ekman 60 Faces test. ∗percentage of patients obtaining an impaired/borderline performance
was computed according to the Italian normative values. TABLE 1 | Demographic, clinical, and neuropsychological features of the sample. The table reports, (A) demographic information for patients and controls, as well as clinical characteristics of the patient group. (B) Illustrates scores obtained from
the cognitive and behavioral assessment in patients, reporting mean and standard deviation (SD), the proportion of patients scoring out of the cutoff score and that of
patients obtaining a borderline score. ALS, amyotrophic lateral sclerosis; HC, healthy control; ALS-FRSr, ALS functional rating scale revised; ECAS, Edinburgh cognitive
and behavioral ALS screen battery; SET, story-based empathy task; Ek-60F, Ekman 60 Faces test. ∗percentage of patients obtaining an impaired/borderline performance
was computed according to the Italian normative values. proved to be sensitive on ALS patients (Girardi et al., 2011;
Crespi et al., 2014). alternation, and sentence completion tasks), social cognition
(Yoni task), fluency (verbal fluency tasks for words beginning
with the letter “S” and for four-letter words starting with
the letter “C”), and language (naming, comprehension, and
spelling) tasks, ECAS explored also cognitive domains not
typically affected in ALS but common in pathological aging [ALS
Non-specific domains: memory (immediate recall and delayed
recognition) and visuospatial (dot counting, cube counting, and
number location)] (Poletti et al., 2016). Neuropsychological
assessment also included the MMSE (Folstein et al., 1975)
to assess global cognitive efficiency. Primary caregivers were
asked to complete the frontal behavioral inventory (FBI)
(Kertesz et al., 1997; Alberici et al., 2007) to assess behavioral
changes in patients. All subjects gave informed consent to the experimental
procedure, which was approved by the local ethics committee. See Table 1 for details on demographic variables. Frontiers in Psychology | www.frontiersin.org Experimental Moral Task The task of moral dilemmas includes eight scenarios derived from
Lotto et al. (2014). Each dilemma was presented as text in two
phases. The first phase described the scenario in which different
kinds of threats were going to cause death to a group of people. The second phase described a hypothetical resolution in which
the participant, identifying him/herself as the main character,
can choose to kill or not one individual to save the others, who
otherwise would have died. Dilemmas were subgrouped into
two conditions: “instrumental” (i.e., the death of one person is
a mean to save more people) and “incidental” (i.e., the death
of one person is a foreseen but unintended consequence of
the action aimed at saving more people). Additionally, half of
dilemmas involved directly the main character because its life was
at risk (i.e., self-involvement). See Supplementary Table S1 for
stimulus examples. g
p
A brief social cognition battery exploring basic emotion
recognition and emotion and intention attribution in others
was also administered including the Ekman-60-Faces test (Ek-
60F) (Dodich et al., 2014) and the Story-based Empathy Task
(SET) (Dodich et al., 2015). The EK-60F consists of 60 b/w
pictures from the Ekman and Friesen series of pictures of facial
affect. Pictures depict the faces of 10 actors, each displaying
one of the six basic emotions (i.e., happiness, sadness, anger,
fear, surprise, and disgust). The SET is a non-verbal cartoon
task that consists of two main experimental conditions, i.e.,
identifying intentions (SET-IA) and emotional states (SET-EA),
plus a control condition entailing the inference of causality
reaction based on the knowledge of the physical properties of
objects and human bodies (SET-CI). These tasks have been Participants were asked to indicate whether they would do the
proposed action. Then, they were asked to judge how morally
acceptable was the resolution (0 = not at all, 7 = completely), to
attribute an emotional valence to the moral action (0 = totally
unpleasant, 8 = totally pleasant), and to rate the emotional
arousal (0 = totally calm, 8 = totally involved). Outcome variables August 2020 | Volume 11 | Article 2083 3 Moral Cognition in ALS Crespi et al. TABLE 2 | Performance at moral judgment task in patients and healthy controls. Experimental Moral Task A
Type of Dilemma
Significant results
Incidental
Instrumental
Group
ALS
HC
ALS
HC
Affirmative response%
14.28%
88.8%
7.14%
47.22%
Group difference (Incidental): χ2(4) = 36.70, p < 0.001
Group difference (Instrumental): χ2(4) = 14.65, p = 0.005
Moral permissibility
3.15 ± 1.65
2.24 ± 1.50
2.05 ± 2.31
1.07 ± 1.08
Group effect: F(1,62) = 6.09, p < 0.001 Type of dilemma
effect: F(1,62) = 46.58, p < 0.001
Emotional arousal
4.93 ± 2.34
6.88 ± 1.32
5.06 ± 2.70
7.59 ± 1.28
Group effect: F(1,62) = 22.33, p < 0.001 Type of dilemma
effect: F(1,62) = 13.162, p = 0.001 Interaction effect:
F(1,62) = 6.40, p = 0.014
Emotional valence
2.20 ± 1.40
2.22 ± 1.06
2.00 ± 1.20
1.74 ± 0.79
Type of dilemma effect: F(1,62) = 19.96, p < 0.001
B
Involvement
Self
No self
Group
ALS
HC
ALS
HC
Affirmative response%
10.71%
61.11%
10.71%
86.11%
Group difference (Self): χ2(4) = 18.25, p = 0.001 Group
difference (No self): χ2(4) = 38.38, p < 0.001
Moral permissibility
2.48 ± 1.84
1.73 ± 1.39
2.72 ± 2.06
1.58 ± 1.08
Group effect: F(1,62) = 6.09, p < 0.001
Emotional arousal
5.03 ± 2.60
7.12 ± 1.19
4.96 ± 2.45
7.35 ± 1.37
Group effect: F(1,62) = 22.33, p < 0.001
Emotional valence
2.16 ± 1.37
2.06 ± 0.88
2.04 ± 1.23
1.90 ± 1.02
–
The table reports performances at the moral judgment task and significant results obtained from the analysis of variables of interest (i.e., proportion of affirmative responses,
moral permissibility, emotional arousal, and emotional valence). While (A) shows results from mixed ANOVA considering the type of dilemma (incidental vs. instrumental)
as between variable, (B) reports results from mixed ANOVA considering the personal involvement (self vs. no self-involvement) as between variables. ALS, amyotrophic
lateral sclerosis; HC, healthy control. TABLE 2 | Performance at moral judgment task in patients and healthy controls. The table reports performances at the moral judgment task and significant results obtained from the analysis of variables of interest (i.e., proportion of affirmative responses,
moral permissibility, emotional arousal, and emotional valence). While (A) shows results from mixed ANOVA considering the type of dilemma (incidental vs. instrumental)
as between variable, (B) reports results from mixed ANOVA considering the personal involvement (self vs. no self-involvement) as between variables. ALS, amyotrophic
lateral sclerosis; HC, healthy control. Experimental Moral Task Analyses were conducted using IBM SPSS Statistics for
Windows v24.0 (IBM Corporation, Armonk, NY, United States). were (i) the rate of yes/no responses to the proposed resolution
(i.e., moral judgment); the rating of (ii) moral acceptability, (iii)
emotional valence, and (iv) emotional arousal experienced during
the decision-making process. Frontiers in Psychology | www.frontiersin.org Moral Cognition Disorders in
Amyotrophic Lateral Sclerosis Patients
Compared to Healthy Controls The impairments
of decision-making processing of moral actions ascribe ALS
to a more extensive social cognition dysfunction (Lulé et al.,
2005; Papps et al., 2005; Zimmerman et al., 2007; Meier
et al., 2010; Palmieri et al., 2010; Cavallo et al., 2011; Girardi
et al., 2011; Cerami et al., 2014; Crespi et al., 2014), as
also proved by the neuropsychological performances of our
sample. About a third of the patient group displayed in fact
impaired or borderline performances at emotion recognition
and socio-emotional processing tasks. Impairments of such
a
multifaceted cognitive
domain
may be
underestimated
by ECAS battery alone. According to this, an in-depth Concerning the judgment of moral permissibility, the result
of a 2 × 2 (type of dilemma condition × group) mixed ANOVA
showed a significant effect of both group [F(1,62) = 6.1, p = 0.016]
and type of dilemma [F(1,62) = 46.58, p < 0.001] with no
interaction effect, indicating significantly higher ratings in ALS
patients than HC; higher ratings for incidental compared to
instrumental dilemmas characterized both groups (Figure 1). Results from personal involvement × group mixed ANOVA
highlighted a significant effect of group [F(1,62) = 6.1, p = 0.016],
with ALS patients showing higher ratings than HC. We did
not find a significant effect of personal involvement nor an
interaction effect with group. About
ratings
of
emotional
valence,
the
result
of
a
2
×
2
(type
of
dilemma
condition
×
group)
mixed
ANOVA showed a significant effect of the type of dilemma
[F(1,62) = 19.96, p < 0.001], describing low emotional valence
for instrumental dilemmas compared to incidental ones in
both groups, while we did not observe significant group and
interaction effects. We found no significant effects in personal
involvement × group mixed ANOVA. About evaluation of emotional arousal, the result of a 2 × 2
(type of dilemma condition × group) mixed ANOVA showed
a significant effect of both group [F(1,62) = 22.33, p < 0.001]
and type of dilemma [F(1,62) = 13.16, p = 0.001], as well as
a significant interaction effect [F(1,62) = 6.403, p = 0.014],
revealing lower ratings in ALS patients than HC and higher
ratings for instrumental than incidental dilemmas in both groups,
with HC showing a greater difference between instrumental
and incidental conditions than ALS (Figure 1). DISCUSSION See Table 1 for further details. Moral cognition is still a poorly investigated domain in ALS. Although previous results suggested that ALS patients presented
deficits in integrating their moral values into situational
conditions, even if their knowledge of rules and ethics dogma is
preserved (Semler et al., 2019), the impact of changes in affective
processing on decision-making, and specifically related to the
moral judgment, has not been estimated yet. Moral Cognition Disorders in
Amyotrophic Lateral Sclerosis Patients
Compared to Healthy Controls Overall, the ALS sample showed impairments in moral judgment
ability. Chi-Square Independence test highlighted that the global
rate of yes/no response in resolution of moral dilemmas was
significantly different in ALS compared to HC (Incidental
dilemmas: χ2 = 36.70, p < 0.001; Instrumental dilemmas:
χ2 = 14.65, p = 0.005; No self-involvement: χ2 = 38.39,
p < 0.001; Self-involvement: χ2 = 18.25, p < 0.001). These
results were confirmed by one-way ANOVA results, with ALS
patients showing significantly lower percentages of utilitarian
responses than HC in each condition, both factors considered
[i.e., type of dilemma: incidental: F(1,62) = 38.16, p < 0.001;
instrumental: F(1,62) = 3.81, p = 0.05; personal involvement: no
self-involvement: F(1,62) = 20.36, p < 0.001; self-involvement:
F(1,62) = 13.02, p = 0.001]. In the present study, we explored moral cognition in ALS
patients using one of the most reliable moral tasks (Lotto et al.,
2014) and extensively applied on neuropsychiatric populations
(Pletti et al., 2017) and healthy aging (McNair et al., 2018), aiming
at assessing moral judgment controlling for different variables
(i.e., moral acceptability, emotional valence, and arousal). p
y
Our findings provided evidence of different moral judgment
behaviors in patients compared to HC. In detail, ALS showed
significantly lower yes/no rate of responses both in incidental
and
instrumental
conditions,
also
considering
dilemmas
on the basis of personal involvement. At the same time,
patients
displayed
higher
levels
of
moral
permissibility
than HC, reflecting an overall tendency to judge as more
acceptable actions that they would not actually perform. Such a performance may reflect a defective evaluation of
the
consequences
of
the
moral
transgression
committed
in
the
scenario. Indeed,
patients
showed
less
utilitarian
behaviors and more emotion-based evaluations compared to
healthy subjects. Although moral judgment is considered to
occur quickly as an intuitive automatic response, based on
deontological principles rather than being a conscious and
rational reasoning process that follows a careful evaluation
of the moral situation (Haidt, 2001), the definition of the
acceptability of moral actions entails the engagement of specific
decision-making
competences
to
calculate
the
preferable
solution
(Bretz
and
Sun,
2018). Rational
calculation
of
moral actions usually results in utilitarian solutions (the
greatest good for the greatest number), while emotion-based
evaluations imply taking non-utilitarian behaviors, as seen in
our patient sample. Preserved social cognition skills are thus
crucial to maintain the best efficiency in affective decision-
making processes and in moral cognition. Statistical Analyses Basic Cognitive and Social Impairments
in Amyotrophic Lateral Sclerosis Patients
Six out of the 28 patients (21.4%) had an impaired performance
on the MMSE, scoring under the cutoff. Four out of 28 ALS
patients (14.28%) showed significant impairments in the ECAS
global score, and one patient had a borderline score. Four of
these five patients with impaired and/or borderline ECAS global
score presented impaired and/or reduced performance at ECAS
ALS-specific cognitive domains and at social cognition tests. Seven additional patients (25%) showed isolated impaired or
borderline scores at the ECAS ALS-specific cognitive domains. Only one patient showed significant impairments at the ECAS
ALS non-specific cognitive domains, while three others obtained
a borderline performance at ALS non-specific tasks. Impairments
of Ek-60F and SET occurred either isolated or combined
in patients. Global EK-60F or SET scores were impaired or
borderline in half of the patient sample (50%, 14/28). FBI
was completed by 20/28 caregivers, showing mild-to-moderate
behavioral changes in patients. Increased negative (6.42 ± 6.38)
and positive (3.11 ± 3.17) symptoms were both reported, with
a prevalence of symptoms as apathy, emotional flatness, loss We explored patients’ performances at the moral dilemmas task
in comparison to the control group. Preliminarily, we explored
the distribution for each variable with the Kolmogorov–Smirnov
test. Although some variables did not show a normal distribution,
we analyzed data by testing parametric models as well (one-way
ANOVA, mixed ANOVA) according to the results from Blanca
et al. (2017). First, Chi-Square Independence test was used to analyze group
differences in rate of yes/no responses at moral dilemmas for the
diverse task conditions of both type of dilemma (instrumental
or incidental conditions) and personal involvement factors (self-
involvement or no self-involvement conditions). We additionally
performed one-way ANOVA on percentages of yes/no responses
to provide a better description of the pattern of utilitarian
responses in ALS vs. HC. Mixed-ANOVA models were then computed to detect
differences in the rating of moral permissibility, emotional
valence, and emotional arousal experienced during the decision-
making process, considering the type of dilemma and the
personal involvement as within-subject variables and the group
(ALS and HC) as a between-subject variable. August 2020 | Volume 11 | Article 2083 Frontiers in Psychology | www.frontiersin.org 4 Crespi et al. Moral Cognition in ALS of insight, and inflexibility. According to the criteria by Strong
et al. Statistical Analyses (2017), six patients (21.5%) were classified as pure ALS, six
(21.5%) as ALSci, four (14%) as ALSbi, and four (14%) as ALScbi. Eight patients (29%) were not classifiable as their caregivers did
not complete the FBI. not find a significant effect of the personal involvement nor
an interaction effect with group. See Table 2 for further
details. Frontiers in Psychology | www.frontiersin.org Moral Cognition Disorders in
Amyotrophic Lateral Sclerosis Patients
Compared to Healthy Controls Results from
personal involvement × group mixed ANOVA highlighted
a significant effect of group [F(1,62) = 22.33, p < 0.001]
with ALS patients showing lower ratings than HC. We did August 2020 | Volume 11 | Article 2083 Frontiers in Psychology | www.frontiersin.org 5 Moral Cognition in ALS Crespi et al. FIGURE 1 | Significant differences in moral permissibility and emotional arousal in amyotrophic lateral sclerosis (ALS) patients compared to healthy controls (HCs). The figure depicts, on the left, the significant group (*) and type of dilemma (**) effects for the moral permissibility variable, showing significantly higher ratings in ALS
patients than HC and higher ratings for incidental compared to instrumental dilemmas in both groups. On the right, we report significant group (*), type of dilemma
(**) and group × type of dilemma interaction (***) effects for the emotional arousal variable, highlighting lower ratings in ALS patients than HC and higher ratings for
instrumental than incidental dilemmas in both groups, with HC showing a greater difference between instrumental and incidental conditions than ALS patients. FIGURE 1 | Significant differences in moral permissibility and emotional arousal in amyotrophic lateral sclerosis (ALS) patients compared to healthy controls (HCs). The figure depicts, on the left, the significant group (*) and type of dilemma (**) effects for the moral permissibility variable, showing significantly higher ratings in ALS
patients than HC and higher ratings for incidental compared to instrumental dilemmas in both groups. On the right, we report significant group (*), type of dilemma
(**) and group × type of dilemma interaction (***) effects for the emotional arousal variable, highlighting lower ratings in ALS patients than HC and higher ratings for
instrumental than incidental dilemmas in both groups, with HC showing a greater difference between instrumental and incidental conditions than ALS patients. assessment of social cognition performances in ALS should
be systematically promoted. alterations
in
brain
regions
normally
recruited
for
the
elaboration of socio-emotional stimuli and thus impacting
on judgments about moral permissibility of fictitious scenarios
(Bzdok et al., 2012). Our evidence on the emotion arousal subtask of moral
dilemmas further confirmed what was previously stated. HC
showed higher emotional arousal ratings for both incidental and
instrumental conditions compared to ALS patients, with a greater
arousal for incidental vs. instrumental dilemmas. Patients showed
similar levels of emotional engagement in both conditions. CONCLUSION Although the small sample size, the cross-sectional design,
and the lack of neuroimaging markers of moral changes
limit the generalizability of our findings, we expand current
literature (Semler et al., 2019) suggesting for the first time
that the presence of deficits in emotion recognition and
mental state attribution abilities, and emotional flattening
(socio-affective
component
of
moral
decision-making)
may influence the attitude in evaluating a moral situation
(socio-cognitive component of moral decision-making) in
ALS patients. Moreover, this study contributes to define
the extent and boundaries of the frontotemporal cognitive
syndrome in ALS. Although the small sample size, the cross-sectional design,
and the lack of neuroimaging markers of moral changes
limit the generalizability of our findings, we expand current
literature (Semler et al., 2019) suggesting for the first time
that the presence of deficits in emotion recognition and
mental state attribution abilities, and emotional flattening
(socio-affective
component
of
moral
decision-making)
may influence the attitude in evaluating a moral situation
(socio-cognitive component of moral decision-making) in
ALS patients. Moreover, this study contributes to define
the extent and boundaries of the frontotemporal cognitive
syndrome in ALS. From a cognitive neuroscience perspective, brain substrates
underlying cognitive and affective facets of moral cognition
(e.g.,
temporoparietal
junction,
medial
prefrontal
cortex,
middle temporal gyrus) are common to nodes included in
brain networks of both theory of mind (ToM) and empathy
(Bzdok et al., 2012). In particular, brain atrophy and changes
in white matter integrity in structures supporting socio-
affective skills (i.e., ventromedial prefrontal and temporo-limbic
cortex and right ventral associative bundles) have proven to
occur early in ALS (Cerami et al., 2014; Crespi et al., 2014,
2016). These findings are in line with neuropsychological
findings
of
the
present
and
previous
studies
indicating
reduced emotional engagement, emotion recognition, and
attribution in ALS patients (Lulé et al., 2005; Papps et al., 2005;
Zimmerman et al., 2007; Palmieri et al., 2010; Girardi et al.,
2011; Crespi et al., 2014). Indeed, significant differences
in
patients
compared
to
controls
in
the
socio-affective
component of moral reasoning might reflect anatomo-functional Besides its theoretical interest, deficits in the judgment
of moral actions entail crucial practical implications for
the management of neurological patients and should be
appropriately taken into account in the evaluation of therapeutic
approaches and critical life choices. Moral Cognition Disorders in
Amyotrophic Lateral Sclerosis Patients
Compared to Healthy Controls These
findings are suggestive of an incipient emotional blunting, which
can be clinically observed in a proportion of ALS patients, also
at early stages (Phukan et al., 2007; Strong et al., 2009), and that
can interfere with the ability to modulate the application of moral
principles in a consistent and nuanced manner to the different
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a practical method for grading the cognitive state of patients for the clinician. J. Psychiatr. Res. 12, 189–198. doi: 10.1016/0022-3956(75)90026-6 Brooks, B. R., Miller, R. G., Swash, M., and Munsat, T. L. (2000). El Escorial
revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph. Lateral Scler. Other Motor Neuron Disord. 1, 293–299. doi: 10.1080/
146608200300079536 Gibbons, Z. C., Snowden, J. S., Thompson, J. C., Happe, F., Richardson, A., and Neary, D. (2007). Inferring thought and action in motor neurone disease. Neuropsychologia 45, 1196–1207. doi: 10.1016/j.neuropsychologia.2006.10.008 Bzdok, D., Schilbach, L., Vogeley, K., Schneider, K., Laird, A. R., Langner, R., et al. (2012). Parsing the neural correlates of moral cognition: ALE meta-analysis
on morality, theory of mind, and empathy. Brain Struct. Funct. 217, 783–796. doi: 10.1007/s00429-012-0380-y Girardi, A., MacPherson, S. E., and Abrahams, S. (2011). Deficits in emotional and
social cognition in amyotrophic lateral sclerosis. Neuropsychology 25, 53–65. doi: 10.1037/a0020357 Gleichgerrcht, E., Torralva, T., Roca, M., Pose, M., and Manes, F. (2011). The role of
social cognition in moral judgment in frontotemporal dementia. Soc. Neurosci. 6, 113–122. doi: 10.1080/17470919.2010.506751 Cavallo, M., Adenzato, M., MacPherson, S. E., Karwig, G., Enrici, I., and Abrahams,
S. (2011). Evidence of social understanding impairment in patients with
amyotrophic lateral sclerosis. PLoS One 6:e25948. doi: 10.1371/journal.pone. 0025948 Haidt, J. (2001). The emotional dog and its rational tail: a social intuitionist
approach to moral judgment. Psychol. Rev. 108, 814–834. doi: 10.1037/0033-
295x.108.4.814 Cerami, C., Dodich, A., Canessa, N., Crespi, C., Iannaccone, S., Corbo, M., et al. (2014). Emotional empathy in amyotrophic lateral sclerosis: a behavioural
and voxel-based morphometry study. Amyotroph. Lateral Scler. Frontotemporal
Degener. 15, 21–29. doi: 10.3109/21678421.2013.785568 Hardiman, O., Al-Chalabi, A., Chio, A., Corr, E. M., Logroscino, G., Robberecht,
W., et al. (2017). Amyotrophic lateral sclerosis. Nat. Rev. Dis. Primers 3:17071. doi: 10.1038/nrdp.2017.71 Consonni, M., Iannaccone, S., Cerami, C., Frasson, P., Lacerenza, M., Lunetta,
C., et al. (2013). The cognitive and behavioural profile of amyotrophic lateral
sclerosis: application of the consensus criteria. Behav. Neurol. 27, 143–153. doi: 10.3233/BEN-2012-110202 Kertesz, A., Davidson, W., and Fox, H. (1997). Frontal behavioral inventory:
diagnostic criteria for frontal lobe dementia. Can. J. Neurol. Sci. 24, 29–36. doi: 10.1017/s0317167100021053 Khin Khin, E., Minor, D., Holloway, A., and Pelleg, A. (2015). Decisional capacity
in amyotrophic lateral sclerosis. J. Am. Acad. Psychiatry Law 43, 210–217. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation. SUPPLEMENTARY MATERIAL The studies involving human participants were reviewed and
approved by the San Raffaele Hospital Ethics Committee. The
patients/participants provided their written informed consent to
participate in this study. The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fpsyg. 2020.02083/full#supplementary-material CONCLUSION Further studies based
on clinical settings are needed to confirm our data and
estimate in ALS the weight of moral judgment alterations
and their relationship with the quality of life and socio-
affective disorders at the individual level, particularly with
regard to gender effect and disease subtype. Overall, the
variable
vulnerability
to
social
and
non-social
cognitive
changes
observed
in
ALS
patients
indicates
that
larger August 2020 | Volume 11 | Article 2083 Frontiers in Psychology | www.frontiersin.org 6 Moral Cognition in ALS Crespi et al. AUTHOR CONTRIBUTIONS longitudinal studies are also needed to estimate the impact of
moral cognition impairments in real-life situations (e.g., end-
life decisions). CCr: conceptualization, formal analysis, writing – original draft,
and visualization. GS: investigation, formal analysis, and writing – reviewing and editing. AD: investigation and writing –
reviewing and editing. FL and LG: investigation and resources. TP
and CL: investigation and supervision. CCe: conceptualization,
project administration, supervision, investigation, data curation,
and writing – reviewing and editing. All authors contributed to
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an fMRI study. J. Neurol. 257, 1970–1978. doi: 10.1007/s00415-010-5640-2 Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest. Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest. Papps, B., Abrahams, S., Wicks, P., Leigh, P. N., and Goldstein, L. H. (2005). Changes in memory for emotional material in amyotrophic lateral sclerosis
(ALS). Neuropsychologia 43, 1107–1114. doi: 10.1016/j.neuropsychologia.2004. 11.027 Copyright © 2020 Crespi, Santi, Dodich, Lupo, Greco, Piccoli, Lunetta and Cerami. 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. Frontiers in Psychology | www.frontiersin.org August 2020 | Volume 11 | Article 2083 REFERENCES No use, distribution or reproduction is permitted which does not
comply with these terms. Copyright © 2020 Crespi, Santi, Dodich, Lupo, Greco, Piccoli, Lunetta and Cerami. 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. Phukan, J., Pender, N. P., and Hardiman, O. (2007). Cognitive impairment in
amyotrophic lateral sclerosis. Lancet Neurol. 6, 994–1003. Pletti, C., Lotto, L., Buodo, G., and Sarlo, M. (2017). It’s immoral, but I’d do
it! Psychopathy traits affect decision-making in sacrificial dilemmas and in
everyday moral situations. Br. J. Psychol. 108, 351–368. doi: 10.1111/bjop.12205 August 2020 | Volume 11 | Article 2083 Frontiers in Psychology | www.frontiersin.org 8
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https://www.cambridge.org/core/services/aop-cambridge-core/content/view/A7E9A9D4F2E53EE3E1BD75144AF8F940/S0022112024003057a.pdf/div-class-title-propagation-of-a-viscous-gravity-current-beneath-a-granular-mush-div.pdf
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English
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Propagation of a viscous gravity current beneath a granular mush
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Journal of fluid mechanics
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J. Fluid Mech. (2024), vol. 985, A18, doi:10.1017/jfm.2024.305 J. Fluid Mech. (2024), vol. 985, A18, doi:10.1017/jfm.2024.305 (
),
,
,
j Propagation of a viscous gravity current beneath
a granular mush Edward M. Hinton1,† and Anja C. Slim2,3
1School of Mathematics and Statistics, The University of Melbourne, Victoria 3010, Australia
2School of Mathematics, Monash University, Victoria 3800, Australia
3School of Earth, Atmosphere and Environment, Monash University, Victoria 3800, Australia
(Received 8 September 2023; revised 24 March 2024; accepted 25 March 2024) Edward M. Hinton1,† and Anja C. Slim2,3
1School of Mathematics and Statistics, The University of Melbourne, Victoria 3010, Australia
2School of Mathematics, Monash University, Victoria 3800, Australia
3School of Earth, Atmosphere and Environment, Monash University, Victoria 3800, Australia
(Received 8 September 2023; revised 24 March 2024; accepted 25 March 2024) (Received 8 September 2023; revised 24 March 2024; accepted 25 March 2024) The two-dimensional gravity-driven motion of a relatively dense viscous liquid at the base
of a granular mush is investigated using a model that exploits the relative shallowness of
the flow. The granular mush obeys a μ(I)-rheology, and we assume that the two phases
are segregated throughout the motion. The viscous liquid spreads under gravity, carrying
the granular mush above and transporting it outwards as levees at either end of the flow. The accumulation of granular material away from the centre of the deposit produces
hydrostatic pressure gradients that retard the viscous gravity current. At later times, the
granular mush is quasi-static relative to the moving liquid owing to the balance of outward
granular transfer by the liquid and inward hydrostatic pressure gradients associated with
the granular free surface. The viscous liquid exhibits a Poiseuille-like flow structure with
negligible velocity at both the base and the granular interface. The flow of a fixed volume
of viscous liquid becomes self-similar with the effective viscosity quadrupled relative to
a classical viscous gravity current owing to the retarding effects of the granular mush. The case of constant input flux of viscous liquid is also analysed. The qualitative features
are akin to the fixed volume case with the granular mush forming levees and slowing the
viscous spreading. The case in which the upper medium is a Bingham material rather than
a granular mush is also discussed, and the same features are observed, demonstrating the
importance of the yield criterion in the upper medium. Key words: gravity currents, magma and lava flow © The Author(s), 2024. Published by Cambridge University Press. This is an Open Access article,
distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/
licenses/by/4.0), which permits unrestricted re-use, distribution and reproduction, provided the original
article is properly cited.
985 A18-1 1. Introduction Models of the gravity-driven motion of a viscous liquid have been used to describe and
interpret a range of geophysical phenomena, including lava flows, magma chambers and † Email address for correspondence: edward.hinton@unimelb.edu.au † Email address for correspondence: edward.hinton@unimelb.edu.au 985 A18-1 985 A18-1 E.M. Hinton and A.C. Slim
xˆ
g
Levee
Granular mush
Viscous liquid
hˆu (xˆ, tˆ)
Hˆ (xˆ, tˆ)
hˆl (xˆ, tˆ)
zˆ
Figure 1. Schematic diagram illustrating two-dimensional gravity-driven flow of a viscous liquid beneath a
granular mush. The red lines and arrows show a typical flow profile. E.M. Hinton and A.C. Slim E.M. Hinton and A.C. Slim Levee Granular mush Figure 1. Schematic diagram illustrating two-dimensional gravity-driven flow of a viscous liquid beneath a
granular mush. The red lines and arrows show a typical flow profile. glaciers (Huppert 1986). The classical model of the spreading of a viscous liquid on a
horizontal or inclined plane (Huppert 1982; Lister 1992), has been extended to account for
many geophysically relevant features, including cooling (Lyman, Kerr & Griffiths 2005),
a yield stress (Balmforth, Craster & Sassi 2002), and the effect of a second overlying fluid
that can deform during the motion (Kowal & Worster 2015). Here, we analyse the gravity-driven spreading of a viscous liquid at the base of a
granular mush of finite thickness, which is transported and deformed by the viscous
motion; see figure 1. This problem has fundamental fluid dynamical interest as a novel
two-fluid gravity current of materials with distinct rheological properties. However, the
study is also motivated by the formation of massive sulphide ores in magmatic systems
(Hinton & Slim 2023), and has a secondary application to layered mafic intrusions (Maier,
Barnes & Groves 2013; Liu et al. 2014). Massive sulphide ore deposits form small portions of basaltic magmatic intrusions
within the Earth’s crust. They are thought to have formed from metal-sulphide-rich liquid
droplets within the dominant silicate melt, coalescing and depositing in particular parts
of the magmatic plumbing system (Robertson, Barnes & Le Vaillant 2016). Transport of
the sulphide-rich liquid occurs as the system cools, and hence involves interaction with a
mush of silicate melt and silicate crystals. Of particular interest is the final location of this
sulphide-rich liquid, thus understanding the transport within the crystal-rich environment
is key. 1. Introduction y
To capture the dominant processes associated with a coalesced volume of dense
sulphide-rich liquid interacting with a mixture of silicate crystals and liquid, we model
the system as a two-dimensional shallow isothermal flow. The sulphide-rich liquid is
represented by a relatively dense viscous liquid, whilst the silicate crystals within the
silicate liquid are modelled by a continuum granular mush (see figure 1). Importantly, the
two liquids are different, and the silicate crystals within the magma are non-wetting to the
sulphide liquid (Mungall & Su 2005). Hence we assume that there is no interpenetration
across the sharp interface between the viscous liquid and the granular mush. The granular
mush is assumed to obey a μ(I)-rheology, which has been shown to capture accurately
many experimental features of granular flows (GDR-MiDi 2004; Gray & Edwards 2014). Recently, Hinton & Slim (2023) investigated the spreading of a viscous gravity current
atop a dense granular mush. This was motivated by the same magmatic system but with
the sulphide liquid remaining atop the silicate mush and unable to penetrate downwards
owing to the capillary entry pressure. They found that the viscous liquid initially erodes
the underlying granular mush into levees. Subsequently, the top part of the levee is pushed 985 A18-2 Viscous gravity current beneath a granular mush outwards by the viscous liquid, with the remnants of the levees trapping a significant
fraction of the liquid. In the present work, we consider the opposite configuration, with the relatively dense
viscous liquid lying at the bottom of the granular mush. The lower boundary (at ˆz = 0)
is impermeable and corresponds to the host country rock bounding the magma chamber. Both configurations are geologically relevant as the dense liquid sulphide may penetrate
the mush or become trapped above, depending on its thickness (Naldrett 1973; Chung &
Mungall 2009). Although this leads to substantially different dynamics, there are some
analogies between the two flows, particularly the relatively fast emergence of granular
levees either side of the liquid, which subsequently decay in size. The shallow model that we deploy builds upon studies of two-liquid gravity-driven flows
(Kliakhandler & Sivashinsky 1997; Balmforth, Craster & Toniolo 2003), which have been
used recently to investigate the lubrication of ice sheets (Kowal & Worster 2015; Kumar
et al. 2021; Christy & Hinton 2023). 1. Introduction These inertialess two-liquid flows have the common
feature that the volume flux in each liquid is driven by hydrostatic pressure gradients
associated with the liquid thicknesses. The flux in the upper fluid also includes a term
associated with the lubrication provided by the lower liquid (the lower liquid effectively
carries the upper liquid along with it), which is key in the present work. Two-liquid flows
with different rheology or densities of the two media can give rise to various instabilities
(Balmforth et al. 2003; Leung & Kowal 2022), but here we neglect any such behaviour,
noting that our solutions could be used as the base state for future stability analysis. g
y
y
The model is presented in § 2. In § 3, we analyse the evolution of a fixed volume of
viscous liquid, and show that the behaviour is self-similar at late times, with the fluid
spreading at a quarter of the rate of a classical viscous gravity current (Huppert 1982). The case of constant input of viscous liquid is explored in § 4, and conclusions are given
in § 5. Appendix A provides a scaling analysis for the model, and Appendix B discusses
the case of an overlying Bingham material (rather than a granular mush), for which almost
identical dynamics occurs. 2. Model formulation We analyse the two-dimensional motion of a viscous liquid of density ˆρl and viscosity ˆη
displacing a granular mush with μ(I)-rheology; see figure 1. There is no interpenetration
of the viscous liquid into the granular mush. The granular mush consists of ambient liquid
of density ˆρa and grains of density ˆρg > ˆρa. The solids fraction is denoted by φ so that the
bulk density ˆρu of the granular mush is ˆρu = ˆρa(1 −φ) + ˆρgφ,
(2.1) (2.1) and we assume that ˆρu < ˆρl (i.e. the granular mush is lighter than the viscous liquid). There
is an impermeable horizontal boundary at ˆz = 0. The viscous liquid occupies 0 < ˆz <
ˆhl(ˆx,ˆt), whilst the granular mush occupies ˆhl(ˆx,ˆt) < ˆz < ˆH(ˆx,ˆt). The vertical extent of
the granular mush is ˆhu(ˆx,ˆt) = ˆH(ˆx,ˆt) −ˆhl(ˆx,ˆt). Throughout the paper, we use subscripts
u and l to refer to quantities related to the upper and lower media, respectively, and we use
ˆ· to indicate dimensional or unscaled quantities. The flow is assumed to be shallow (a scaling analysis is given in Appendix A). The
pressure in each medium is thus hydrostatic and given by (Huppert 1982) ˆpu = ˆρuˆg( ˆH −ˆz),
for ˆhl < ˆz < ˆH,
(2.2)
ˆpl = ˆρuˆgˆhu + ˆρlg(ˆhl −ˆz),
for0 < ˆz < ˆhl. (2.3) (2.2) (2.3) 985 A18-3 E.M. Hinton and A.C. Slim E.M. Hinton and A.C. Slim E.M. Hinton and A.C. Slim The leading-order momentum balance (∂ˆτ/∂ˆz = ∂ˆp/∂ˆx) then furnishes the shear stress ˆτ
in each medium: ˆτu = −ˆρuˆg( ˆH −ˆz) ∂ˆH
∂ˆx ,
ˆhl < ˆz < ˆH,
(2.4)
ˆτl = ˆη ∂ˆul
∂ˆz = −ˆρuˆg
ˆhu
∂ˆH
∂ˆx +
D ∂ˆhl
∂ˆx + ∂ˆH
∂ˆx
(ˆhl −ˆz)
,
0 < ˆz < ˆhl,
(2.5) (2.4) (2.5) where we have used continuity of the shear stress at the interface between the media,
ˆz = ˆhl, and vanishing shear stress at the free surface, ˆz = ˆH, and introduced the density
ratio D = ˆρl −ˆρu
ˆρu
> 0. (2.6) (2.6) Integrating (2.5) with respect to ˆz furnishes the velocity within the viscous liquid (Kowal
& Worster 2015): ˆul = −ˆρuˆg
ˆη
∂ˆH
∂ˆx
ˆhuˆz +
D ∂ˆhl
∂ˆx + ∂ˆH
∂ˆx
ˆz
ˆhl −1
2 ˆz
,
(2.7) (2.7) where we have used no slip at ˆz = 0. 2. Model formulation The constitutive model for the granular mush is provided by the μ(I)-rheology with
(GDR-MiDi 2004; Gray & Edwards 2014) The constitutive model for the granular mush is provided by the μ(I)-rheology with
(GDR-MiDi 2004; Gray & Edwards 2014) ˆτu = μ(I) ˆpu sgn
∂ˆuu
∂ˆz
. (2.8) (2.8) For the relatively slow flows considered in the present work, the relationship between the
inertial number I and the effective friction coefficient μ is linearised to obtain (Da Cruz
et al. 2005; Kamrin & Koval 2012) For the relatively slow flows considered in the present work, the relationship between the
inertial number I and the effective friction coefficient μ is linearised to obtain (Da Cruz
et al. 2005; Kamrin & Koval 2012) I = max(0, μ −μs)
b
,
where I =
∂ˆuu
∂ˆz
ˆm/ˆpu,
(2.9) (2.9) and μs is the minimum friction coefficient for which the granular mush deforms; μs
is assumed to be small (for further discussion, see Appendix A). The parameter b is a
dimensionless constant, and ˆm is the grain mass (per unit length in the third dimension). Substituting (2.2) and (2.4) into (2.8), we find that μ(I) is independent of the ˆz coordinate
and given by (Gray & Edwards 2014) μ(I) =
∂ˆH
∂ˆx
. (2.10) (2.10) The definition of I in (2.9) then furnishes the relation The definition of I in (2.9) then furnishes the relation
∂ˆuu
∂ˆz
ˆm
ˆpu
=
max
0,
∂ˆH
∂ˆx
−μs
b
. (2.11)
985 A18-4 (2.11) 985 A18-4 Viscous gravity current beneath a granular mush Equation (2.11) is integrated with respect to ˆz to obtain the velocity profile in the granular
mush: Equation (2.11) is integrated with respect to ˆz to obtain the velocity profile in the granular
mush: ˆuu = −2
ˆρuˆg
3b
√
ˆm
ˆh3/2
u
−( ˆH−ˆz)3/2
ˆF
∂ˆH
∂ˆx
−ˆρuˆg
ˆη
∂ˆH
∂ˆx
ˆhuˆhl +
D ∂ˆhl
∂ˆx + ∂ˆH
∂ˆx
1
2
ˆh2
l
,
(2 12) where we have used continuity of velocity at the interface, ˆz = ˆhl, utilising (2.7) (there is
no interpenetration of viscous liquid into the granular medium), and we have introduced
the function ˆF
∂ˆH
∂ˆx
= sgn
∂ˆH
∂ˆx
max
0,
∂ˆH
∂ˆx
−μs
. 2.1. Non-dimensionalisation 2.1. Non-dimensionalisation Initially, the two media combined have thickness ˆa, which motivates the non-
dimensionalisation Initially, the two media combined have thickness ˆa, which motivates the non-
dimensionalisation (z, hl, hu, H) = (ˆz, ˆhl, ˆhu, ˆH)/ˆa,
x = ˆxμs/ˆa,
t = ˆt ˆρuˆgˆaμ2
s/ˆη,
(2.18a–c (2.18a–c) (2.18a–c) where ˆt has been scaled with the time scale for viscous deformation. The horizontal length
scale is chosen to remove the parameter μs from the dimensionless model, and thus make
the critical slope for yielding of the granular layer equal to unity. The dimensionless
velocities in each medium ((2.7) and (2.12)) take the forms ul = −1
2
2 ∂H
∂x huz +
D ∂hl
∂x + ∂H
∂x
z (2hl −z)
,
(2.19)
uu = −5
3 K
h3/2
u
−(H −z)3/2
F
∂H
∂x
−1
2
2 ∂H
∂x huhl +
D ∂hl
∂x + ∂H
∂x
h2
l
,
(2.20) (2.19) 3
∂x
2
∂x
∂x
∂x
(2.20)
(2.20)
where where where
F
∂H
∂x
= sgn
∂H
∂x
max
0,
∂H
∂x
−1
(2.21) and and K =
2ˆη
5b
ˆm ˆρuˆgˆa
,
(2.22) (2.22) which is the ratio of the viscosity of the Newtonian liquid, ˆη, to the effective ‘viscosity’
of the granular mush (which quantifies the resistance of the granular mush to deformation
when the yield criterion is exceeded, i.e. |∂H/∂x| > 1). The dimensionless volume fluxes
((2.14) and (2.15)) become Ql = −
∂H
∂x
huh2
l
2
+
D ∂hl
∂x + ∂H
∂x
h3
l
3
,
(2.23) Ql = −
∂H
∂x
huh2
l
2
+
D ∂hl
∂x + ∂H
∂x
h3
l
3
,
(2.23)
Qu = −Kh5/2
u
F1
∂H
∂x
−
∂H
∂x h2
uhl +
D ∂hl
∂x + ∂H
∂x
huh2
l
2
. (2.24) (2.23) Qu = −Kh5/2
u
F1
∂H
∂x
−
∂H
∂x h2
uhl +
D ∂hl
∂x + ∂H
∂x
huh2
l
2
. (2.24) (2.24) This is combined with dimensionless mass conservation (2.16): ∂hi
∂t + ∂Qi
∂x = 0,
for i = u, l. 2. Model formulation (2.13) (2.13) The volume flux in each medium is calculated by integrating the velocity over the
thickness: ˆQl = −ˆρuˆg
6ˆη
3 ∂ˆH
∂ˆx
ˆhuˆh2
l + 2
D ∂ˆhl
∂ˆx + ∂ˆH
∂ˆx
ˆh3
l
,
(2.14)
ˆQu = −2
ˆρuˆg
5b
√
ˆm
ˆh5/2
u
ˆF
∂ˆH
∂ˆx
−ˆρuˆg
2ˆη
2 ∂ˆH
∂ˆx
ˆh2
uˆhl +
D ∂ˆhl
∂ˆx + ∂ˆH
∂ˆx
ˆhuˆh2
l
. (2.15) (2.14) The flux in the viscous liquid, ˆQl, is driven by viscous shearing arising from hydrostatic
pressure gradients associated with the thickness variations of the two media. The flux in
the granular mush, ˆQu, consists of two terms: the first term is associated with shearing of
the granular mush, whilst the second term arises from ‘lubrication’ by the viscous liquid
(motion in the liquid can carry the granular mush) (cf. Kowal & Worster 2015). Hence the
granular mush can move even when |∂ˆH/∂ˆx| does not exceed μs owing to the motion of
the underlying viscous liquid; see figure 1. For further discussion regarding motion of the
quasi-rigid mush, see Appendix A. Mass conservation in each medium is written as ∂ˆhi
∂ˆt + ∂ˆQi
∂ˆx = 0,
for i = u, l. (2.16) (2.16) The model is completed with appropriate initial and boundary conditions; we generally
take a constant total thickness ˆH(x, 0) ≡ˆa and consider different starting shapes of the
viscous deposit. The model is completed with appropriate initial and boundary conditions; we generally
take a constant total thickness ˆH(x, 0) ≡ˆa and consider different starting shapes of the
viscous deposit. p
We analyse two distinct cases: (i) a fixed volume of viscous liquid (§ 3), and (ii) a
constant input flux of viscous liquid (§ 4). In each case, global mass conservation of the
viscous liquid takes the form https://doi.org/10.1017/jfm.2024.305 Pu ˆxf
−ˆxf
ˆhl dˆx =
ˆA0
(fixed volume),
ˆq0ˆt
(constant input flux),
(2.17) (2.17) where ˆxf (ˆt) is the distance of the tip of the viscous layer from the origin. where ˆxf (ˆt) is the distance of the tip of the viscous layer from the origin. 985 A18-5 985 A18-5 E.M. Hinton and A.C. Slim E.M. Hinton and A.C. Slim 2.1. Non-dimensionalisation Viscous gravity current beneath a granular mush –1
0
x
1
–1
0
x
1
–1
0
x
1
–1
0
1
–1
0
1
–1
0
1
–1
0
1
–1
0
1
–1
0
1
1.0
(a)
(b)
(c)
(d)
(e)
( f )
(g)
(h)
(i)
0.5
Granular
mush
Granular
mush
z
0
Liquid
1.0
0.5
z
0
1.0
0.5
z
0
1.0
0.5
0
1.0
0.5
0
1.0
0.5
0
1.0
0.5
0
1.0
0.5
0
1.0
0.5
0
Figure 2. Evolution of a fixed volume of viscous liquid beneath a granular mush with ‘viscosity’ ratio K =
1 and density ratio D = 0.5. (a) Initial condition (t = 0) given by (3.1). (b–i) The interface shapes at t =
1, 5, 20, 30, 35, 40, 100, 250. The red arrows in (e,h) show the flow directions schematically; see also figure 3
and the discussion in the text. –1
0
1
(b)
(e)
1.0
0.5
0 –1
0
1
(c)
( f )
1.0
0.5
0 ( f ) –1
0
1
( f )
(i)
1.0
0.5
0 (i) –1
0
x
1
(g)
1.0
0.5
z
0 –1
0
x
1
1.0
0.5
0 Figure 2. Evolution of a fixed volume of viscous liquid beneath a granular mush with ‘viscosity’ ratio K =
1 and density ratio D = 0.5. (a) Initial condition (t = 0) given by (3.1). (b–i) The interface shapes at t =
1, 5, 20, 30, 35, 40, 100, 250. The red arrows in (e,h) show the flow directions schematically; see also figure 3
and the discussion in the text. 2.1. Non-dimensionalisation (2.25) (2.25) Equation (2.25) is integrated numerically using finite differences with an appropriate
initial condition; the method is described in the appendix of Hinton & Slim (2023). Equation (2.25) is integrated numerically using finite differences with an appropriate
initial condition; the method is described in the appendix of Hinton & Slim (2023). Finally, global mass conservation (2.17) becomes Finally, global mass conservation (2.17) becomes xf
−xf
hl dx =
A
(fixed volume), see § 3,
Qt
(constant input flux), see § 4,
(2.26) (2.26) where A =
ˆA0μs
ˆa2 ,
Q =
ˆq0 ˆη
ˆa3 ˆρuˆgμs
. (2.27a,b) (2.27a,b) 985 A18-6 Viscous gravity current beneath a granular mush 3. Fixed volume of viscous liquid Parameter values and
initial condition are as in figure 2, with (a) t = 5 corresponding to figure 2(c), (b) t = 40 corresponding to
figure 2(g), and (c) t = 250 corresponding to figure 2(i). the origin and the crest of the levee in the combined height profile. This provides an
adverse contribution to the hydrostatic pressure gradient, decelerating the viscous flow
and in particular reducing the outward flow velocity near the viscous–granular interface;
see figure 3. Eventually, the slumping liquid detaches from the upper free surface as the central
granular material moves slowly inwards; see figures 2 and 3. (This was handled
numerically by initially adding an artificial film of granular material of thickness 10−4
over the entire region of the viscous liquid, which ensures that in the numerical method,
there is no transition from two-layer flow to one-layer flow; the resulting solution was not
sensitive to the thickness of this granular film.) Subsequently, the viscous liquid continues
to thin and spread laterally, pushing the levees outwards. The perturbation to the upper
free surface diminishes in time owing to the lateral spreading, and the levees are also
reduced in size. The small gradients in the upper free surface continue to retard the viscous
deformation, even at late times. The system becomes self-similar at late times, which is
analysed in § 3.1. To investigate the sensitivity to the details of the initial condition, figure 4 shows the
flow evolution with a semicircular initial condition (and D = 0.5, K = 1) given by hl(x, 0) =
√
1 −x2,
|x| < 1,
0,
otherwise,
(3.2) (3.2) which is shown in figure 4(a). Comparison of figures 2 and 4 demonstrates that the system
generally passes through the same stages regardless of the initial shape. Indeed, figures 2(e)
and 4(e) are very similar, and the evolution is almost identical at later times. (This is
associated with late-time self-similar behaviour that ‘forgets’ the initial conditions; see
§ 3.1.) The effect of varying the density ratio D is demonstrated in figure 5. The qualitative
behaviour of the flow is unchanged with different values of D. Larger D is associated with
faster flow because the motion of both media is driven primarily by the gravity-driven
spreading of the liquid. However, there is no simple quantitative rescaling of time with
D. 3. Fixed volume of viscous liquid Numerical results for the spreading of a fixed volume of relatively dense viscous liquid
beneath a granular mush are shown in figure 2 with D = 0.5, K = 1. The initial shape
takes the form hl(x, 0) = 1
2 [1 −tanh(50(|x| −0.5))] ,
(3.1) (3.1) which is shown in figure 2(a). This is an approximation to the Heaviside function. (The
Heaviside function is slightly smoothed for efficient numerical integration, but we note
that steeper initial profiles lead to imperceptible changes in the results.) The initial shape
of the upper free surface is H(x, 0) = hl(x, 0) + hu(x, 0) = 1. Figure 2 shows that initially, the viscous liquid spreads outwards driven by hydrostatic
pressure gradients associated with the thickness of the viscous liquid, D ∂hl/∂x. Although
there is negligible deformation in the granular mush initially (with |∂H/∂x| < 1), the
outward motion of the liquid carries the granular mush outwards; see figures 2(a,b). Granular material is piled into levees above the edges of the viscous liquid; see figure 2(b). These levees grow initially with their outer slope being quasi-rigid, |∂H/∂x| ≈1. Figure 3 shows the horizontal velocity fields for figures 2(c,g,i). The outward carrying
of the crest of the levees by the liquid can be seen in the thin vertical slices of high velocity
at edges of the liquid in figure 3. The combination of outward transfer of granular material in the levee and inward
collapse of granular material above the viscous liquid creates a positive slope between 985 A18-7 E.M. Hinton and A.C. Slim 1.0
(a)
0.02
0
–0.02
0.5
z
0
–0.5
0x
0.5 1.0
(a)
(b)
(c)
0.02
0
–0.02
0.02
0
–0.02
2
0 u
–2
0.5
z
0
–0.5
0x
0.5
–1
0x
1
–1
0x
1
1.0
0.5
0
1.0
0.5
0
(×10–3)
Figure 3. Horizontal flow velocity u in each medium obtained using (2.19) and (2.20). Parameter values and
initial condition are as in figure 2, with (a) t = 5 corresponding to figure 2(c), (b) t = 40 corresponding to
figure 2(g), and (c) t = 250 corresponding to figure 2(i). (c)
2
0 u
–2
–1
0x
1
1.0
0.5
0
(×10–3) (b)
0.02
0
–0.02
–1
0x
1
1.0
0.5
0 (b) (c) (a) Figure 3. Horizontal flow velocity u in each medium obtained using (2.19) and (2.20). 3. Fixed volume of viscous liquid Changes in the relative granular viscosity K have negligible influence on the results
because the granular material is rigid or quasi-rigid everywhere. Viscous gravity current beneath a granular mush 1.0
(a)
(d)
0.5
z
0
–2
0
2 1.0
(a)
(d)
(g)
(b)
(e)
(c)
( f )
(h)
(i)
0.5
z
0
1.0
0.5
z
0
1.0
0.5
z
0
–2
0
x
2
–2
0
x
2
–2
0
x
2
–2
0
2
–2
0
2
–2
0
2
–2
0
2
–2
0
2
–2
0
2
1.0
0.5
0
1.0
0.5
0
1.0
0.5
0
1.0
0.5
0
1.0
0.5
0
1.0
0.5
0
Figure 4. Evolution of a fixed volume of viscous liquid beneath a granular mush with ‘viscosity’ ratio K =
1 and density ratio D = 0.5. (a) Initial condition (t = 0) given by (3.2). (b–i) The interface shapes at t =
1, 5, 20, 40, 60, 80, 150, 300. The red arrows in (a,d,g) show the flow directions schematically. (b)
(e)
(c)
( f )
2
–2
0
2
–2
0
2
1.0
0.5
0
1.0
0.5
0 (a) (d) (e) ( )
( f )
(h)
(i)
2
–2
0
2
–2
0
2
1.0
0.5
0
1.0
0.5
0 (h) (i) (g) (i)
–2
0
x
2
1.0
0.5
0 (g)
1.0
0.5
z
0
–2
0
x
2 )
–2
0
x
2
1.0
0.5
0 Figure 4. Evolution of a fixed volume of viscous liquid beneath a granular mush with ‘viscosity’ ratio K =
1 and density ratio D = 0.5. (a) Initial condition (t = 0) given by (3.2). (b–i) The interface shapes at t =
1, 5, 20, 40, 60, 80, 150, 300. The red arrows in (a,d,g) show the flow directions schematically. 1.0
(a)
(b)
(c)
0.5
z
0
–1
0
x
1
–1
0
x
1
–1
0
x
1
1.0
0.5
0
1.0
0.5
0
Figure 5. Flow evolution for three different values of the density ratio D with K = 1: D = 0.25 (green lines),
D = 0.5 (black lines) and D = 1 (red lines). The initial condition is (3.1) (as in figure 2). Times are (a) t = 1,
(b) t = 10, and (c) t = 100. 1.0
(a)
(
0.5
z
0
–1
0
x
1 (c)
–1
0
x
1
1.0
0.5
0 b)
–1
0
x
1
1.0
0.5
0 (a) (c) (b) Figure 5. 3.1. Late-time self-similar behaviour For a wide range of initial conditions and parameter values, the late-time evolution
becomes self-similar, with the viscous liquid spreading outwards and the perturbation
985 A18-8 For a wide range of initial conditions and parameter values, the late-time evolution
becomes self-similar, with the viscous liquid spreading outwards and the perturbation Viscous gravity current beneath a granular mush Viscous gravity current beneath a granular mush Thus the velocity at the top of the viscous liquid is approximately zero. y
p
q
pp
y
Using (2.19), the flow velocity at the interface is Using (2.19), the flow velocity at the interface is ul(z = hl) = −∂H
∂x huhl −
D ∂hl
∂x + ∂H
∂x
h2
l
2 ≈0. (3.3) (3.3) This condition provides a relation between the two thickness gradients, ∂hl/∂x and ∂H/∂x,
which ensures that the granular mush is quasi-static. Equation (3.3) can be used to rewrite
the velocity in the viscous liquid (2.19) as This condition provides a relation between the two thickness gradients, ∂hl/∂x and ∂H/∂x,
which ensures that the granular mush is quasi-static. Equation (3.3) can be used to rewrite
the velocity in the viscous liquid (2.19) as ul = −D ∂hl
∂x
1
2 z(hl −z),
(3.4) (3.4) (3.4) where we have also assumed that |∂H/∂x| ≪D |∂hl/∂x|, which we confirm a posteriori
(this assumption also implies that hl ≪1; see (3.3)). Equation (3.4) is analogous to a
Poiseuille flow with no-slip at both the top and bottom of the viscous liquid; see figure 6. The Poiseuille flow structure contrasts with an ‘unconfined’ viscous gravity current with
no overlying granular mush, which has a velocity field with no-slip at z = 0, and zero
vertical gradient at z = hl. The flux in the viscous liquid is obtained by integrating (3.4) across its thickness to
obtain ∂hl
∂t + ∂Ql
∂x = 0,
where Ql = −D ∂hl
∂x
h3
l
12. (3.5) (3.5) The flux in the viscous liquid is driven by the same hydrostatic pressure gradients as a
classical (or ‘unconfined’) viscous gravity current (Huppert 1982), but the flux is reduced
by a factor of four owing to the different boundary condition at the top of the viscous liquid
(arising from the retarding influence of the overlying granular mush). The flux in the viscous liquid is driven by the same hydrostatic pressure gradients as a
classical (or ‘unconfined’) viscous gravity current (Huppert 1982), but the flux is reduced
by a factor of four owing to the different boundary condition at the top of the viscous liquid
(arising from the retarding influence of the overlying granular mush). Viscous gravity current beneath a granular mush Flow evolution for three different values of the density ratio D with K = 1: D = 0.25 (green lines),
D = 0.5 (black lines) and D = 1 (red lines). The initial condition is (3.1) (as in figure 2). Times are (a) t = 1,
(b) t = 10, and (c) t = 100. to the upper free surface thinning and extending laterally; see figures 2(i) and 4(i). At
first sight, it appears that ∂H/∂x ≈0 at late times, so the viscous liquid should spread
independently of the overlying granular mush as the classical self-similar gravity current
of Huppert (1982). As we show below, this description is incorrect; the flow is self-similar,
but the small gradients in the upper free surface retard the outward propagation, and this
leads to the rate of spreading being exactly a quarter of that of a classical (or ‘unconfined’)
viscous gravity current. The hydrostatic pressure gradient associated with ∂H/∂x competes with the outwards
carrying of the granular mush by the gravity-driven spreading liquid as discussed in § 3. 985 A18-9 E.M. Hinton and A.C. Slim E.M. Hinton and A.C. Slim
1.0
0.5
z
0
1.10
1.15
1.20
x
1.25
15
10
5 u
0
(×10–4)
Figure 6. A slice 1.1 ≤x ≤1.25 from the horizontal velocity field at t = 250 shown in figure 3(c). The
granular mush is quasi-static, and the viscous liquid moves with a parabolic profile for the horizontal velocity
(Poiseuille flow). 1.0
0.5
z
0
1.10
1.15
1.20
x
1.25
15
10
5 u
0
(×10–4) Figure 6. A slice 1.1 ≤x ≤1.25 from the horizontal velocity field at t = 250 shown in figure 3(c). The
granular mush is quasi-static, and the viscous liquid moves with a parabolic profile for the horizontal velocity
(Poiseuille flow). At late times, the granular mush is in a quasi-static balance; any substantial velocity at
the top of the viscous liquid drives significant movement of granular material, which then
immediately suppresses the motion through the increased reverse gradient of the upper
free surface. Thus the velocity at the top of the viscous liquid is approximately zero. At late times, the granular mush is in a quasi-static balance; any substantial velocity at
the top of the viscous liquid drives significant movement of granular material, which then
immediately suppresses the motion through the increased reverse gradient of the upper
free surface. Viscous gravity current beneath a granular mush The self-similar flow of the viscous liquid is calculated by combining (3.5) with global
mass conservation (2.26), which motivates the similarity solution hl = A2/5D−1/5t−1/5 fl(ξ),
ξ =
x
(A3Dt)1/5 . (3.6) (3.6) 985 A18-10 Viscous gravity current beneath a granular mush Viscous gravity current beneath a granular mush
–0.6
0.4
0.2
0
F(ξ)
–0.2
1.0
(a)
(b)
0.5
fl (ξ)
0
–0.4
–0.2
0
ξ
0.2
0.4
0.6
–0.6
–0.4
–0.2
0
0.2
0.4
0.6
Figure 7. Late-time self-similar interface shapes. (a) Comparison of the numerical solutions for hl(x, t) at
t = 100, 250, 1000, 5000, 50 000 (solid lines) with the similarity solution (3.7) (dot-dashed magenta line). Parameters and initial conditions are as in figure 2. (b) Comparison for the upper free surface H(x, t). The
similarity solution is given by (3.11). –0.6
0.4
0.2
0
F(ξ)
–0.2
1.0
(a)
(b)
0.5
fl (ξ)
0
–0.4
–0.2
0
ξ
0.2
0.4
0.6
–0.6
–0.4
–0.2
0
0.2
0.4
0.6 (a) 1.0
0.5
(ξ)
0
–0.6
–0.4
–0.2
0
0.2
0.4
0.6 Figure 7. Late-time self-similar interface shapes. (a) Comparison of the numerical solutions for hl(x, t) at
t = 100, 250, 1000, 5000, 50 000 (solid lines) with the similarity solution (3.7) (dot-dashed magenta line). Parameters and initial conditions are as in figure 2. (b) Comparison for the upper free surface H(x, t). The
similarity solution is given by (3.11). The self-similar shape function is given by The self-similar shape function is given by The self-similar shape function is given by The self-similar shape function is given by The self-similar shape function is given by fl(ξ) =
18
5
1/3
ξ2
0 −ξ21/3
,
ξ0 = 1
2
⎡
⎣5
π1/2
10
9
1/3 Γ
5
6
Γ
1
3
⎤
⎦
3/5
≈0.566, (3.7) where we have used global mass conservation of viscous liquid (2.26) to determine ξ0. The solution (3.7) is compared to the numerical results at various late times in figure 7(a). where we have used global mass conservation of viscous liquid (2.26) to determine ξ0. The solution (3.7) is compared to the numerical results at various late times in figure 7(a). Once hl(x, t) has been determined, the evolution of the upper free surface H(x, t) is
obtained via (3 3) which we rewrite as where we have used global mass conservation of viscous liquid (2.26) to determine ξ0. The solution (3.7) is compared to the numerical results at various late times in figure 7(a). Once hl(x, t) has been determined, the evolution of the upper free surface H(x, t) is
obtained via (3.3), which we rewrite as Once hl(x, t) has been determined, the evolution of the upper free surface H(x, t) is
obtained via (3.3), which we rewrite as −∂H
∂x hl −D h2
l
2
∂hl
∂x ≈0,
(3.8) (3.8) since hl ≪1, hu ≈1 and |∂H/∂x| ≪D |∂hl/∂x|. Equation (3.8) reflects the balance of
the competing hydrostatic pressure gradients associated with the two interfaces. Given the
self-similar scalings for hl and x (3.6), we obtain the following solution for H(x, t): since hl ≪1, hu ≈1 and |∂H/∂x| ≪D |∂hl/∂x|. Equation (3.8) reflects the balance of
the competing hydrostatic pressure gradients associated with the two interfaces. Given the
self-similar scalings for hl and x (3.6), we obtain the following solution for H(x, t): H(x, t) = 1 + A4/5D3/5t−2/5 F(ξ). (3.9) (3.9) Equation (3.8) is recast in the similarity variables as Equation (3.8) is recast in the similarity variables as −fl
dF
dξ = 1
2 f 2
l
dfl
dξ ,
(3.10) (3.10) https://doi.org/10.1017/jfm.2024.305 Pu which we integrate to obtain which we integrate to obtain F(ξ) =
9
20
2/3
F0 −
ξ2
0 −ξ22/3
,
F0 = 10
7
25π3
12
1/30 ⎡
⎣
Γ
5
6
Γ
1
3
⎤
⎦
9/5
≈0.346,
(3 11) 985 A18-11 E.M. Hinton and A.C. Slim E.M. The self-similar shape function is given by The key feature that gives
rise to the self-similar behaviour is a yield criterion in the upper layer. y
pp
y
Finally, we observe that since hl ∼t−1/5 and H −1 ∼t−2/5, our assumptions that
|∂H/∂x| ≪D |∂hl/∂x| and hl ≪1 are valid. It is worth noting that although ∂H/∂x is
relatively small, it is non-zero and cannot be neglected when determining the leading-order
behaviour. The self-similar shape function is given by Hinton and A.C. Slim
1.0
(a)
(b)
(c)
(d)
(e)
( f )
0.01
0
–0.01
0.5
z
0
–0.5
0
0.5
1.0
0.05
0
–0.05
0.5
0
–1
0
1
1.5
0.05
0
u
–0.05
0.5
1.0
0
–2
0
2
2
0.05
0
–0.05
1
z
0
–2
0
x
2
2
0.1
0
–0.1
1
0
–2
–4
0
x
4
2
2
0.1
0
u
–0.1
1
0
–5
0
x
5
Figure 8. Interface evolution and horizontal flow velocity u of the granular mush and viscous liquid for a
constant input flux Q = 0.1, of viscous liquid with K = 1 and D = 0.5, for (a) t = 1, (b) t = 5, (c) t = 15,
(d) t = 30, (e) t = 50, and ( f) t = 80. The red dashed lines show the numerical integration of (3.5) with the
same input flux condition as for the two-media flow. (c)
5
1.5
0.05
0
u
–0.05
0.5
1.0
0
–2
0
2 1.0
(a)
(d)
0.01
0
–0.01
0.5
z
0
–0.5
0
0.5 (b)
( )
1.0
0.05
0
–0.05
0.5
0
–1
0
1 (b) (c) (a) (d) ( f ) (e) (d)
(
2
0.05
0
–0.05
1
z
0
–2
0
x
2 f )
2
0.1
0
u
–0.1
1
0
–5
0
x
5 )
2
0.1
0
–0.1
1
0
–2
–4
0
x
4
2 Figure 8. Interface evolution and horizontal flow velocity u of the granular mush and viscous liquid for a
constant input flux Q = 0.1, of viscous liquid with K = 1 and D = 0.5, for (a) t = 1, (b) t = 5, (c) t = 15,
(d) t = 30, (e) t = 50, and ( f) t = 80. The red dashed lines show the numerical integration of (3.5) with the
same input flux condition as for the two-media flow. where we have used global mass conservation of the combined media,
∞
−∞(H −1) dx =
0, to determine F0. The solution (3.11) is compared to the numerical results at various late
times in figure 7(b). We note that this similarity solution also applies to the gravity-driven spreading of a
viscous liquid beneath a viscoplastic material; see Appendix B. Viscous gravity current beneath a granular mush 1.5
(a)
(b)
(c)
(d)
(e)
( f )
(g)
(h)
(i)
1.0
0.5
z
0
2
5.0
2.5
0
5.0
2.5
0
5.0
2.5
0
1
0
2
1
0
2
1
0
1.5
1.0
0.5
z
0
1.5
1.0
0.5
z
0
–1
0
x
1
–4
–2
0
2
x
4
–10
0
10
–1
0
1
–4
–2
0
2
4
–10
0
10
–1
0
1
–4
–2
0
2
4
–10
0
10
x
Figure 9. Constant input flux of viscous liquid with K = 1. (a–c) Thicknesses at t = 0.5, 5, 50 for Q = 0.5
and D = 0.25. (d–f) Corresponding plots for Q = 0.5 and D = 1. (g–i) Corresponding plots for Q = 1.5 and
D = 1. 1.5
(a)
(d)
1.0
0.5
z
0 –1
0
1 (b)
( )
2
1
0–4
–2
0
2
4 (e) (i) (g)
1.5
1.0
0.5
z
0
–1
0
x
1 i)
5.0
2.5
0
–10
0
10
x (h)
2
1
0
–4
–2
0
2
x
4 Figure 9. Constant input flux of viscous liquid with K = 1. (a–c) Thicknesses at t = 0.5, 5, 50 for Q = 0.5
and D = 0.25. (d–f) Corresponding plots for Q = 0.5 and D = 1. (g–i) Corresponding plots for Q = 1.5 and
D = 1. figure 8 show the numerical integration of (3.5) with the same input flux condition as for
the two-media flow. There is good agreement at early and intermediate times. At later times, the viscous liquid penetrates the upper free surface owing to continued
input of viscous liquid; see figure 8( f). The viscous gravity current becomes segregated
into different regions separated by the contact points with the granular mush: (i) an
‘unconfined’ region in the centre where the liquid is not bounded above by granular mush
and the flow is driven solely by the liquid’s weight, and (ii) outer regions on either side
in which the gravity-driven spreading of the liquid is retarded by the hydrostatic pressure
gradients associated with the upper granular interface; see figure 8( f). This resistance to
the liquid motion is evidenced in the discrete change in the gradient of the liquid interface
across the contact point with the granular mush. The influence of varying the parameters D and Q is indicated in figure 9. 4. Constant input flux of viscous liquid In this section, we analyse the evolution in the case that the volume of viscous liquid
increases linearly in time at a rate Q; see (2.26). The viscous liquid is injected uniformly
in the interval −0.5 ≤x ≤0.5. Initially, hl = 0 and hu = 1 everywhere. y
l
u
y
Numerical results for the interface shapes (black lines) and the velocity field are shown
in figure 8 at six different times, with K = 1, Q = 0.1 and D = 0.5. At early times, the
viscous liquid is entirely enclosed by the granular mush and the upper free surface develops
an ‘M’ shape (e.g. figure 8c). This is because the early-time input of viscous liquid has two
key effects: (i) the upper free surface is lifted around x = 0, and (ii) granular material is
transferred horizontally outwards from x = 0 into two levees as it is carried by the liquid;
see the horizontal velocity fields in figure 8. The ‘M’ shape of the upper free surface leads to hydrostatic pressure gradients that
retard the viscous gravity current, in a fashion identical to the evolution of a fixed volume
release; see § 3. Indeed, the horizontal velocity has an approximately Poiseuille-like flow
structure in the liquid until the liquid penetrates the upper free surface; see figure 8(c). This suggests that the viscous liquid evolves as a gravity current with a quarter of the
flux of an ‘unconfined’ viscous gravity current (so (3.5) applies). The red dashed lines in Viscous gravity current beneath a granular mush Viscous gravity current beneath a granular mush Figures 9(a–c)
and 9(d–f) show identical results but with two values of the density difference D. At
early times, the density difference has little effect on the motion because the input flux
dominates the evolution. At later times, a larger density difference is associated with
greater gravity-driven spreading of the viscous liquid in the lateral direction. g
g
y
p
g
q
Figures 9(g–i) demonstrate the effect of a larger input flux Q, which is predominantly
to reduce the time scale of the flow (with the qualitative features unchanged). Varying K
leads to negligible change in the results. In the absence of a granular mush, the constant input of viscous liquid leads to a
self-similar gravity current with increasing thickness (h ∼t1/5) and extent (x ∼t4/5)
(Huppert 1982). This scaling does not apply to the present configuration at intermediate
times as demonstrated in figure 10, which shows the solution from figures 9(d–f) in
rescaled coordinates. If the ‘unconfined’ scalings applied, then the volume of displaced
granular material either side of the liquid would increase in proportion to t4/5 because 985 A18-13 E.M. Hinton and A.C. Slim
–0.3
–0.2
–0.1
0
0.1
0.2
0.3
0
0.5
z/t1/5
x/t4/5
1.0
1.5
Figure 10. Thicknesses for constant input flux in rescaled coordinates at t = 10 (blue), t = 100 (red) and t =
1000 (black). Parameter values as in figures 9(d–f). Although the gradients appear to be getting steeper in terms
of these scaled coordinates, in (x, z) coordinates, the interface gradients are diminishing so the flow remains
shallow. E.M. Hinton and A.C. Slim –0.3
–0.2
–0.1
0
0.1
0.2
0.3
0
0.5
z/t1/5
x/t4/5
1.0
1.5 Figure 10. Thicknesses for constant input flux in rescaled coordinates at t = 10 (blue), t = 100 (red) and t =
1000 (black). Parameter values as in figures 9(d–f). Although the gradients appear to be getting steeper in terms
of these scaled coordinates, in (x, z) coordinates, the interface gradients are diminishing so the flow remains
shallow. initially hu = 1, but the volume of viscous liquid is proportional to t so it is not possible to
have the same length and thickness scalings in the granular mush and the viscous liquid. At
very late times, the granular volume and thickness will become negligible in comparison
to the viscous liquid, so the ‘unconfined’ similarity solution will apply (results not shown). Declaration of interests. The authors report no conflict of interest. Author ORCIDs.
Edward M. Hinton https://orcid.org/0000-0002-2204-1204. Declaration of interests. The authors report no conflict of interest.
Author ORCIDs.
Edward M. Hinton https://orcid.org/0000-0002-2204-1204. 5. Discussion and conclusion This contribution has analysed the gravity-driven spreading of viscous liquid at the base of
a dense granular mush. Numerical integration of a lubrication model has revealed the key
features of the flow of a fixed volume of viscous liquid. At early times, there is a relatively
fast outward transfer of granular material owing to the viscous spreading, which carries
the overlying granular mush. This leads to the formation of levees of granular material at
either side of the liquid. In addition, it produces a hydrostatic pressure gradient against
the viscous flow because the granular mush is thicker away from the centre of the liquid
deposit. The counter-pressure gradient retards the flow. At late times, the flow becomes
self-similar, with the spreading rate quartered relative to a classical viscous gravity current
due to the evolution of the upper free surface (which is also self-similar). We have also analysed the case in which viscous liquid is injected at a constant rate
at the base of the granular mush. At early times, the liquid is contained entirely by the
granular mush, and the mush is carried into levees. A Poiseuille velocity field develops in
the liquid, and the flux is a quarter of that for an unconfined viscous gravity current. At
later times, the liquid penetrates the upper free surface, but the granular levees continue to
be pushed outwards. We have used a thin-film model for the flow, but we note that if the system is initially
not shallow, then it will still spread under gravity and eventually become shallow owing
to the outward spreading. Given the insensitive nature of the flow to the initial shape, the
dynamics at later times will be qualitatively unchanged. Although we focused on a two-dimensional geometry, the qualitative flow features
would be identical for an axisymmetric configuration. This includes the quartering of the
flux relative to a classical viscous gravity current and the late-time self-similar evolution. Finally, we have shown that the dynamics is unchanged if the overlying medium is a
Bingham material rather than a granular mush; see Appendix B. 985 A18-14 Viscous gravity current beneath a granular mush Appendix A. Scaling analysis In this appendix, we analyse the validity of the shallow-flow approximation. We assume
that μs ≪1. 5. Discussion and conclusion μ
The governing equations in each medium are conservation of mass and conservation of
momentum in the ˆx and ˆz directions, given by 0 = ∂ˆui
∂ˆx + ∂ˆwi
∂ˆz ,
(A1)
0 = −∂ˆpi
∂ˆx + ∂ˆτxx,i
∂ˆx
+ ∂ˆτxz,i
∂ˆz ,
(A2)
0 = −∂ˆpi
∂ˆz + ∂ˆτxz,i
∂ˆx
+ ∂ˆτzz,i
∂ˆz
−ˆρiˆg,
(A3) (A1) (A2) (A3) where i = u, l. These equations are combined with the Newtonian constitutive law in the
viscous liquid and the μ(I)-rheology in the granular mush. where i = u, l. These equations are combined with the Newtonian constitutive law in the
viscous liquid and the μ(I)-rheology in the granular mush. We follow a standard thin-film analysis (Ockendon & Ockendon 1995) and scale
thicknesses (ˆz, ˆh) with ˆH and lengths ˆx with ˆL, where we assume that ϵ = ˆH/ ˆL ≪1. Horizontal velocities are scaled with ˆu ∼ˆU, where this scale is determined later (see
(A9)). Continuity (A1) furnishes the vertical velocity scale: ˆw ∼ϵ ˆU. The ˆz-momentum
equation (A3) motivates the pressure scale ˆp ∼ρug ˆH in both materials, where we have
assumed that the two densities ˆρu and ˆρl have the same order of magnitude. In the (Newtonian) viscous liquid, the constitutive law furnishes the following scalings
for the stresses: ˆτxx ∼ˆη ∂ˆu
∂ˆx ∼ˆη ϵ ˆU
ˆH
,
(A4)
ˆτxz ∼ˆη ∂ˆu
∂ˆz ∼ˆη
ˆU
ˆH
,
(A5)
ˆτzz ∼ˆη ∂ˆw
∂ˆz ∼ˆη ϵ ˆU
ˆH
. (A6) (A4) (A5) (A6) The leading-order terms in the momentum equations in the viscous liquid ((A2) and (A3))
are (Huppert 1982) 0 = −∂ˆp
∂ˆx + ∂ˆτxz
∂ˆz ,
(A7)
0 = −∂ˆp
∂ˆz −ˆρlˆg,
(A8) (A7) (A8) and for a balance in (A7), the horizontal velocity scale is set to and for a balance in (A7), the horizontal velocity scale is set to ˆU = ϵ ˆρuˆg ˆH2
ˆη
. (A9) ˆU = ϵ ˆρuˆg ˆH2
ˆη
. (A9) This furnishes a viscous shear stress scale This furnishes a viscous shear stress scale This furnishes a viscous shear stress scale ˆτxz ∼ϵ ˆρuˆg ˆH. (A10) ˆτxz ∼ϵ ˆρuˆg ˆH. (A10)
985 A18-15 ˆτxz ∼ϵ ˆρuˆg ˆH. 985 A18-15 E.M. Hinton and A.C. Slim We next consider the stresses in the granular mush, and we assume that μ(I) ∼μs ∼ϵ. 5. Discussion and conclusion The dominant contribution to the second strain-rate invariant ˙γ is ˙γ ∼∂ˆu
∂ˆz ,
(A11) (A11) provided that there is deformation of the granular material; the case of quasi-rigid material
(∂ˆu/∂ˆz ≈0) is discussed in § A.1. The μ(I)-rheology furnishes the following scaling for
the stresses: ˆτxx ∼μ(I) ˆp ∂ˆu/∂ˆx
˙γ
∼μ(I) ϵ ˆρuˆg ˆH,
(A12)
ˆτxz ∼μ(I) ˆp ∂ˆu/∂ˆz
˙γ
∼μ(I) ˆρuˆg ˆH,
(A13)
ˆτzz ∼μ(I) ˆp ∂ˆw/∂ˆz
˙γ
∼μ(I) ϵ ˆρuˆg ˆH. (A14) (A12) (A14) Since μ(I) ∼ϵ, the shear stress in the granular mush has the same order of magnitude as
in the Newtonian liquid (A10). The stresses τxx and τzz also have the same magnitude as
in the Newtonian liquid. Hence the momentum equations for the granular mush reduce to
a shallow form identical to that in the Newtonian liquid, given by (A7) and (A8). Since μ(I) ∼ϵ, the shear stress in the granular mush has the same order of magnitude as
in the Newtonian liquid (A10). The stresses τxx and τzz also have the same magnitude as
in the Newtonian liquid. Hence the momentum equations for the granular mush reduce to
a shallow form identical to that in the Newtonian liquid, given by (A7) and (A8). A.1. Pseudo-rigid granular material For shallow flows of viscoplastic fluids, the reduction of the governing equations to the
leading-order shallow form leads to an apparent inconsistency; there are regions that
appear rigid at leading order, but they can deform in the streamwise direction (Lipscomb
& Denn 1984; Putz, Frigaard & Martinez 2009). For free-surface flows, this so-called
‘lubrication paradox’ was resolved by Balmforth & Craster (1999), who showed that the
regions in which ∂u/∂z vanishes at leading order are in fact ‘pseudo-plugs’ that are weakly
yielded owing to the importance of extensional variations in the velocity. A similar feature occurs for a granular mush carried by an underlying viscous liquid. The granular mush can appear to be rigid at leading order but is in fact deforming owing
to streamwise variations as it is carried by the spreading viscous liquid below. As for
viscoplastic pseudo-plugs, the velocity in these regions can be written as u = u0(x, t) + ϵ u1(x, z, t) + · · ·,
(A15) (A15) u = u0(x, t) + ϵ u1(x, z, t) + · · ·, so the leading-order velocity appears plugged since ∂u0/∂z = 0. The dominant
contributions to ˙γ then arise from both ϵ ∂u1/∂z and ∂u0/∂x, which are of the same order
of magnitude, and (A11) must be adjusted. The key conclusion is that the leading-order
velocity profiles from the shallow theory remain correct. Hence, for μs ∼ϵ ≪1, the model (and the velocity profiles) in § 2 are justified. The non-dimensionalisation with the horizontal length scale being order 1/μs times the
thickness scale (2.18) is also valid. 985 A18-16 Viscous gravity current beneath a granular mush Appendix B. Viscous gravity current beneath a Bingham material
The flux in each medium is (Balmforth et al. 2002; Christy & Hinton 2023) Appendix B. Viscous gravity current beneath a Bingham material
The flux in each medium is (Balmforth et al. A.1. Pseudo-rigid granular material 2002; Christy & Hinton 2023) ˆQl = −ˆρuˆg
6ˆη
3 ∂ˆH
∂ˆx
ˆhuˆh2
l + 2
D ∂ˆhl
∂ˆx + ∂ˆH
∂ˆx
ˆh3
l
,
(B1) (B1) 6η
∂x
∂x
∂x
ˆQu = −ˆρuˆg
6ˆν
ˆY2(3ˆhu −ˆY) ∂ˆH
∂ˆx −ˆρuˆg
2ˆη
2 ∂ˆH
∂ˆx
ˆh2
uˆhl +
D ∂ˆhl
∂ˆx + ∂ˆH
∂ˆx
ˆhuˆh2
l
,
(B2)
ˆY = max
⎛
⎜⎜⎜⎜⎝
0, ˆhu −
ˆτY
ˆρuˆg
∂ˆH
∂ˆx
⎞
⎟⎟⎟⎟⎠
,
(B3) (B2) (B3) where ˆτY is the yield stress of the Bingham material, and ˆν is the plastic shear viscosity. These forms of the flux are identical to (2.14) and (2.15), except for the different first term
in the Bingham flux, associated with its plastic behaviour. g
p
We non-dimensionalise using the following scalings (cf. (2.18)): (z, hl, hu, H) = (ˆz, ˆhl, ˆhu, ˆH)/ˆa,
x =
ˆxˆτY
ˆρuˆgˆa2 ,
t = ˆt ˆρuˆgˆa
ˆη
ˆτY
ˆρuˆgˆa
2
. (B4a–c) (B4a–c) The shallow approximation requires the vertical length scale to be much smaller than the
horizontal length scale, which becomes ˆτY
ˆρuˆgˆa ≪1,
(B5) (B5) which is equivalent to the Bingham number being small. This condition is analogous
to μs ≪1 for the granular mush; the yield criterion must be triggered at relatively
small gradients in the free surface so that the outer slopes of the levees are small. The
dimensionless fluxes in each medium are Ql = −
∂H
∂x
huh2
l
2
+
D ∂hl
∂x + ∂H
∂x
h3
l
3
,
(B6) (B6) Qu = −M
6 Y2(3hu −Y) ∂H
∂x −
∂H
∂x h2
uhl +
D ∂hl
∂x + ∂H
∂x
huh2
l
2
,
(B7 Qu = −M
6 Y2(3hu −Y) ∂H
∂x −
∂H
∂x h2
uhl +
D ∂hl
∂x + ∂H
∂x
huh2
l
2
,
(B7)
Y = max
⎛
⎜⎜⎝0, hu −
1
∂H
∂x
⎞
⎟⎟⎠,
(B8) (B7) Y = max
⎛
⎜⎜⎝0, hu −
1
∂H
∂x
⎞
⎟⎟⎠,
(B8) (B8) where M = ˆη
ˆν
(B9) (B9) is the ‘viscosity’ ratio, analogous to K; see (2.22). Mass conservation is given by (2.16). A.1. Pseudo-rigid granular material The system is integrated numerically, and results are shown in figure 11 for M = 1,
985 A18-17 is the ‘viscosity’ ratio, analogous to K; see (2.22). Mass conservation is given by (2.16). The system is integrated numerically, and results are shown in figure 11 for M = 1,
985 A18-17 is the ‘viscosity’ ratio, analogous to K; see (2.22). Mass conservation is given by (2.16). The system is integrated numerically, and results are shown in figure 11 for M = 1, E.M. Hinton and A.C. Slim E.M. Hinton and A.C. Slim
1.0
(a)
(b)
(c)
(d)
(e)
( f )
0.5
z
0
–1
0
1
1.0
0.5
0
–1
0
1
1.0
0.5
0
1.0
0.5
z
0
1.0
0.5
0
1.0
0.5
0
–1
0
1
–1
0
x
x
x
1
–2
0
2
–2
0
2
Figure 11. Gravity-driven flow of a fixed volume of viscous liquid beneath a Bingham material with M = 1
and D = 0.5. The initial condition is given by (3.1). Times are (a) t = 1, (b) t = 5, (c) t = 20, (d) t = 100,
(e) t = 1000, and ( f) t = 10 000. In (a–c), the blue dashed lines correspond to the granular case from figure 2. In (e, f), the red dashed line shows the similarity solution ((3.6) and (3.7)) for the lower layer. 1.0
(a)
0.5
z
0
–1
0
1 (b)
1.0
0.5
0
–1
0
1 (c)
1.0
0.5
0
–1
0
1 (b) (a) (e)
1.0
0.5
0
x
–2
0
2 (d)
1.0
0.5
z
0
–1
0
x
1 (d) (e) ( f )
1.0
0.5
0
x
–2
0
2 Figure 11. Gravity-driven flow of a fixed volume of viscous liquid beneath a Bingham material with M = 1
and D = 0.5. The initial condition is given by (3.1). Times are (a) t = 1, (b) t = 5, (c) t = 20, (d) t = 100,
(e) t = 1000, and ( f) t = 10 000. In (a–c), the blue dashed lines correspond to the granular case from figure 2. In (e, f), the red dashed line shows the similarity solution ((3.6) and (3.7)) for the lower layer. D = 0.5, and a fixed volume release of viscous liquid with initial shape given by (3.1). A.1. Pseudo-rigid granular material The evolution is qualitatively similar to the case of an overlying granular mush. Indeed,
the results from figure 2 for a granular mush with the same density difference are overlain
as blue dotted lines in figures 11(a–c) with excellent agreement. At late times, an identical
similarity solution, given by ((3.6) and (3.7)), applies, and it is shown as a red dashed line
in figure 11(e, f). This demonstrates that the key feature that controls the dynamics is the
yield criterion in the overlying material. Viscous gravity current beneath a granular mush N, K. & KOVAL, G. 2012 Nonlocal constitutive relation for steady granular flow. Phys. Rev. Lett. 108
178301. KLIAKHANDLER, I.L. & SIVASHINSKY, G.I. 1997 Viscous damping and instabilities in stratified liquid film
flowing down a slightly inclined plane. Phys. Fluids 9 (1), 23–30. KOWAL, K.N. & WORSTER, M.G. 2015 Lubricated viscous gravity currents. J. Fluid Mech. 766, 626–655. S
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power-law fluids. J. Fluid Mech. 916, A33. L.T. & KOWAL, K.N. 2022 Lubricated viscous gravity currents of power-law fluids. Part 2. Stability
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Fluid Mech. 14, 337–346. ,
LISTER, J.R. 1992 Viscous flows down an inclined plane from point and line sources. J. Fluid Mech. 242,
631–653. LIU, P.-P., ZHOU, M.-F., WANG, C.Y., XING, C.-M. & GAO, J.-F. 2014 Open magma chamber processes in
the formation of the Permian Baima mafic–ultramafic layered intrusion, SW China. Lithos 184, 194–208. the formation of the Permian Baima mafic–ultramafic layered intrusion, SW China. Lithos 184, 194–208. LYMAN, A.W., KERR, R.C. & GRIFFITHS, R.W. 2005 Effects of internal rheology and surface cooling on
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the emplacement of lava flows. J. Geophys. Res. 110, B08207. MAIER, W.D., BARNES, S.-J. & GROVES, D.I. 2013 The Bushveld Complex, South Africa: formation of
platinum–palladium, chrome- and vanadium-rich layers via hydrodynamic sorting of a mobilized cumulate
slurry in a large, relatively slowly cooling, subsiding magma chamber. Miner. Deposita 48, 1–56. MUNGALL, J.E. & SU, S. 2005 Interfacial tension between magmatic sulfide and silicate liquids: constraints
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OCKENDON, H. & OCKENDON, J.R. 1995 Viscous Flow, vol. 13. Cambridge Texts in Applied Mathematics,
Cambridge University Press. PUTZ, A., FRIGAARD, I.A. & MARTINEZ, D.M. REFERENCES BALMFORTH, N.J. & CRASTER, R.V. 1999 A consistent thin-layer theory for Bingham plastics. J. Non-Newtonian Fluid Mech. 84 (1), 65–81. BALMFORTH, N.J., CRASTER, R.V. & SASSI, R. 2002 Shallow viscoplastic flow on an inclined plane. J. Fluid Mech. 470, 1–29. BALMFORTH, N.J., CRASTER, R.V. & TONIOLO, C. 2003 Interfacial instability in non-Newtonian fluid
layers. Phys. Fluids 15 (11), 3370–3384. CHRISTY, I. & HINTON, E.M. 2023 Two-layer gravity currents of generalized Newtonian fluids. Proc. R. Soc. A 479 (2279), 20230429. CHUNG, H.-Y. & MUNGALL, J.E. 2009 Physical constraints on the migration of immiscible fluids through
partially molten silicates, with special reference to magmatic sulfide ores. Earth Planet. Sci. Lett. 286
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Mech. 969, R3. HUPPERT, H.E. 1982 The propagation of two-dimensional and axisymmetric viscous gravity currents over a
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HUPPERT, H.E. 1986 The intrusion of fluid mechanics into geology. J. Fluid Mech. 173, 557–594. 985 A18-18 985 A18-18 Viscous gravity current beneath a granular mush Viscous gravity current beneath a granular mush 2009 On the lubrication paradox and the use of
regularisation methods for lubrication flows. J. Non-Newtonian Fluid Mech. 163 (1–3), 62–77. ROBERTSON, J.C., BARNES, S.J. & LE VAILLANT, M. 2016 Dynamics of magmatic sulphide droplets
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2445–2472. 985 A18-19 985 A18-19
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Serum Biomarkers for Early Detection of Gynecologic Cancers
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Cancers
| 2,010
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cc-by
| 8,000
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cancers
ISSN 2072-6694
www.mdpi.com/journal/cancers
OPEN ACCESS cancers
ISSN 2072-6694
www.mdpi.com/journal/cancers
OPEN ACCESS cancers
ISSN 2072-6694
www.mdpi.com/journal/cancers Yutaka Ueda, Takayuki Enomoto *, Toshihiro Kimura, Takashi Miyatake, Kiyoshi Yoshino,
Masami Fujita and Tadashi Kimura Yutaka Ueda, Takayuki Enomoto *, Toshihiro Kimura, Takashi Miyatake, Kiyoshi Yoshino,
Masami Fujita and Tadashi Kimura Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, 2-2,
Yamadaoka, Suita, Osaka, 565-0871, Japan; E-Mails: ZVF03563@nifty.com (Y.U.);
kimbinbin@gmail.com (T.K.); tmiyatake@aim.com (T.M.); yosino-ki@mc.pref.osaka.jp (K.Y.);
fujita@gyne.med.osaka-u.ac.jp (M.F.); tadashi@gyne.med.osaka-u.ac.jp (T.K.) * Author to whom correspondence should be addressed; E-Mail: enomoto@gyne.med.osaka-u.ac.jp;
Tel: +81-66879-3351; Fax: +81-66879-3359. Received: 8 March 2010; in revised form: 31 May 2010 / Accepted: 3 June 2010 /
Published: 14 June 2010 Received: 8 March 2010; in revised form: 31 May 2010 / Accepted: 3 June 2010 /
Published: 14 June 2010 Received: 8 March 2010; in revised form: 31 May 2010 / Accepted: 3 June 2010 /
Published: 14 June 2010 Abstract: Ovarian, endometrial, and cervical cancers are three of the most common
malignancies of the female reproductive organs. CA 125, historically the most reliable
serum marker for ovarian cancer, is elevated in 50% of early-stage ovarian tumors. For
endometrial cancers, there are no established serum markers. SCC, which is the best
studied serum marker for squamous cell carcinomas, has been unreliable; SCC is elevated
in cervical squamous cell carcinomas ranging from 28–85% of the time. Recent
proteomics-based analyses show great promise for the discovery of new and more useful
biomarkers. In this review, we will discuss the currently utilized serum tumor markers for
gynecologic cancers and the novel biomarkers that are now under investigation. Keywords: ovarian; endometrial; cervical; cancer; serum; tumor marker; CA 125; SCC;
proteomics; biomarkers Cancers 2010, 2, 1312-1327; doi:10.3390/cancers2021312 Cancers 2010, 2, 1312-1327; doi:10.3390/cancers2021312 1. Introduction Endometrial, cervical, and ovarian cancers are the three most common malignancies of the female
reproductive tract. In the United States alone, roughly 12,000 women are diagnosed with uterine
cervical cancer annually, and 4,000 will die from the disease [1]. The relatively low incidence of
cervical cancer in the US is largely attributable to the effectiveness of Papanicolaou’s cytological
cervical screening test (the Pap test). The importance of screening for early tumor markers is supported
by the finding that 60% of cervical cancers occur in women who have never received a Pap test, or
who have not been tested in the past five years [1]. For comparison of effectiveness, the serum marker
SCC, which is the best studied serum marker for squamous cell carcinomas, is elevated in only
22–60% of early-stage cervical squamous cell carcinomas [2,3]. Endometrial cancer is even more common than cervical cancer in the US, with 37,000 diagnosed
with it and 7,000 deaths attributable to it in 2005, making it the fourth most common cancer in
women [1]. In approximately 75% of endometrial cancer cases, the tumor remains confined to the
uterus (FIGO stage I) and has a favorable prognosis. The prognosis, however, worsens dramatically as
the disease progresses [4]. Despite this, screening for endometrial cancer is not currently done because
of the lack of an appropriate, cost-effective, and acceptable test [5]. Intermediate in frequency between cervical and endometrial cancers, but by far the most fatal, is
ovarian cancer. In 2005, 20,000 women in the US were diagnosed with ovarian cancer, but a
heartbreaking 15,000 (75%) of these women died from the disease [1]. Roughly three-quarters of
ovarian cancer cases present at an advanced disease stage, with the disease spread well beyond the
ovaries [6]. In advanced-stage disease, patients most often have first symptoms related to the presence
of an enlarging tumor and ascites fluid. However, in early- and mid-stage disease, most patients are
asymptomatic for a prolonged period [5]. Serum cancer antigen-125 (CA 125) levels and transvaginal
ultrasonography (TV-USG) can contribute to the early detection of ovarian cancer. Unfortunately, these
tests are not currently cost-effective; they are thus not used routinely to screen for ovarian cancer [5]. For ovarian, endometrial, and cervical cancers, it is critical to detect the disease at the earliest
possible stage. The discovery of useful serum biomarkers for the early detection of gynecologic
cancers has thus been a high priority. Keywords: ovarian; endometrial; cervical; cancer; serum; tumor marker; CA 125; SCC;
proteomics; biomarkers Abbreviations:
ApoA-1:
apolipoprotein
A-1;
CA
15-3:
cancer
antigen-15-3;
CA 19-9: cancer antigen-19-9; CA 72-4: cancer antigen-72-4; CA 125: cancer antigen-125;
CEA: cancer embryonic antigen; CIN: cervical intraepithelial neoplasia; CYFRA 21-1:
cytokeratin 19 fragments; HE4: (WFDC2), human epididymis-specific 4-disulfide core
protein; H4: inter-α-trypsin inhibitor heavy chain fragment; IAP: immunosuppressive
acidic protein; IGFII: insulin-like growth factor II; MIF: macrophage inhibitory factor;
LPA: lysophosphatidic acid; MLRM: multiple logistic regression model; M-CSF: macrophage 1313 Cancers 2010, 2 colony-stimulating factor; Pap test: Papanicolaou’s test; SAA: human serum amyloid A;
SCC: squamous cell carcinoma antigen; SELDI-TOF-MS: surface-enhanced laser
desorption-ionization time-of-flight mass spectrometry; sFas: serum soluble Fas;
TF: transferrin; TK: thymidine kinase; TTR: transthyretin; TV-USG: transvaginal
ultrasonography; USG: ultrasonography; VEGF-C: serum isoform of vascular endothelial
growth factor; YKL-40: (aka CHI3L1) human cartilage glycoprotein-39 Cancers 2010, 2 Cancers 2010, 2 Cancers 2010, 2 silenced genes or as an alternative mRNA splicing expression of an already expressed gene product. Some glycoproteins produced by cancer cells have altered glycan structures, although the proteins
themselves are ubiquitous [7]. Tumor markers might be unique extracellular matrix or cell adhesion
molecules, receptors, growth factors, cytokines, or products of abnormal metabolism. Rarely, the
marker molecules can be released by other tissues and organs in response to signals from the tumor. Even the body’s own antibodies against tumor markers can be markers. Tumor markers are secreted, released, or leaked into the interstitial fluids, and thus into the lymph,
and finally (or directly) into the bloodstream, where they become detectable in serum samples. To be
able to enter the bloodstream directly, larger molecules, often proteins, are cleaved into truncated
forms or fragments, which are sometimes specific to the protease micro-environment of the tumor. Tumor markers can be associated with patient diagnosis, prognosis, clinical management, and
follow-up. Ideally, a serum marker would only appear in the blood of patients with a true malignancy;
the marker would correlate with tumor stage and response to treatment, and it could be easily, cheaply,
and reproducibly measured. The serum marker would be used for the screening of healthy populations
and of specific groups with higher risk factors. The marker would enable a diagnosis for a specific type
of cancer, help determine prognostic factors, and be used to monitor the course of treatment, remission,
and recurrence, while receiving surgery, radiation, chemical, and immunological treatments. Recent advances in clinical proteomics have propelled us into an exciting period of discovery of
new cancer biomarkers, although the available proteomic technologies have their limitations. The
principles of proteomic technology require stringent guidelines for the collection of clinical material,
the application of analytical techniques, and for our interpretation of the data. In this review, we present an overview of the serum tumor markers in current use. A lack of
sensitivity and specificity has, so far, given most of the tumor markers in current use an unsatisfactory
predictive value. We will discuss the novel biomarkers of the future, where there is great hope for the
better detection and management of gynecologic cancers, including ovarian, endometrial, and
cervical cancers. 1. Introduction Such tumor markers will be molecules arising from the presence
of a tumor, which can appear in the surrounding tissue, and then within the blood and excretions. Tumor markers can be secreted or shed by the tumor in excess of the normal tissue or cell
phenotype. Sometimes, the molecule is uniquely specific to the tumor phenotype, often as embryonic,
fetal (i.e., AFP), undifferentiated, or stem-cell phenotypes. These can occur as re-expression of 1314 Cancers 2010, 2 Cancers 2010, 2 1315 Table 1. Diagnostic serum markers for cervical cancer in clinical use. Table 1. Diagnostic serum markers for cervical cancer in clinical use. Serum markers
SCC
CYFRA 21-1
CA 125
CA 19-9
CEA
IAP
Positive rates
squamous
squamous
adeno
adeno
adeno
43–51%
28–85%
42–52%
27–75%
35–42%
26–48%
Positive rates (elevated serum levels) detected for the indicated serum markers, in cases of
squamous cell carcinoma (squamous), adenocarcinoma (adeno), or for all histological types. squamous cell carcinoma (squamous), adenocarcinoma (adeno), or for all histological types. Serum SCC levels have also been shown to parallel the responses to radiotherapy and
chemotherapy [13,19–21]. Elevated pre-radiotherapy SCC levels were detected in 60% of 72 squamous
cell carcinoma patients, whereas post-treatment SCC levels were above the cut-off value in only 2% of
the patients whose disease was considered completely treated [13]. The serum SCC level also proved
to be an independent predictor of response to neoadjuvant chemotherapy in locally advanced cervical
cancer patients who received neoadjuvant chemotherapy and radical surgery [19]. SCC has also been
used in the follow-up examination of cervical cancer patients. Increased serum SCC was shown to
precede the clinical detection of recurrence of the disease [12,13,22]. The marker CYFRA 21-1 (serum fragments of cytokeratin 19) is also being used as a serum tumor
marker for cervical cancer, especially for squamous cell carcinoma. Elevated CYFRA 21-1 levels have
been detected in 42–52% of patients with squamous cell carcinoma of the uterine cervix [18,23,24]
(Table 1). Similar to the usefulness of SCC, pre-treatment levels of CYFRA 21-1 are related to stage of
the disease, size of the tumor, depth of the stromal invasion, the lymph-vascular space involvement,
and lymph node metastasis [25–27]. However, raised CYFRA 21-1 levels were not demonstrated to be
of predictive value for prognosis in some studies [18]. Serum CYFRA 21-1 levels were reported to be useful for monitoring the response to radiotherapy
and chemotherapy [12,24]. The CYFRA 21-1 assay was also used in the follow-up examination of
cervical cancer patients. Additionally, increased serum CYFRA 21-1 was shown to precede the clinical
detection of recurrence of the disease [12]. Adenocarcinoma accounts for 10–15% of cervical cancers [4]. SCC, CYFRA 21-1, CA 125,
CA 19-9 and CEA are positive in 20–25%, 33–63%, 27–75%, 34–42% and 26–48%, respectively, of
such tumors [9–11,20,23,28–34] (Table 1). 2. Serum Markers for Cervical Cancer Screening for cervical cancer with cervical cytology reduced the incidence of cervical cancer by
more than 50% over the past 30 years in the United States [8]. However, it is estimated that 50% of the
women in whom cervical cancer is diagnosed each year will have never had cervical cytology
testing [8]. One approach for further reducing the incidence and the mortality of cervical cancer would
be to increase the screening rates among groups of women at highest risk, who currently are not being
screened. Another would be the establishment of appropriate serum testing for the early detection of
cervical cancer. The squamous cell carcinoma antigen, SCC, is the most commonly used serum marker
for squamous cell cervical carcinoma, which makes up 85–90% of all cervical carcinomas [4]. Elevated serum SCC levels have been detected in 28–88% of cervical squamous cell carcinomas [9–15]
(Table 1). Pre-treatment levels of SCC have been shown to be related to the stage of the disease, size of
the tumor, depth of the stromal invasion, the lymph-vascular space involvement, and lymph node
metastasis [9,11,13,14,16–19]. Elevated SCC levels were also demonstrated to have predictive value
for prognosis in some studies [16,17,19]. Cancers 2010, 2 Cancers 2010, 2 cell carcinomas when compared to that of healthy women (p < 0.0001) [36]. Vascular endothelial
growth factor (VEGF), especially the VEGF-C isoform, was revealed to be elevated in the serum of
patients with squamous cell carcinoma and cervical intraepithelial neoplasia (CIN) when compared to that
of healthy women [37,38]. In cervical squamous cell carcinoma patients, serum VEGF levels were
associated with the stage of the disease, but not with prognosis [38]. The serum level of thymidine
kinase (TK) was demonstrated to be significantly higher in patients with cervical carcinoma than in
normal women and patients with carcinoma in situ (p < 0.01 and p < 0.05, respectively) [39]. Table 2. Diagnostic serum markers for cervical cancer currently under investigation. Serum
markers
M-CSF
YKL-40
sFas
VEGF
TK
Positive rates
27%
squamous adeno
squamous
squamos
N/A
75% 78%
N/A
N/A
Positive rates (elevated serum levels) detected for the indicated serum markers, in cases of
squamous cell carcinoma (squamous), adenocarcinoma (adeno), or for all histological types. able 2. Diagnostic serum markers for cervical cancer currently under investigation. ostic serum markers for cervical cancer currently under investigation. Cancers 2010, 2 Raised serum CA 125 is associated with the stage of the
cervical disease and is of some prognostic significance [29]. Another novel marker (that is also a target for new drug development) is immunosuppressive acidic
protein (IAP), which is elevated in 43–51% of cervical carcinomas [33,34] (Table 1). Battaglia et al. found pre-treatment-elevation of serum IAP in 53% of squamous cell carcinoma cases, and in 40% of
adenocarcinoma patients [34]. IAP level is related to disease stage and lymph node metastasis, and is
of predictive value for prognosis [34]. There are some newer serum markers still under investigation (Table 2). Suzuki et al. showed that
serum M-SCF was elevated in 27% of cervical cancer cases [35]. YKL-40, also known as CHI3L1 or
human cartilage glycoprotein-39, was shown to be a potential biomarker in the detection and
management of cervical cancer [28]. Elevated serum YKL-40 was found in 75% of squamous cell
carcinoma patients and 78% of adenocarcinoma patients. Moreover, elevated pre-treatment-levels of
YKL-40 were shown to predict an unfavorable prognosis, independent of the stage of the disease. Significantly elevated serum levels of circulating soluble Fas (sFas) were demonstrated in squamous 1316 Cancers 2010, 2 Cancers 2010, 2 demonstrated to provide 46% sensitivity for endometrioid adenocarcinoma of the endometrium in all
stages at 95% specificity [54]. For stage I disease, HE4 yielded a 17% improvement in sensitivity
compared with that of CA 125. Significantly elevated levels of serum sFas were demonstrated in
endometrioid adenocarcinoma of the endometrium compared to that of healthy women (p < 0.0001) [36]. Human serum amyloid A (SAA) is a high-density lipoprotein that has recently been proposed as a
useful biomarker for several kinds of tumors, including gastric, lung, pancreatic, and nasopharyngeal
cancers [55–59]. Cocco et al. showed that SAA was overexpressed and actively secreted by Grade-3
endometrioid adenocarcinoma and serous papillary carcinoma of the endometrium, and that SAA was
present at high concentration in the serum of these patients [55,60]. Table 4. Diagnostic serum markers for endometrial cancer under investigation. Serum markers
M-CSF
HE4
sFas
SAA
Positive rates
25–73%
E
E
G3-E/S
46%
N/A
N/A
Positive rates (elevated serum levels) of each serum marker detected in cases of squamous cell
carcinoma, adenocarcinoma, and for all histological types, are shown. E: endometrioid adenocarcinoma;
G3-E: endometrioid adenocarcinoma Grade-3; S: serous adenocarcinoma; N/A: not assessed Table 4. Diagnostic serum markers for endometrial cancer under investigation. Recently, panels of novel biomarkers have been developed to better detect cancers, including
endometrial tumors (Table 5). Farias-Eisner et al. constructed a multiple logistic regression model
(MLRM) with the use of the values for ApoA-1 (apolipoprotein A-1), TF (transferrin), and TTR
(transthyretin) for the detection of endometrial cancer [61]. This panel distinguished normal samples
from early-stage endometrial cancer with a sensitivity of 71% and a specificity of 88% Additionally,
the panel distinguished normal samples from late stage endometrial cancer with a sensitivity of 82%
and a specificity of 86%. Table 5. Novel biomarker panels for detection of endometrial cancer. Reference
Biomarkers
Sensitivity
Specificity
Zhu et al. (2006)[63]
13 proteins
93%
100%
Farias-Eisner et al. (2009) [61]
3 proteins
Early Late
Early Late
71% 82%
88% 86%
Takano et al. (2009) [62]
2 proteins
82%
86%
Shown here is the sensitivity and specificity of each serum biomarker panel used in the detection of
endometrial cancer; Early: early-stage cases; Late: late-stage cases Table 5. Novel biomarker panels for detection of endometrial cancer. Recent proteomic techniques, which can identify differentially expressed proteins in a large set of
samples, have been applied to the discovery of new biomarkers in many diseases [62]. 3. Serum Markers for Endometrial Cancer In current practice, screening for endometrial cancer is not undertaken because of the lack of an
appropriate, cost-effective, and acceptable test that actually reduces mortality [5]. Routine use of an
endometrial cytological test, comparable to the Pap test for cervical cancer, is too insensitive and
nonspecific to be useful in screening for endometrial cancer [5]. Currently used serum markers and
novel biomarkers under investigation for endometrial cancer are discussed below. Elevated serum CA 125 levels have been detected in 11–43% of endometrial cancers [40–46] (Table 3). Pre-treatment CA 125 levels were shown to be related to the stage of the disease, the depth of
myometrial invasion, peritoneal cytology, and lymph node metastasis [42–48]. Raised CA 125 levels
were also demonstrated to be of predictive value for prognosis in some studies [42,47]. The serum CA
125 level usually parallels the clinical course of the disease [43,45,49]; however, the fact that serum
CA 125 levels are often elevated in disease-free endometrial cancer patients who have undergone
abdominal radiation should be kept in mind [50]. Table 3. Diagnostic serum markers for endometrial cancer in clinical use. Serum
markers
CA 125 CA 19-9 CA 15-3
CA 72-4
CEA
IAP
Positive rates
11–43% 22–24% 24–32%
22–32%
14–22% 55–76%
Positive rates (elevated serum levels) detected for the indicated serum marker in cases of endometrial
cancer are shown. Table 3. Diagnostic serum markers for endometrial cancer in clinical use. Table 3. Diagnostic serum markers for endometrial cancer in clinical use. The serum markers CA 19-9, CA 15-3, CA 72-4, and CEA levels are raised in endometrial cancer
patients in 22–24%, 24–32%, 22–32% and 14–22% of the cases, respectively [11,33,40,42,45,51,52]
(Table 3). Serum CA 15-3 levels were shown to be associated with prognosis [42,53]. Elevated serum
IAP levels have been detected in 55–76% of endometrial cancer patients [17,33] (Table 3). The serum markers CA 19-9, CA 15-3, CA 72-4, and CEA levels are raised in endometrial cancer
patients in 22–24%, 24–32%, 22–32% and 14–22% of the cases, respectively [11,33,40,42,45,51,52]
(Table 3). Serum CA 15-3 levels were shown to be associated with prognosis [42,53]. Elevated serum
IAP levels have been detected in 55–76% of endometrial cancer patients [17,33] (Table 3). There are other serum markers for endometrial cancer now under investigation (Table 4). Raised
serum M-SCF levels were detected in 25–73% of endometrial cancer cases [35,43]. HE4 was 1317 4. Serum Markers for Ovarian Cancer Establishment of an appropriate screening test for ovarian cancer has long been sought. This disease
is the leading cause of death from gynecologic malignancies in the US, with its poor prognosis
resulting from the lack of reliable, sensitive screening tests and our limited understanding of the
mechanisms of chemoresistance and relapse. More than two-thirds of cases of ovarian cancers are
diagnosed only after the disease has progressed to stage III or IV and involve the peritoneal cavity or
other organs. Symptoms that are associated with ovarian cancer are typically nonspecific and the association is
often not recognized until the disease is advanced [64]. Previous studies showed that ultrasonography
(USG), with or without power Doppler, provided high sensitivity. However, ultrasonography’s
specificity and positive predictive values were unsatisfactory [65,66]. Serum markers and novel
biomarkers for early detection of ovarian cancer that are currently used or under investigation are
discussed below. Elevated serum CA 125 levels have been detected in 50% and 92% of ovarian cancers in early and
late stages, respectively [67] (Table 6). According to a review by Nossov et al., the positive predictive
value of the CA 125 assay for the early detection of ovarian cancer is 57% [68]. Elevated CA 125
occurs in other cancers, such as in endometrial, breast, pancreatic, gastrointestinal, and lung cancers. Raised CA 125 levels are sometimes found in patients with benign gynecologic conditions, such as
menstruation, pregnancy, endometriosis, and pelvic inflammatory disease, and even in
non-gynecologic conditions, such as hepatitis and pancreatitis [49]. The predictive value of
pre-treatment CA 125 levels for prognosis is controversial; however, changes in CA 125 levels
correlate with the regression, stability, and progression of the disease in 87–94% of instances [49]. Table 6. Diagnostic serum markers for ovarian cancer in clinical use. Serum markers
CA 125
CA 19-9
CA 15-3
CA 72-4
IAP
Positive rates
Early Late
M non-M
50–56%
63–71%
70–93%
50% 92%
68–83% 28–29%
Positive rates detected for each serum marker in cases of ovarian cancer are shown. Early: early-stage cases; Late: late-stage cases; M: mucinous adenocarcinoma; Non-M: histological
types of ovarian carcinoma other than mucinous adenocarcinoma. Table 6. Diagnostic serum markers for ovarian cancer in clinical use. Serum levels of CA 19-9 (a monosialoganglioside antigen widely used in GI adenocarcinoma
diagnostics) are elevated in 68–83% of mucinous ovarian cancers, and only in 28–29% of non-mucinous
types. Cancers 2010, 2 Cancers 2010, 2 proportion of cancer patients than would be found in a general population, and thus a significantly
lower proportion of healthy controls, had methodological limitations, as previously described [64]. The
prevalence of endometrial cancer was 50% in the study by Takano et al. [62] and 57% in the study by
Zhu et al. [63]. Thus, these results might have overstated the sensitivity of the tests. However, because
one of the candidate proteins discovered in a study by Takano et al. [62], apolipoprotein A-1, was also
a constituent of the biomarker panel constructed by Farias-Eisner et al. [61], this protein may be a very
promising biomarker for endometrial cancer. Cancers 2010, 2 Zhu et al. has
established a diagnostic system with 13 novel potential biomarkers, using surface-enhanced laser
desorption-ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to differentiate endometrial
cancer patients from healthy women. The technique had a sensitivity of 93% and a specificity of
100% [63]. Takano et al. also used SELDI-TOF-MS to identify candidate markers for endometrial
cancer [62]. Two of the candidates turned out to be apolipoprotein A-1 and apolipoprotein C-1. Dual-biomarker analysis for the detection of endometrial cancer yielded a sensitivity of 82% and a
specificity of 86%. These studies, which analyzed test samples consisting of a significantly higher 1318 Cancers 2010, 2 Serum CA 15-3, CA 72-4, and CEA levels are raised in 50–56%, 24–32%, 63–71% and 70–93% of the
ovarian cancer patients, respectively [71,73–78] (Table 6). According to Gadducci et al., CA 19-9,
CA 15-3, and CA 72-4 correlated worse than CA 125 with the clinical course of the disease. Additionally, these markers did not offer additional clinical benefit for monitoring ovarian cancer,
suggesting that the serial measurement of these markers may play a role only in the management of
patients with a normal CA 125 assay [49]. There are serum markers for ovarian cancer that are under active investigation (Table 7). In a
review by Li et al., HE4 displayed the highest sensitivity among single markers, including CA 125, in
the detection of ovarian cancer in both the early (62–83%) and late (75–93%) stages, respectively [79]. Elevated serum lysophosphatidic acid (LPA) levels were found in 90% and 98% of ovarian cancer in
early and late stages, respectively. However, serum levels of LPA do not correlate with the stage of the
disease, and nonspecific elevation of LPA was detected in healthy and benign gynecologic
conditions [68,80,81]. Significantly elevated sFas levels were detected in ovarian cancer patients when
compared to that of healthy women. Additionally, the serum sFas level was demonstrated to be a
statistically significant indication factor for survival, as well as for the histological grading of ovarian
carcinomas [36]. Table 7. Diagnostic serum markers for ovarian cancer under investigation. Serum markers
HE4
LPA
sFas
Positive rates
Early Late
Early Late
N/A
62–83% 75–93%
90% 98%
Positive rates detected for each serum marker in cases of ovarian cancer are shown. Early: early-stage cases; Late: late-stage cases; N/A: not assessed Table 7. Diagnostic serum markers for ovarian cancer under investigation. Novel biomarker panels have also been investigated for the early detection of ovarian cancers
(Table 8). Zhang et al. identified a panel that consisted of three proteins, including ApoA-1, a truncated
form of TTR, and a cleavage fragment of H4 (inter-α-trypsin inhibitor heavy chain), to detect
early-stage ovarian cancer with a sensitivity of 83% and a specificity of 94% [82]. Table 8. Novel biomarker panels for the detection of ovarian cancer. Reference
Biomarkers
Sensitivity
Specificity
Zhang et al. (2004) [82]
3 proteins
early
early
83%
94%
Su et al. (2007) [83]
4 proteins
early, M early, all
early, M early, all
95% 89%
92% 97%
Nosov et al. 4. Serum Markers for Ovarian Cancer In contrast, whereas CA 125 is elevated in 80% of non-mucinous ovarian tumors [69–72]. 1319 The sensitivity and specificity of each serum biomarker panel used for the detection of ovarian
cancer are shown; Early: early-staged cases; Late: late-staged cases; All: cases in all stages; S:
serous adenocarcinoma; M: mucinous adenocarcinoma. 5. Conclusions For gynecologic cervical, endometrial, and ovarian cancers, only a small handful of
tumor-associated antigens, such as SCC and CA 125, have been routinely used as tumor markers. Some markers are useful, not only as diagnostic tools, but also as a predictive marker for the prognosis
and the clinical course after treatment. Some recently investigated new serum markers seem to be
clinically useful, such as YKL-40 for cervical cancer and HE4 for endometrial and ovarian cancers. Recent breakthroughs in proteomics and bioinformatics technology will expand our understanding of
tumor-specific biomarkers. Such investigations will establish newer and more useful biomarkers for
the more accurate detection and management of ovarian, endometrial, and cervical cancers. Cancers 2010, 2 1320 Su et al. utilized an MLRM with values for CA 125, ApoA-I, TF, and TTR for the early detection of
ovarian cancer [83]. This model provided a sensitivity of 89% and a specificity of 97% for the
detection of early stage ovarian cancer. Furthermore, the sensitivity and the specificity of this panel in
distinguishing between normal and mucinous ovarian cancer samples were 95% and 92%, respectively. Nosov et al. applied this same MLRM model and marker panel to serous and endometrioid histological
types of ovarian carcinomas and demonstrated a sensitivity of 94% and a specificity of 94% for serous
ovarian carcinoma in its early stage, and a sensitivity of 98% and a specificity of 98% for endometrioid
ovarian carcinoma in its early stage [84]. Visintin et al. proposed a set of serum biomarkers that consisted of leptin, prolactin, osteopontin,
insulin-like growth factor II (IGFII), macrophage inhibitory factor (MIF), and CA 125 to discriminate
between ovarian cancer patients and healthy women. The panel had a sensitivity of 95% and a
specificity of 99% [85]. Not surprisingly, this panel provided a significant improvement over CA 125
alone. However, these studies had the same methodological limitations, of excessive numbers of tumor
cases versus matched population controls, described above in the endometrial cancer section. With that
being said, novel proteomics-based investigations and bioinformatics analyses still provide the greatest
promise of using existing approaches to find ever more accurate and useable biomarkers for
gynecological cancers. One important question remains: how long before the diagnosis of an ovarian cancer does the serum
level of various markers begin to rise above background levels as a tumor grows? Anderson et al. [86]
studied concentrations of CA125, HE4, and mesothelin in serum samples collected from 0–18 years before
the diagnosis of a tumor, as part of an unrelated study. They found that the markers may provide some
evidence of ovarian cancer up to 3 years before clinical diagnosis, but the more likely lead time for the
detection of a change associated with these markers appears to be less than 1 year. Cancers 2010, 2 (2009) [84]
4 proteins
early, S early, E
early, S early, E
94% 98%
94% 98%
Visintin et al. (2008) [85]
6 proteins
95%
99%
The sensitivity and specificity of each serum biomarker panel used for the detection of ovarian
cancer are shown; Early: early-staged cases; Late: late-staged cases; All: cases in all stages; S:
serous adenocarcinoma; M: mucinous adenocarcinoma. Table 8. Novel biomarker panels for the detection of ovarian cancer. The sensitivity and specificity of each serum biomarker panel used for the detection of ovarian
cancer are shown; Early: early-staged cases; Late: late-staged cases; All: cases in all stages; S:
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of selected ovarian cancer biomarkers: a nested case-control study. J. Natl. Cancer Inst. 2010, 102,
26–38. © 2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an Open Access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/). © 2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an Open Access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).
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Somatotype of Top-Level Serbian Rhythmic Gymnasts
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Journal of Human Kinetics
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by Tijana Purenović-Ivanović1, Ružena Popović1 Body size and build influence performance in many sports, especially in those belonging to the group of female
aesthetic sports (rhythmic gymnastics, artistic gymnastics, and figure skating). These sports pose high specific demands
upon the functional, energy, motor and psychological capacities of athletes, but also upon the size, body build and
composition of the performers, particularly of the top-level female athletes. The study of the top athletes (rhythmic
gymnasts, in this case) may provide valuable information on the morphological requirements for achieving success in
this sport. Therefore, the main objective of this research was to analyze the somatotype of 40 Serbian top-level rhythmic
gymnasts, aged 13.04±2.79, and to form the five age group categories. The anthropometric variables included body
height, body mass, the selected diameters, girths and skinfolds, and the Heath-Carter anthropometric somatotype. All of
the anthropometric data were collected according to International Biological Programme, and then processed in the
Somatotype 1.2. The applied analysis of variance indicated an increase in endomorphic component with age. The
obtained results show that the balanced ectomorph is a dominant somatotype, being similar for all of the athletes that
took part in the research (3.54-3.24-4.5). These results are in line with the ones obtained in previous studies. Key words: rhythmic gymnastics, selection, kinanthropometry, constitution, elite athletes. 181 Journal of Human Kinetics volume 40/2014, 181-187 DOI: 10.2478/hukin-2014-0020
Section III – Sports Training 1 - University of Niš, Faculty of Sport and Physical Education, Serbia. Authors submitted their contribution of the article to the editorial board.
Accepted for printing in Journal of Human Kinetics vol. 40/2014 on March 2014. Somatotype of Top-Level Serbian Rhythmic Gymnasts by
Tijana Purenović-Ivanović1, Ružena Popović1 Introduction Athletes are characterized by combination of body
composition/body size traits which are believed to
influence the chance of success in any given sport. Athletes are characterized by combination of body
composition/body size traits which are believed to
influence the chance of success in any given sport. Therefore, it is suggested that the measurement of
kinanthropometry is a crucial tool in the search
for information that would assist coaches and
athletes in the quest for success at the highest
level in sport, i.e. in talent identification (Sánchez-
Muñoz et al., 2012). This discipline imposes high demands upon body
size,
body
build and
composition
of
the
performers, particularly of elite female athletes. A
vast majority of the authors (Lapieza et al., 1993;
Menezes and Filho, 2006; Amigo et al., 2009;
Poliszczuk and Broda, 2010; Quintero et al., 2011)
have proposed one, the most common model of
the somatotype that rhythmic gymnasts (RGs)
pertain to - the balanced ectomorph - which
implies that the ectomorphic component is the
dominant one and the remaining two have equal
prevalence. However, there are some other
models which should not be overlooked, such as
the mesoectomorph (Lopéz-Benedicto et al., 1991;
Amigo et al., 2009), the mesomorph-ectomorph
(Vernetta et al., 2011), as well as the central
somatotype (Quintero et al., 2011; Vernetta et al.,
2011),
and
even
the
balanced
endomorph
(Quintero et al., 2011). Therefore, it is suggested that the measurement of
kinanthropometry is a crucial tool in the search
for information that would assist coaches and
athletes in the quest for success at the highest
level in sport, i.e. in talent identification (Sánchez-
Muñoz et al., 2012). Literature
describes
desirable
model
characteristics of the elite athletes in the form of
basic
anthropometric
dimensions,
their
interrelations, body composition components, and
somatotypes. Body size and body build contribute
significantly to performance in many sports,
particularly
in
aesthetic
sports
(Misigoj-
Durakovic, 2012). Within the group of the so-
called female aesthetic sports, the rhythmic
gymnastics (RG) is one of the most demanding. Authors submitted their contribution of the article to the editorial board. Accepted for printing in Journal of Human Kinetics vol. 40/2014 on March 2014. 182 Somatotype of top-level Serbian rhythmic gymnasts The body mass was measured with a digital scale
Omron BF511 (Kyoto, Japan). Measures and Procedures The measures were taken during 2012
National Championships held in Belgrade (Serbia)
on December 16th 2012, and testing was conducted
in agreement with the principles stated in the
Declaration
of
Helsinki
(WMA,
2002). An
anthropometric method was used for obtaining
the RGs’ somatic type and it included 10
following variables: body height (in cm), body
mass (in kg), four skinfolds (over triceps,
subscapular, supraspinale, and calf; in mm), and
biceps girth (flexed 90° and tensed; in cm),
standing calf girth (in cm), humerus breadth (in
cm) and femur breadth (in cm). Analysis The somatotype was determined according
to the methodology of Heath-Carter (Carter and
Heath, 1990), applying the statistical data analysis
(Descriptive statistics and one-way ANOVA)
using the computer program Somatotype 1.2. Results The obtained data are presented in tables
and graphs (by somatoplots). The sample of 40
top-level Serbian RGs showed that the mean
somatotype was: 3.54 - 3.24 - 4.5 (values for the
endomorphy, mesomorphy and ectomorphy,
respectively; Table 2). The descriptive statistics of
all the measurements are presented in Table 2,
and all the forty profiles (squares) with the mean
somatotype (circle) are presented in Figure 2. Participants Forty rhythmic gymnasts (age: 13.04±2.79
years, body height: 153.64±13.16 cm, body mass:
40.55±11.31 kg) volunteered to participate in the
study. A written request was promptly sent to the
Expert Committee of Gymnastics Federation of
Serbia, and, after being informed about the study,
its scientific value and multiple benefits, the
approval was given for the testing to be
conducted
during
the
2012
National
Championships. All clubs participating in the
National Championships were informed about
the conducted research and four out of ten RG
clubs have given their consent. All participants
are an “A” program, group routines competitors. The baseline characteristics of the sample in total,
and sub-samples (age categories) are presented in
Table 1. Analyzing the somatoplots presented in
Figure 1, a variety of somatotype categories can be
noticed,
with
one
major
category
(the
ectomorphy) prevailing. It is obvious that an
increase in age entails an increase in the value of
the endomorphic component while the other two
components
remain
rather
stable. This
is
confirmed by ANOVA due to the presence of
statistically significant differences (F=3.6, p=0.015)
among the five age categories. The discriminatory
factor (apart from the age, height and body mass)
was one somatotype component (endomorphy,
p<0.001). Such results are common in many other
sports. What can be said about the range of RGs’
somatotype
components
is
that
they
are
moderate. Introduction Taking into account the fact that the
relevant literature has not established one
morphological prototype of rhythmic gymnasts as
the dominant one and the fact that there are no
relevant data on the somatotype of Serbian RGs,
the purpose of the present study was to assess the
body constitution of the top-level rhythmic
gymnasts in Serbia. The main objective of the
study was to determine somatic characteristics of
Serbian girls of different age who were competing
at the top-level RG and thus provide valuable
information for the future selection process. Discussion Apart from talent, the adequate body
constitution is a prerequisite for achieving success
in sports. Body build is, to a large extent,
determined by the human genotype, but within
the
defined
limits
it
is
also
subject
to
environmental influence. The extent of sensitivity
to the external environment is also hereditary
conditioned. The type of body constitution in the
observed RGs was mainly characterized by the
prevalence of the ectomorphic component, with
moderate values of the other two components
(3.54-3.24-4.5). This
balanced
ectomorph
constitution is seen in other RG studies. For All of the measurements were taken by
both authors in the optimal climatic conditions,
with the participants in underwear, and according
to the methods proposed by the International
Biological Programme (Weiner and Lourie, 1969). Journal of Human Kinetics volume 40/2014 http://www.johk.pl by Purenović-Ivanović T. and Popović R. 183 example, Lapieza et al. (1993) analyzed the
somatotype of 18 female rhythmic gymnasts from
Spain, aged 12 to 16 (belonging to cadet and
junior teams) and achieved the following mean
profile: 2.28–2.45–3.7. What can be noticed are the
low values of the first and the second component,
which are, at the same time, lower than the values
we obtained from our cadet team gymnasts (2.93-
3.63-4.4), as well as the junior team gymnasts
(3.91-3.06-4.38). Menezes
and
Filho
(2006)
analyzed the somatotype of 24 female Brazilian
rhythmic gymnasts (seven from the National RG
Team, 10 participants from the 2003 National
Championships, and seven participants from the
Regional Championships from Rio de Janeiro),
and obtained the following profiles: 2.33-2.83-4.17,
2.72-2.65-4.17 and 2.88-3.16-3.51. Even here the
low values of the first two components are
noticeable as was the case with the previously
mentioned study. Poliszczuk and Broda (2010) analyzed 19 female rhythmic gymnasts, aged 8 to
11 and found the following somatotype: 2.65-2.45-
3.95, which, when compared to the somatotype of
our novices (2.76-3.6-4.74), shows the low values
of
the
mesomorphic
and
the
ectomorphic
components. Lopéz-Benedicto et al. (1991) in the
sample of 21 Spanish junior RGs of a regional and
national level (11.1 to 15.8 ages), found the meso-
ectomorph somatotype profile (2.3-3.1-4.5) as the
result of their study. Vernetta et al. (2011) did
their research with 20 Andalusian RGs, 9 to 15
years of age, and divided them into two sub-
samples: A(N=12): 9-11 years of age (1.804-3.694-
3.701, mesomorphic ectomorph) and B(N=8): 12-
15
years
of
age
(2.059-3.161-4.115,
central
somatotype). N – number of study participants, SD – standard deviation. N number of study participants, SD standard deviation.
Age is presented in years, body height in cm, and body mass in kg. Discussion The total sample showed a central
mean somatotype (2.906-3.481-3.866). Table 1
The baseline characteristics of top-level Serbian RGs
Age Categories
Variable
Mean±SD
Median
Range
Novices
(N=5)
Age
8.99±1.16
8.93
8.07 – 10.92
Body Height
133.32±6.17
133
127.2 – 142.5
Body Mass
25.3±3.4
24.7
22.3 – 30.8
Young juniors
(N=12)
Age
11.07±0.73
11.17
9.94 – 11.92
Body Height
145.64±7.81
145.9
134 – 158.4
Body Mass
32.37±5.65
31.3
25.9 – 44.5
Cadets
(N=6)
Age
12.84±0.65
12.9
12 – 13.57
Body Height
152.97±7.36
151.25
143.7 – 164
Body Mass
39.27±4.23
39.35
33.7 – 44.3
Juniors
(N=12)
Age
14.51±0.78
14.24
13.48 – 16.59
Body Height
164.09±4.27
163.1
158.5 - 171
Body Mass
48.63±4.64
48.05
41.3 – 56.3
Seniors
(N=5)
Age
18.13±1.18
17.57
16.93 – 19.45
Body Height
168.88±6.22
168.2
161 – 175.1
Body Mass
57.6±3.69
56.2
54 – 61.6
Total (N=40)
Age
13.04±2.79
12.9
8.07 – 19.45
Body Height
153.64±13.16
156.75
127.2 – 175.1
Body Mass
40.55±11.31
40.4
22.3 – 61.6
N – number of study participants, SD – standard deviation. Age is presented in years, body height in cm, and body mass in kg. © Editorial Committee of Journal of Human Kinetics © Editorial Committee of Journal of Human Kinetics Somatotype of top-level Serbian rhythmic gymnasts 184 Figure 1
Somatoplots of top-level Serbian RGs, according to five age categories. The squares are the individual somatotypes, and the circle is the mean profile. 1. Novices (N=5)
2. Young juniors (N=12)
3. Cadets (N=6)
2.76 - 3.6 - 4.74
(mesomorphic ectomorph)
3.33 - 2.96 - 4.99
(balanced ectomorph)
2.93 - 3.63 - 4.4
(mesomorphic ectomorph)
4. Juniors (N=12)
5. Seniors (N=5)
6. Sample in total (all five age
categories)
3.91 - 3.06 - 4.38
(endomorph-ectomorph)
4.64 - 3.5 - 3.46
(balanced endomorph)
3.54 - 3.24 - 4.5
(balanced ectomorph) Figure 1
Somatoplots of top-level Serbian RGs, according to five age categories. The squares are the individual somatotypes, and the circle is the mean profile. 1. Novices (N=5)
2. Young juniors (N=12)
3. Cadets (N=6)
2.76 - 3.6 - 4.74
(mesomorphic ectomorph)
3.33 - 2.96 - 4.99
(balanced ectomorph)
2.93 - 3.63 - 4.4
(mesomorphic ectomorph)
4. Juniors (N=12)
5. Seniors (N=5)
6. Discussion i
i l C
i
f J
l f H
Ki
i
Table 2
The descriptive statistics of the measured anthropometric
variables of the sample in total (N=40)
Variable
Mean±SD
Median
Range
Triceps SF
12.74±3.87
11.9
6 – 21.2
Subscapular SF
9±2.48
8.5
4.2 – 14.2
Supraspinale SF
10.03±3.74
9.6
4.2 – 18.8
Calf SF
8.79±3.81
8.1
3 – 17.8
Flexed arm G
22.95±2.68
23.1
17.5 – 28.2
Calf G
30.91±3.62
30.8
24 – 38.1
Humerus B
6.11±0.6
6.17
4.75 – 7.02
Femur B
7.84±0.7
8
6.53 – 8.82
HWR
45.17±1.23
45.05
42.41 – 48
SAD
1.32±0.68
1.32
0.08 – 2.89
Endomorphy
3.54±0.82
3.5
2.1 – 5.3
Mesomorphy
3.24±0.86
3.2
1.8 – 5.5
Ectomorphy
4.5±0.91
4.4
2.5 – 6.6
SF – skinfold, G – girth, B – breadth, HWR – height-weight ratio,
SAD – somatotype attitudinal distance, SD – standard deviation. Table 3
The evolution of somatotype with age (Spanish RGs)
Study
Age
N
Endo
Meso
Ecto
Amigo et al. (2009)
10
12
1.4
2.6
5.5
11
11
1.4
2.5
5.4
12
15
1.4
2.4
5.5
13
23
1.5
2.4
5.5
14
27
1.6
2.7
5.2
15
25
1.7
2.5
5.1
16
14
1.7
2.6
4.8
17
14
1.9
2.7
4.8
18
10
1.8
2.6
4.9
Quintero et al. (2011)
8 - 11
12
4.25
3.28
4.8
11 - 13
11
4.13
3.01
4.64
13 - 14
23
4.59
3.06
3.89
15 - 19
25
4.45
3.33
3.57
N – number of study participants, Endo – endomorphy, Meso – mesomorphy,
Ecto – ectomorphy. Age is presented in years. Table 2
The descriptive statistics of the measured anthropometric
variables of the sample in total (N=40)
Variable
Mean±SD
Median
Range
Triceps SF
12.74±3.87
11.9
6 – 21.2
Subscapular SF
9±2.48
8.5
4.2 – 14.2
Supraspinale SF
10.03±3.74
9.6
4.2 – 18.8
Calf SF
8.79±3.81
8.1
3 – 17.8
Flexed arm G
22.95±2.68
23.1
17.5 – 28.2
Calf G
30.91±3.62
30.8
24 – 38.1
Humerus B
6.11±0.6
6.17
4.75 – 7.02
Femur B
7.84±0.7
8
6.53 – 8.82
HWR
45.17±1.23
45.05
42.41 – 48
SAD
1.32±0.68
1.32
0.08 – 2.89
Endomorphy
3.54±0.82
3.5
2.1 – 5.3
Mesomorphy
3.24±0.86
3.2
1.8 – 5.5
Ectomorphy
4.5±0.91
4.4
2.5 – 6.6
SF – skinfold, G – girth, B – breadth, HWR – height-weight ratio,
SAD – somatotype attitudinal distance, SD – standard deviation. Discussion Sample in total (all five age
categories)
3.91 - 3.06 - 4.38
(endomorph-ectomorph)
4.64 - 3.5 - 3.46
(balanced endomorph)
3.54 - 3.24 - 4.5
(balanced ectomorph) Journal of Human Kinetics volume 40/2014
http://www.johk.pl
Figure 1
Somatoplots of top-level Serbian RGs, according to five age categories. The squares are the individual somatotypes, and the circle is the mean profile. Figure 2
Somatotype distribution of top-level Serbian RGs (N=40). The squares are the individual somatotypes, and the circle is the mean profile. 1. Novices (N=5)
2. Young juniors (N=12)
3. Cadets (N=6)
2.76 - 3.6 - 4.74
(mesomorphic ectomorph)
3.33 - 2.96 - 4.99
(balanced ectomorph)
2.93 - 3.63 - 4.4
(mesomorphic ectomorph)
4. Juniors (N=12)
5. Seniors (N=5)
6. Sample in total (all five age
categories)
3.91 - 3.06 - 4.38
(endomorph-ectomorph)
4.64 - 3.5 - 3.46
(balanced endomorph)
3.54 - 3.24 - 4.5
(balanced ectomorph) Figure 1
Somatoplots of top-level Serbian RGs, according to five age categories. The squares are the individual somatotypes, and the circle is the mean profil Figure 2
Somatotype distribution of top-level Serbian RGs (N=40). The squares are the individual somatotypes, and the circle is the mean profile. igure 2
Somatotype distribution of top-level Serbian RGs (N=40). The squares are the individual somatotypes, and the circle is the mean profile. Somatotype distribution of top-level Serbian RGs (N=40). The squares are the individual somatotypes, and the circle is the mean profile. Journal of Human Kinetics volume 40/2014 Journal of Human Kinetics volume 40/2014 http://www.johk.pl by Purenović-Ivanović T. and Popović R. 185 urenović-Ivanović T. and Popović R. Discussion Table 3
The evolution of somatotype with age (Spanish RGs)
Study
Age
N
Endo
Meso
Ecto
Amigo et al. (2009)
10
12
1.4
2.6
5.5
11
11
1.4
2.5
5.4
12
15
1.4
2.4
5.5
13
23
1.5
2.4
5.5
14
27
1.6
2.7
5.2
15
25
1.7
2.5
5.1
16
14
1.7
2.6
4.8
17
14
1.9
2.7
4.8
18
10
1.8
2.6
4.9
Quintero et al. (2011)
8 - 11
12
4.25
3.28
4.8
11 - 13
11
4.13
3.01
4.64
13 - 14
23
4.59
3.06
3.89
15 - 19
25
4.45
3.33
3.57
N – number of study participants, Endo – endomorphy, Meso – mesomorphy,
Ecto – ectomorphy. Age is presented in years. Table 2
The descriptive statistics of the measured anthropometric
variables of the sample in total (N=40)
Variable
Mean±SD
Median
Range
Triceps SF
12.74±3.87
11.9
6 – 21.2
Subscapular SF
9±2.48
8.5
4.2 – 14.2
Supraspinale SF
10.03±3.74
9.6
4.2 – 18.8
Calf SF
8.79±3.81
8.1
3 – 17.8
Flexed arm G
22.95±2.68
23.1
17.5 – 28.2
Calf G
30.91±3.62
30.8
24 – 38.1
Humerus B
6.11±0.6
6.17
4.75 – 7.02
Femur B
7.84±0.7
8
6.53 – 8.82
HWR
45.17±1.23
45.05
42.41 – 48
SAD
1.32±0.68
1.32
0.08 – 2.89
Endomorphy
3.54±0.82
3.5
2.1 – 5.3
Mesomorphy
3.24±0.86
3.2
1.8 – 5.5
Ectomorphy
4.5±0.91
4.4
2.5 – 6.6
SF – skinfold, G – girth, B – breadth, HWR – height-weight ratio,
SAD – somatotype attitudinal distance, SD – standard deviation. f
g
g
g
SAD – somatotype attitudinal distance, SD – standard deviation. Table 3
The evolution of somatotype with age (Spanish RGs)
Study
Age
N
Endo
Meso
Ecto
Amigo et al. (2009)
10
12
1.4
2.6
5.5
11
11
1.4
2.5
5.4
12
15
1.4
2.4
5.5
13
23
1.5
2.4
5.5
14
27
1.6
2.7
5.2
15
25
1.7
2.5
5.1
16
14
1.7
2.6
4.8
17
14
1.9
2.7
4.8
18
10
1.8
2.6
4.9
Quintero et al. (2011)
8 - 11
12
4.25
3.28
4.8
11 - 13
11
4.13
3.01
4.64
13 - 14
23
4.59
3.06
3.89
15 - 19
25
4.45
3.33
3.57
N – number of study participants, Endo – endomorphy, Meso – mesomorphy,
Ecto – ectomorphy. Age is presented in years. © Editorial Committee of Journal of Human Kinetics Somatotype of top-level Serbian rhythmic gymnasts 186 Amigo et al. Discussion (2009) (N=151 RGs, aged 10-
18) and Quintero et al. (2011) (2008 Tenerife
Championships, N=70 RGs, aged 8-19) conducted
longitudinal
studies
with
national
and
international level Spanish RGs (results are
presented in Table 3) and obtained quite different
results, which can be noted when the somatotype
components’ values of the gymnasts of the same
age are compared. Namely, Spanish gymnasts
from the study by Amigo et al. (2009) are of great
somatotype stability across ages, and they have
the meso-ectomorphic and balanced ectomorph
body constitution, i.e. very low endomorphic and
mesomorphic component values and very high
ectomorphic
values. In
the
second
study
(Quintero et al., 2011), one can notice the
moderate values for all of the three somatotype
components, but with an endomorphic value as
the highest one (an increase of the endomorphic
component with age). However, in the study by
Quintero et al. (2011), authors did not specify the
competition level of studied RGs, which could be
possible
explanation
for
the
discrepancies
between the obtained results of these two studies. amongst the five age group categories of the top-
level Serbian RGs were established, and they
indicate
that
the
endomorphic
component
increases
with
age,
while
the
other
two
components are rather stable (this could be due to
maturation, or even genetics and nutrition factor). Having also in mind the fact that there is no
evidence of this two-way causality between the
body constitution and sport (is the somatic type a
cause or a consequence?), this interpretation of
our findings still does not represent sufficient
information for the talent identification process in
RG. It could, however, inform about where the
Serbian RG was positioned in relation to more
successful RG countries. Study Limitations Having in mind the fact that results in
sports do not depend solely on the physique of an
athlete, at this point we can detect the main flaw
of the study: it does not take into consideration
other abilities that influence performance in sports
(such as motor abilities, psychological state,
functional abilities, etc.). Another area that could
be improved would be the sample of participants
and a study design type, which hinders the
authors
from
more
significant
conclusions. Maturity, of which the tempo and the period of
time of its occurrence varies and is associated
with an increase of the body height and body
mass, could also represent the key points for a
variety of body constitution types. Therefore, it is
recommended that the status of the Serbian
rhythmic
gymnasts
should
undergo
multidisciplinary analysis on the basis of a larger
and more representative sample, monitored over
a period of few years. Conclusions All of the previous research results mostly
indicate graceful and thin (ectomorphic) body
build of rhythmic gymnasts, characterized by
long,
slim
and
thin
limbs,
with
small
circumferences of muscles and thin and light
bones, made for subtle and graceful movements. However,
top-level
Serbian
RGs
can
be
characterized as balanced ectomorph, with not so
low values for endomorphic and mesomorphic
components, which is not seen as consistent with
the somatotype of RGs from other countries (the
ones that have higher ranking in rhythmic
gymnastics). Apart from that, the differences Acknowledgements: The authors would like to thank the Gymnastics Federation of Serbia, the organizer of the 2012
National Championships, the rhythmic gymnasts and the coaches of RG Serbian clubs: “Ritam-Pinki”,
“Ritam”, “Radnički” and “Paraćin”, as well as the coach and Junior RGs Group routines national team of
Serbia, who made this study possible. This research is a part of the project of the Ministry of Science and Technological Development of the
Republic of Serbia (No: 179019, Head researcher: Prof. R. Stanković). References Amigo A, Sala V, Faciabén A, Evrard M, Marginet M, Zamora L. Height, weight, somatotype and body
composition trends in rhythmic Spanish elite gymnasts from childhood to adulthood. Apunts. Journal of Human Kinetics volume 40/2014 http://www.johk.pl 187 by Purenović-Ivanović T. and Popović R. Educacíon Física y Deportes, 2009; 95(1): 64-74 Educacíon Física y Deportes, 2009; 95(1): 64-74 Carter JEL, Heath BH. Somatotyping. Development and Applications. Cambridge: Cambridge University Press;
1990 Lapieza MG, Nuviala RJ, Castillo MC, Giner A. Morphological characteristics in adolescent rhythmic gymnasts and
swimmers. Apunts. Medicina de l'Esport, 1993; 30(118): 255-263 Lopéz-Benedicto A, Franco L, Terreros J. Rhythmic gymnastics. Physiological and anthropometrical
evolution in the season. Archivos de medicina del deporte, 1991; 8(30): 127-133 Menezes LS, Filho JF. Identification and comparison of dermatoglyfics, somatotype and basic physical
aptitude characteristics of rhythmic gymnasts of different qualification levels. Fitness & Performance
Journal, 2006; 5(6): 393-401 Misigoj-Durakovic M. Anthropometry in Premenarcheal Female Esthetic Sports Athletes and Ballerinas. In
V. R. Preedy (Ed.), Handbook of Anthropometry (pp. 1817-1836). New York: Springer; 2012 Poliszczuk T, Broda D. Somatic constitution and the ability to maintain dynamic body equilibrium in girls
practicing rhythmic gymnastics. Pediatric Endocrinology, Diabetes and Metabolism, 2010; 16(2): 94-99 Quintero B, Martín A, Henríquez J. The anthropometric profile of rhythmic gymnasts. Apunts. Educación
Física y Deportes, 2011; 103(1): 48-55 Sánchez-Muñoz C, Zabala M, Williams K. Anthropometric Variables and its Usage to Characterise Elite
Youth Athletes. In V. R. Preedy (Ed.), Handbook of Anthropometry (pp. 1865-1888). New York: Springer;
2012 Vernetta M, Fernández E, López-Bedoya J, Gómez-Landero A, Oña A. The relations between morphological
profile and body esteem of Andalusian rhythmic gymnastics team. Motricidad. European Journal of
Human Movement, 2011; 26: 77-92 Weiner JS, Lourie JA. Human Biology, A guide to Field methods. International Biological Programme. Edinburgh:
Blackwell Scientific Publications; 1969 World Medical Association. World Medical Association Declaration of Helsinki: Ethical Principles for
Medical
Research
Involving
Human
Subjects,
2002. Available
at
http://www.fda.gov/ohrms/dockets/dockets/06d0331/06D-0331-EC20-Attach-1.pdf;
accessed
on
01.05.2013 © Editorial Committee of Journal of Human Kinetics © Editorial Committee of Journal of Human Kinetics
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https://openalex.org/W2481698804
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http://www.scielo.org.za/pdf/tvg/v56n2-2/06.pdf
|
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Kapasiteitsbou van informele gemeenskapsgebaseerde organisasies in die Suid-Afrikaanse welsynsektor: Die bydrae deur gevestigde maatskaplike diensorganisasies
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Tydskrif vir geesteswetenskappe
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Kapasiteitsbou van informele gemeenskapsgebaseerde
organisasies in die Suid-Afrikaanse welsynsektor: Die
bydrae deur gevestigde maatskaplike diensorganisasies
Capacity-building of informal community-based organisations in the South African welfare
sector: The contribution of established social service organisations Shanie Boshoff
PhD-gegradueerde
Departement Maatskaplike Werk
Stellenbosch Universiteit
E-pos: headoffice@acvv.org.za
Lambert K. Engelbrecht
Departement Maatskaplike Werk
Stellenbosch Universiteit
E-pos: lke@sun.ac.za Shanie Boshoff
PhD-gegradueerde
Departement Maatskaplike Werk
Stellenbosch Universiteit
E-pos: headoffice@acvv.org.za Lambert Engelbrecht
Shanie Boshoff Lambert Engelbrecht Shanie Boshoff Lambert K. Engelbrecht
Departement Maatskaplike Werk
Stellenbosch Universiteit
E-pos: lke@sun.ac.za Shanie Boshoff Lambert Engelbrecht Lambert Engelbrecht Shanie Boshoff is ’n PhD-gegradueerde van die
Departement van Maatskaplike Werk aan die
Universiteit van Stellenbosch. Sy het haar Meesters
graad in Maatskaplike Werk in Maart 2006 aan die
genoemde universiteit behaal. Sy is die hoof-
uitvoerende beampte van die ACVV, die oudste
nasionale welsynsorganisasie in Suid-Afrika wat
uitgebreide ontwikkelings- en beskermingsdienste
aan kinders, hul gesinne en aan ouer persone lewer. Sy het ’n besondere belangstelling in organisasie
ontwikkeling, die ontwikkeling van vrywillger-
besture en die benutting van sterktes in bestuur en
dienslewering. Sy dien op verskeie nasionale en
provinsiale gespreksforums vir maatskaplike diens-
verskaffers. Tans dien sy as visevoorsitter van
NACOSS (National Coalition of Social Services),
’n gespreksforum vir nasionale gevestigde maat
skaplike diensverskaffers in Suid-Afrika, en op die
Uitvoerende Komitee van die Wes-Kaapse Direk
teureforum. Shanie Boshoff is a PhD graduate at the Depart
ment of Social Work at the University of Stellen
bosch. In March 2006 she obtained her MA Degree
in Social Work at the said university. She is the chief
executive officer of the ACVV, the oldest national
welfare organisation in South Africa, rendering
extensive development and protection services to
children, their families and older persons. Her
interests lie especially in the fields of organisational
development, the development of volunteer-
management boards, and the use of strengths in
management and service delivery. She serves on
several national and provincial discussion forums
of social service providers. She is currently serving
as vice-chairperson of NACOSS (National Coalition
of Social Services), a discussion forum of established
social service providers in South Africa, and on the
Executive Committee of the Western Cape Director’s
Forum. Lambert Engelbrecht teaches in the Depart
ment of Social Work at the University of Stellen
bosch. He has extensive experience as a social work
practitioner and community developer in vulnerable
communities. His interests lie in the fields of social
work management and supervision, as well as
strategies for poverty alleviation in social work Lambert Engelbrecht is verbonde aan die
Departement Maatskaplike Werk van die Univer
siteit van Stellenbosch. 592 592 ABSTRACT S
C
Capacity-building of informal community-based organisations in the South African welfare
sector: The contribution by established social service organisations Capacity-building of informal community-based organisations in the South African welfare
sector: The contribution by established social service organisations
Large-scale poverty and increasing needs prevail in South Africa. The National Department of
Social Development has the constitutional mandate to provide sector-specific national leadership
in social development. Despite the Department of Social Development’s intention to restructure the
social service delivery system since 1997 and although established social service organisations, as
well as informal community-based organisations (CBOs), are regarded as being valuable resources
of service delivery to communities at risk, all these organisations are threatened by serious challenges. Established social service organisations that provide and maintain a significant portion of the social
welfare services in South Africa, are at present – despite their efficient management – engulfed in
a grim battle for survival. CBOs which form an integral part of communities at risk are still excluded
from state financing because they do not comply with the basic requirements of management. The
Policy on Financial Awards for Social Service Providers (2012a) of the Department of Social
Development indicates that the State aims to redirect its financing from established social service
organisations to CBOs and to demand that established organisations build the capacity of CBOs in
addition to their own direct service delivery to those at risk. y
An investigation into the capacity-building of CBOs within the South African context is
therefore at the same time both relevant and important. The purpose of this study is to develop
an understanding of the contribution that established social service organisations in South Africa
already make to the capacity-building of CBOs. The investigation not only provides information
on the nature of this capacity-building, but also focuses attention on the successes and many
challenges experienced by established social service organisations in their capacity-building of
CBOs. Qualitative research was chosen as the approach to explore the contributions of established
social service organisations to the capacity-building of CBOs in the social welfare sector and to
establish the link with the social development approach as it is applied in South Africa. At the
same time capacity-building of CBOs by established social service organisations is analysed and
described. Kapasiteitsbou van informele gemeenskapsgebaseerde
organisasies in die Suid-Afrikaanse welsynsektor: Die
bydrae deur gevestigde maatskaplike diensorganisasies
Capacity-building of informal community-based organisations in the South African welfare
sector: The contribution of established social service organisations Hy beskik oor uitgebreide
praktykervaring as maatskaplike werker en gemeen
skapsontwikkelaar in kwesbare gemeenskappe. Sy
belangstellingsvelde is maatskaplikewerkbestuur en
supervisie, sowel as armoedeverligtingstrategieë in Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016
doi.10.17159/2224-7912/2016/v56n2-2a5 593 service delivery. His role as lecturer, his engagement
in research and interaction with the community find
common purpose in his involvement in the
management of non-governmental organisations and
the supervision of social workers. Several of his
books, book chapters, articles and papers on this
subject have been published and presented nationally
and internationally. He is involved in several inter
national research projects on the impact of neolibe
ralism on social welfare services and the manage
ment of social work organisations. service delivery. His role as lecturer, his engagement
in research and interaction with the community find
common purpose in his involvement in the
management of non-governmental organisations and
the supervision of social workers. Several of his
books, book chapters, articles and papers on this
subject have been published and presented nationally
and internationally. He is involved in several inter
national research projects on the impact of neolibe
ralism on social welfare services and the manage
ment of social work organisations. maatskaplikewerk-dienslewering. Hy integreer
akademiese onderrig, sy navorsingsfunksie en
gemeenskapsinteraksie in sy betrokkenheid by die
bestuur van nieregeringsorganisasies en supervisie
van maatskaplike werkers. Verskeie boeke,
boekhoofstukke, artikels en referate oor hierdie
onderwerp is reeds deur hom nasionaal en inter
nasionaal gepubliseer en aangebied. Hy is betrokke
by verskeie internasionale navorsingsprojekte oor
die impak van neoliberalisme op maatskaplike
welsynsdienste en die bestuur van maatskaplikewerk-
organisasies. Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016
doi.10.17159/2224-7912/2016/v56n2-2a5 ABSTRACT An instrumental case study was used as the research strategy by using NACOSS
(National Coalition of Social Services) as a discussion forum of established social service providers
in South Africa. A purposive non-probability sample selection was used to select participants,
Nine NACOSS members indicated in a survey that they are already involved in the capacity-
building of CBOs and agreed to participate in the study. Data was collected on three levels: (a)
Semi-structured interviews were conducted with 9 chief executive officers of 9 established social Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016
doi.10.17159/2224-7912/2016/v56n2-2a5 594 service organisations, (b) 15 social workers who are already building CBOs’ capacities, and (c)
9 representatives of the CBOs whose capacities were being built by the participating service
organisations. service organisations, (b) 15 social workers who are already building CBOs’ capacities, and (c)
9 representatives of the CBOs whose capacities were being built by the participating service
organisations. i g
Based on this study, the following main findings and recommendations are made: •
Capacity-building of CBOs, as it is currently operationalised in developmental social
work by established service organisations, is an inherent part of social development. Therefore, capacity-building of CBOs is rather more a social work intervention than
exclusively one of organisational development and is currently being interpreted and
applied in this way by established service organisations. The focus of capacity-building
of CBOs as a social work intervention is therefore on informal systems or bodies within
communities at risk. •
Capacity-building in practice is far more elementary than what is assumed to be the
case in theory and policy. •
Capacity-building in practice is far more elementary than what is assumed to be the
case in theory and policy. •
CBOs’ urgent need for continuous practice-oriented support from service organisations
is evident, thus confirming the necessity for this kind of support to form an integral part
of the capacity-building process. In this respect established social service organisations
are excellently positioned to make a valuable contribution to the capacity-building of
CBOs. •
Due to the challenges of service delivery, the State should rather view the service
organisations as resources in the restructuring of the South African social service delivery
system than considering the redirection of financing from essential services by established
service organisations to the informal sector. Urgent consideration should be given to
providing state funding for the capacity-building of CBOs. TREFWOORDE: DE:
Kapasiteitsbou, gevestigde maatskaplike diensorganisasies, informele
gemeenskapsgebaseerde organisasies, maatskaplike ontwikkeling,
maatskaplike werk KEY CONCEPTS: TREFWOORDE:
Kapasiteitsbou, gevestigde maatskaplike diensorganisasies, informele
gemeenskapsgebaseerde organisasies, maatskaplike ontwikkeling,
maatskaplike werk ABSTRACT Constructive discussion
between the Department of Social Development and established social service
organisations is crucial for the advancement of an effective social service delivery system. KEY CONCEPTS: Capacity building, established social service organisations, informal
community-based organisations, social development, social work S: Capacity building, established social service organisations, informal
community-based organisations, social development, social work Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016
doi.10.17159/2224-7912/2016/v56n2-2a5 OPSOMMING Ná twintig jaar van demokrasie is Suid-Afrika steeds ’n verdeelde samelewing met grootskaalse
armoede en hoë vlakke van kwesbaarheid. Ontwikkeling van alle sektore van die samelewing word
deur die Regering as oplossing gesien. Deur middel van die Departement Maatskaplike Ontwikkeling
se finansieringsbeleid (Departement van Maatskaplike Ontwikkeling 2012a) plaas die Staat druk
op gevestigde maatskaplike diensorganisasies om tot kapasiteitsbou van informele gemeen
skapsgebaseerde organisasies by te dra. Die doel van hierdie artikel is om begrip te ontwikkel vir
die bydrae van gevestigde maatskaplike diensorganisasies tot die kapasiteitsbou van informele
gemeenskapsgebaseerde organisasies (voortaan in hierdie artikel na verwys as “IGO’s”) ten einde
grondige bevindinge en aanbevelings daarvoor te kan maak. Om hierdie doel te bereik, word die
agtergrond van die onderwerp in meer detail belig, word die navorsingsmetodologie wat gevolg
is, verduidelik en word die bevindinge aangebied deur middel van ʼn geïntegreerde sintese van
deelnemers se beskrywings van gevestigde organisasies en IGO’s, hulle beskouings van suksesse
behaal en hulle reflektering van verdere uitdagings in kapasiteitsboupogings. Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016
doi.10.17159/2224-7912/2016/v56n2-2a5 595 INLEIDING Maatskaplike welsynsdienste aan kwesbare mense in Suid-Afrika word gelewer deur die
regeringsektor sowel as die niewinsgerigte nieregeringsektor (Departement van Maatskaplike
Ontwikkeling 2012a). Daar is egter twee hoofkategorieë van niewinsgerigte maatskaplike diens
verskaffers wat in die burgerlike samelewing bestaan, naamlik gevestigde of formele maatskaplike
diensorganisasies (in die spreektaal dikwels na verwys as “welsynsorganisasies”) en IGO’s
(Departement van Maatskaplike Ontwikkeling 2006; Patel 2005). Beide van hierdie groeperinge
van niewinsgerigte maatskaplike diensverskaffers word as waardevolle hulpbronne vir kwesbare
mense beskou en kan ’n besondere bydrae tot maatskaplike ontwikkeling lewer, maar beide
organisasiegroeperinge ervaar ernstige uitdagings ten opsigte van volhoubare dienslewering. Desondanks blyk dit dat die Departement Maatskaplike Ontwikkeling (2012a) aanvoer dat
gevestigde maatskaplike diensorganisasies ʼn addisionele verantwoordelikheid het om die kapasiteit
van IGO’s te bou ten einde vir voortgesette staatsfinansiering te kan kwalifiseer. Geen empiriese
studie is egter tans beskikbaar oor die werklike bydrae wat gevestigde maatskaplike diensorgani
sasie tot die kapasiteitsbou van IGO’s lewer nie. AGTERGROND Naas IGO’s is gevestigde maatskaplike diensorganisasies wat nieregeringsorganisasies is, een
van die grootste niewinsgerigte maatskaplike diensverskaffers in Suid-Afrika wat beduidende
uitgebreide maatskaplike dienste aan kwesbare individue, gesinne en gemeenskappe lewer
(Departement van Maatskaplike Ontwikkeling 2012a, 2012b; Ministerie van Welsyn en
Bevolkingsontwikkeling 1997; Patel 2005). Die Tesourie Uitgawe Oorsig-dokument vir 2005/2006
tot 2011/2012 toon dat niewinsgerigte organisasies meer as 98% van die maatskaplike welsyns
fasiliteite in Suid-Afrika bedryf en aan meer as 71% van maatskaplike verbruikers dienste lewer
(Departement van Maatskaplike Ontwikkeling 2012b). Oorhoofs onderskei gevestigde
welsynsorganisasies hulself van IGO’s ten opsigte van die volgende aspekte: hulle het professionele
personeel in diens waarvan maatskaplike werkers ’n beduidende rol speel; beskik oor gevestigde
organisasiestrukture; volg bepaalde skriftelike beleide en praktyke; beskik oor ’n eie organisasiekul
tuur; streef bepaalde doelwitte van die organisasie na en ontvang staatsubsidie van die Departement
van Maatskaplike Ontwikkeling (Habib & Kotzé 2002; Hanson 1995; Patel1998; Republiek van
Suid-Afrika 1997; Swilling & Russel 2001). Hierdie eienskappe en gestruktureerdheid bied juis
aan gevestigde welsynsorganisasies of maatskaplike diensorganisasies die vermoë om aan die
vereistes vir die verkryging van staatsfinansiering te kan voldoen. Tog ervaar gevestigde
maatskaplike diensorganisasies – ten spyte van effektiewe bestuur – tans ’n bestaanskrisis om as
maatskaplike diensverskaffers finansieel te oorleef (Davids 2012; Swart 2012; NACOSS 2012a;
2013; NAWONGO 2011). IGO’s is in teenstelling met gevestigde maatskaplike diensorganisasies slegs in dié mate
georganiseerd dat hulle integraal deel van kwesbare gemeenskappe vorm, maar tog hulself in die
gemeenskap as maatskaplike entiteite onderskei wat sekere dienste aan armes en kwesbares lewer. Hierdie dienste word op ’n vrywillige basis gelewer (Govender 2001; Molefe 1996; Patel 1998;
2005; Republiek van Suid-Afrika 1997; Swilling & Russel 2001). Alhoewel IGO’s soms as projekte
van gevestigde maatskaplike diensorganisasies ontstaan, kom daar ’n punt wanneer hul uit vrye
keuse óf amptelik affilieer by die diensorganisasie, óf kies om as onafhanklike entiteite verder te
funksioneer. Ten spyte van die Departement van Maatskaplike Ontwikkeling se fokus om veral
IGO’s te ondersteun in die verkryging van toegang tot staatsfinansiering, is die IGO’s tans steeds Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016
doi.10.17159/2224-7912/2016/v56n2-2a5 596 grootliks uitgesluit van staatsfinansiering omdat hulle nie aan die nodige basiese staatsvereistes
voldoen vir die kwalifisering van staatsfinansiering nie (Departement van Maatskaplike
Ontwikkeling 2012a; 2012b; Ministerie van Welsyn en Bevolkingsontwikkeling 1997; Patel 2005). g
;
;
y
g
g
;
)
Die Staat se beskouing van niewinsgerigte maatskaplike diensverskaffers blyk merendeels
polities-georiënteerd te wees. AGTERGROND Gevestigde maatskaplike diensorganisasies word beskou as daardie
organisasies wat juis in staat is om uitgebreide dienste te lewer, omdat hulle bevoordeel is deur
die staatsondersteuning wat hulle voor 1994 in die apartheidsjare ontvang het (Departement van
Maatskaplike Ontwikkeling 2012a; 2012b). Daarteenoor word IGO’s deur die Staat beskou as
daardie organisasies wat tot 1994 uitgesluit was van staatsfinansiering, maar ’n onontbeerlike
bydrae in die apartheidsjare gelewer het tot die aanspreek van die direkte nood van kwesbare
gemeenskappe (Govender 2001; Ministerie van Welsyn en Bevolkingsontwikkeling 1997; Patel
2005; Swilling & Russel 2001). Die Departement se mees resente Beleid op Finansiële Toekennings
aan Maatskaplike Diensverskaffers (Departement Maatskaplike Ontwikkeling 2012a) (voortaan
in hierdie artikel slegs na verwys as “Finansieringsbeleid”) voer egter die onderskeid tussen
gevestigde maatskaplike diensorganisasies en IGO’s verder en beklemtoon die verantwoordelikheid
van gevestigde maatskaplike diensorganisasies om die kapasiteit van IGO’s te bou sodat hulle vir
voortgesette staatsfinansiering kan kwalifiseer. In teenstelling met die Suid-Afrikaanse Nasionale
Ontwikkelingsplan (National Planning Commission 2011) se voorstel dat befondsing aan
gevestigde maatskaplike diensverskaffers verhoog word, dui die Departement van Maatskaplike
Ontwikkeling se praktyke en Finansieringsbeleid daarop dat die Staat inderwaarheid ten doel het
om hul finansiering van die gevestigde diensverskaffers na IGO’s te herlei. Alhoewel hierdie
herleiding nog nie plaasgevind het nie, sal dit ernstige gevolge vir maatskaplike gevestigde
diensorganisasies inhou wat kan lei tot die ineenstorting van dienslewering aan kwesbares deur
die gevestigde maatskaplike diensorganisasies. g
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Die Staat se beskouing van diensverskaffers, sy siening oor die herstrukturering van die
maatskaplike diensleweringsisteem en gepaardgaande finansieringsbeleid spruit voort uit die Staat
se benadering tot maatskaplike dienslewering. Die Witskrif vir Welsyn (Ministerie vir Welsyn en
Bevolkingsontwikkeling 1997) stel maatskaplike ontwikkeling as die verkose benadering vanaf
1997. Hierdie maatskaplike ontwikkelingsgerigte benadering is gefundeer in reaksie op die land
se vorige apartheidsbestel en fokus onder andere op toeganklikheid van maatskaplike
welsynsdienste vir spesifiek voorheen benadeelde en kwesbare mense (Departement van
Maatskaplike Ontwikkeling 2005, 2012a; Gray 2006; Ministerie van Welsyn en Bevolkings
ontwikkeling 1997; Lombard 2011; Midgley 1997; Patel 2005). Deur hierdie beleidsraamwerk
het die Departement van Maatskaplike Ontwikkeling sy benadering tot maatskaplike dienslewering
van ’n maatskaplike behandelingsmodel na ’n ontwikkelingsgerigte diensleweringsmodel verskuif
en klem geplaas op die daadwerklike herstrukturering van die bestaande maatskaplike
diensleweringsisteem (Departement van Maatskaplike Ontwikkeling 2006; Patel 2005; Ministerie
vir Welsyn en Bevolkingsontwikkeling 1997). AGTERGROND Deur die Wet op Organisasies Sonder Winsoogmerk (Republiek van Suid-Afrika, 1997) poog
die Staat om die minimum vereistes te stel waaraan organisasies moet voldoen om finansiering
te bekom. Registrasie in terme van hierdie Wet sal dus as bewys aan die Staat, ander befondsers
en die publiek dien dat die betrokke organisasies maatskaplike welsynsdienste verantwoordelik
en verantwoordbaar bestuur (Republiek van Suid-Afrika 1997). Die Staat het in die vooruitsig
gestel dat dié Wet die meganisme aan IGO’s sou bied om toegang te verkry tot staatsfinansiering,
wat weer sou lei tot groter volhoubaarheid van IGO’s. Ten spyte van insette deur die Departement
van Maatskaplike Ontwikkeling om die kapasiteit van IGO’s sodanig te ontwikkel dat hulle
kwalifiseer vir staatsfinansiering, erken die Departement van Maatskaplike Ontwikkeling onom Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016
doi.10.17159/2224-7912/2016/v56n2-2a5 597 wonde dat, alhoewel IGO’s 53% van die beraamde totaal van niewinsgerigte nieregeringsorga
nisasies in die Suid-Afrikaanse welsynsektor uitmaak (Swilling & Russel 2001), hierdie
organisasies steeds nie in status gelyk is aan gevestigde maatskaplike diensorganisasies nie en
steeds uitgesluit word van staatsfinansiering juis omdat hulle nie aan die nodige staatsvereistes
voldoen nie (Departement van Maatskaplike Ontwikkeling 2012b). Dit is egter onduidelik waarom
daar nog nie genoegsame vordering gemaak is om IGO’s in staat te stel om aan staatsvereistes
vir staatsfinansiering te voldoen nie. Een van die redes blyk te wees dat IGO’s dikwels nie oor
die nodige finansiële stelsels beskik om staatsfinansiering op ’n verantwoordbare en voorgeskrewe
wyse te hanteer en daarvan boek te hou nie (Departement van Maatskaplike Ontwikkeling 2012a). Hieruit word afgelei dat die kapasiteit van IGO’s nie genoegsaam ontwikkel of versterk word
deur die Wet op Organisasies Sonder Winsoogmerk (Republiek van Suid-Afrika 1997) nie. Meer
deurlopende plaaslike aksies en ondersteuning blyk dus nodig te wees om die kapasiteit van IGO’s
te versterk en dit is juis hiérin waar gevestigde maatskaplike diensorganisasies, wat reeds uitge
breide dienste op grondvlak lewer, ’n bydrae kan lewer. ’n Opname by ’n gespreksforum van nasionale gevestigde maatskaplike diensorganisasies
in 2012 (NACOSS 2012b) het aangedui dat daar reeds ’n beduidende aantal nasionale organisasies
is wat vrywillig die kapasiteit van IGO’s bou. Uit die opname blyk dit duidelik dat kapasiteitsbou
van IGO’s as een van die vorme van maatskaplikewerk-intervensie deur gevestigde maatskaplike
diensorganisasies toegepas word en dat die fokus van maatskaplike ontwikkeling in kapasiteitsbou
op organisasievlak of die vlak van die institusionele instelling lê. AGTERGROND Gevestigde maatskaplike
diensorganisasies het die ervaring ten opsigte van die nakoming van staatsvereistes en
staatsfinansiering. Hulle lewer saam met IGO’s dienste in kwesbare gemeenskappe, wat hulle
gunstig plaas om in bepaalde konteks praktykgerigte ondersteuning aan IGO’s te bied. Teoretiese en empiriese studies oor die bydrae van nasionale gevestigde maatskaplike
diensorganisasies wat reeds die kapasiteit van IGO’s bou, is egter beperk. Studies wat wél beskikbaar
is, soos dié deur Symes (2007) en Yachakaschi (1995), fokus op die kapasiteitsbou van IGO’s deur
spesialiteitsorganisasies en verwys nie na gevestigde maatskaplike diensorganisasies waarvan die
kernbesigheid direkte dienslewering aan kwesbare individue, groepe en gemeenskappe behels nie. Ten spyte daarvan dat die kapasiteitsbou van IGO’s deur gevestigde maatskaplike diensorganisasies
as vereiste in die Departement van Maatskaplike Ontwikkeling se Finansieringsbeleid (Departement
van Maatskaplike Ontwikkeling 2012a) gestel word, is daar tans nie inligting beskikbaar oor die
bydrae wat gevestigde maatskaplike diensorganisasies alrééds lewer tot die kapasiteitsbou van
IGO’s of die uitdagings wat hulle ten opsigte daarvan ervaar nie. Daar is ook nie inligting beskikbaar
oor die wyse waarop die kapasiteit van IGO’s binne die Suid-Afrikaanse konteks en binne die
maatskaplike ontwikkelingsgerigte benadering toegepas word nie. Empiriese navorsing is dus nodig om die bydrae van kapasiteitsbou van IGO’s deur gevestigde
maatskaplike diensorganisasies binne die konteks van die Suid-Afrikaanse maatskaplike
ontwikkelingsgerigte benadering te verken. Sodanige verkenning kan ’n waardevolle perspektief
bied op die rol en bydrae deur die gevestigde maatskaplike diensorganisasies in die kapasiteitsbou
van IGO’s. Navorsingsresultate kan lei tot die ontwikkeling van ’n teoretiese raamwerk vir
kapasiteitsbou van Suid-Afrikaanse IGO’s deur gevestigde maatskaplike diensorganisasies wat
verder in die praktyk getoets kan word. NAVORSINGSMETODOLOGIE In hierdie studie is ’n kwalitatiewe navorsingsbenadering gebruik om kapasiteitsbou van IGO’s
deur gevestigde maatskaplike diensorganisasies as maatskaplike beginsel te verstaan en te
ondersoek, aangesien min inligting daaroor bekend is (Bless, Higson-Smith & Kagee 2006; Fouché Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016
doi.10.17159/2224-7912/2016/v56n2-2a5 598 & Delport 2014; Mouton 2006). ’n Instrumentele gevallestudie is as navorsingstrategie gekies
omdat dit fokus op die bestudering van kapasiteitsbou van IGO’s as fenomeen en hoe gevestigde
diensorganisasies hierdie fenomeen vanuit ’n maatskaplike ontwikkelingsgerigte benadering
ervaar (Creswell 2013; Fouché & Delport 2014; Fouché & Schurink 2014). Beide beskrywende
en verkennende navorsingsontwerpe is in kombinasie gebruik omdat inligting oor die
navorsingsonderwerp beperk is en ’n beduidende insig in ’n bepaalde fenomeen sal bied. Beskrywende vrae verwys na vrae soos “wat is die verhouding tussen gevestigde maatskaplike
diensorganisasies en IGO’s?” en verkennende vrae na vrae soos “watter uitdagings word aan
gevestigde organisasies vir kapasiteitsbou van IGO’s gestel?”. Die 19 gevestigde diensorganisasies
wat lede van NACOSS is (“National Coalition of Social Services”, ’n gespreksliggaam van
gevestigde maatskaplike diensorganisasies in Suid-Afrika, wat as sodanig deur die Departement
van Maatskaplike Ontwikkeling erken word), vorm die universum van die studie (Strydom &
Delport 2014; NACOSS 2012b). Daar sal voortaan in hierdie artikel slegs verwys word na
“NACOSS”. Deur middel van ’n opname is bepaal dat 11 van die 19 lede (organisasies) reeds
betrokke is by die kapasiteitsbou van IGO’s en word sodanige lede as die populasie in hierdie
studie geneem (Strydom & Delport 2014; Tutty, Rothery & Grinnell 1996). ’n Doelbewuste
steekproef (Strydom & Delport 2014; Monette, Sullivan & De Jongh 2008; Oliver 2010) is
uitgevoer en het bestaan uit nege NACOSS-lede (diensorganisasies) wat aangedui het dat hulle
betrokke is by kapasiteitsbou van IGO’s en bereid is om aan die studie deel te neem. ’n Semi-
gestruktureerde onderhoudskedule is as ’n navorsingsinstrument benut sodat ’n meer omvattende
beskrywing van deelnemers se persepsies en gewaarwordinge oor die kapasiteitsbou van IGO’s
verkry kon word (Bless et al. 2006; Greeff 2014; Gochros 2005; Tutty et al. 1996). 1
Bless et al. (2006); Cherry (2000); Darlington & Scott (2002); Goodman (2001); Grinnell & Unrau
(1995); Monette et al. (2008); Strydom (2014); Williams, Tutty & Grinnell (1995). NAVORSINGSMETODOLOGIE y
(
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Data-insameling het op drie vlakke deur middel van semi-gestruktureerde onderhoude
plaasgevind, naamlik met hoofuitvoerende beamptes en maatskaplike werkers van gevestigde
diensorganisasies en ook met verteenwoordigers van IGO’s wie se kapasiteit hulle bou: (a)
Onderhoude is met nege hoofuitvoerende beamptes gevoer (een van elke deelnemende organisasie);
(b) vyftien onderhoude is met maatskaplike werkers gevoer (verteenwoordigend van elkeen van
die deelnemende organisasies wat regstreeks by kapasiteitsbou van IGO’s betrokke is); en (c) nege
onderhoude is met verteenwoordigers van IGO’s gevoer (waarvan die kapasiteit deur die nege
deelnemende organisasies gebou word). Die onderhoude het elk tussen 60 en 75 minute geduur. g
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In hierdie studie het die navorser geloofwaardigheid, oordraagbaarheid en afhanklikheid as
strategieë vir ʼn geldigheidsverklaring gebruik om die proses van data-insameling en data-analise
se geldigheid te staaf (Barusch, Gringeri & George 2011; Creswell 2013; Strydom 2014;
Whittemore, Chase & Mandle 2001). Die volgende etiese oorwegings het in die studie gegeld en
word kortliks genoem:1 Etiese risiko’s is geëvalueer; etiese klaring is deur die Universiteit van
Stellenbosch toegeken; die inligting verkry, is professioneel en vertroulik hanteer ingevolge die
SARMD se Etiese Kode; ingeligte toestemming is van elke deelnemer aan die studie verkry; die
gevolge van deelname aan die studie is deeglik met deelnemers deurgewerk; die skending van
privaatheid is teengewerk; en daar is gelet op deelnemers se behoefte aan ontlonting. 2
Vergelyk Departement van Maatskaplike Ontwikkeling (2006), (2012a) en (2012b); Hanson (1995);
Hepworth, Rooney, Dewberry Rooney & Strom-Gottfried (2013); Ministerie vir Welsyn en
Bevolkingsontwikkeling (1997); Republiek van Suid-Afrika (1997); Zastrow (2013). Omskrywing van gevestigde maatskaplike diensorganisasies Uit die ondersoek is dit duidelik dat die organisatoriese kenmerke wat gevestigde diensorganisasies
gestruktureerd maak, inherent deel van die funksionering van sodanige organisasies is. Hoof
uitvoerende beamptes wat op ’n beleidsvlak betrokke is by gevestigde diensorganisasies, sowel
as maatskaplike werkers wat direkte dienste lewer, was tydens die studie in staat om die kenmerke
wat diensorganisasies as gevestigde entiteite kategoriseer, te identifiseer en te omskryf.2 Dit
beklemtoon dat die kenmerke wat gevestigde diensorganisasies van informele diensverskaffers
onderskei, integraal deel van die organisasies se funksionering is en dat dit op verskeie vlakke in
die organisasies na vore tree. Die aard van dienste wat gelewer word, beïnvloed die fokus en beskikbaarheid van gevestigde
organisasies se interne kapasiteit vir kapasiteitsbou van IGO’s. Die effek van veral die
implementering van die Kinderwet (2005) en die beskikbaarheid van maatskaplikewerk-personeel,
kan kapasiteitsbou van IGO’s deur diensorganisasies strem. Buiten die verwysing na die gevestigde maatskaplike diensorganisasies as “nasionale rade”
(Departement van Maatskaplike Ontwikkeling 2006, 2012a), vind geen verdere groeperinge binne
hierdie kategorie plaas nie. Binne die lidmaatskap van NACOSS (wat as verteenwoordigend van
nasionale diensleweringsorganisasies beskou word) blyk dit uit die ondersoek dat die aard van
dienslewering twee groepe in hierdie kategorie onderskei. Beide hierdie groeperings vorm deel
van hierdie studie: •
Die eerste groep verwys na gevestigde maatskaplike diensorganisasies wat uitgebreide
generiese dienste aan kinders, gesinne en ouer persone insluit. •
Die eerste groep verwys na gevestigde maatskaplike diensorganisasies wat uitgebreide
generiese dienste aan kinders, gesinne en ouer persone insluit. •
Die tweede groep verwys na gevestigde maatskaplike diensorganisasies wat spesialiteits
dienste lewer en waarvan die omvang van werk dikwels meer beperk is as voorgaande
groep juis vanweë die fokus op spesialiteitsvelde. Ten spyte van hul gevestigde aard lewer gevestigde maatskaplike diensorganisasies direk dienste
in kwesbare gemeenskappe en is hulle oorwegend deur middel van strukture teenwoordig en
toeganklik vir sodanige gemeenskappe. Hulle georganiseerdheid skep juis kanale waardeur ’n
wye geografiese omvang van dienste gelewer kan word. Dit weerlê ’n moontlike persepsie dat
gevestigde maatskaplike diensverskaffers nie direk self in gemeenskappe dienste lewer nie en dat
IGO’s beter as gevestigde maatskaplike diensorganisasies geleë is om dienste aan kwesbare
gemeenskappe te lewer. Daar bestaan nie tans statistiese bewys oor die hoeveelheid IGO’s waarvan die kapasiteit
deur gevestigde diensorganisasies gebou word nie – iets wat onderhandelinge met die Staat en
ander befondsers kan bemoeilik. BEVINDINGE Die bevindinge van hierdie studie word as ʼn sintese van die narratiewe van hoofuitvoerende
beamptes van gevestigde organisasies, maatskaplike werkers betrokke by kapasiteitsbou van
IGO’s en verteenwoordigers van IGO’s aangebied. Aangesien ʼn instrumentele gevallestudie, Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016
doi.10.17159/2224-7912/2016/v56n2-2a5 Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2
doi.10.17159/2224-7912/2016/v56n2-2a5 599 gebaseer op verskillende deelnemers in verskillende organisasies se belewenisse, as strategie in
hierdie navorsing benut is, word die geïntegreerde aanbieding van bevindinge meer waardevol
geag as die blote liniêre reflektering van deelnemers se narratiewe. Die verwerkte narratiewe van
deelnemers word vervolgens in terme van vier hooftemas aangebied. Omskrywing van IGO’s Alhoewel IGO’s as maatskaplike diensverskaffers voldoen aan die oorhoofse onderskeidende
kenmerke wat aan hierdie groep diensverskaffers toegeskryf word, verskil hul grootliks van die
gevestigde diensorganisasies ten opsigte van die aard van hul vrywilligergrondslag, ongestruk
tureerdheid en ongeorganiseerdheid in terme van wetlike vereistes en omvang van dienslewering
(vergelyk Departement van Maatskaplike Ontwikkeling 2006, 2012a). Wat wel ooreenstem, is
dat beide hierdie tipe diensverskaffers die nood van kwesbares aanspreek en dat IGO’s meewerk
om die direkte nood (veral bestaansnood) van gemeenskapslede aan te spreek (vergelyk Ministerie
van Welsyn en Bevolkingsontwikkeling 1997). Met inagneming van die wye omvang van IGO’s in die maatskaplike welsynsektor in Suid-
Afrika in vergelyking met die beperkte hoeveelheid deelnemende IGO’s aan hierdie studie, is daar
bevind dat daar twee groeperinge van IGO’s in hierdie studie na vore tree. Die oorgrote meerderheid
van IGO’s het betrekking op informele groeperinge wat ’n beperkte diens in omvang aan kwesbare
gemeenskappe lewer, wat waarskynlik in baie opsigte nie in omvang en grootte sal groei nie en
sal voortgaan om informeel te funksioneer. ’n Klein groepering van IGO’s het sodanig gegroei en
ontwikkel dat ’n meer gesofistikeerde diens as die deursnee-IGO aangebied word. Hier word
spesifiek verwys na pleegsorghuise, waar IGO’s sodanig ontwikkel het dat hulle maatskaplike
werkers in diens neem wat onder toesig van die gevestigde maatskaplike diensorganisasies
funksioneer. As gevolg van die basiese vlak van organisasiestruktuur wat hierdie IGO’s bemeester
en hul dienste uitgebrei het, beweeg hierdie IGO’s inderwaarheid uit die basiese informele sektor. Aangesien die oorgrote meerderheid van IGO’s waarvan die gevestigde maatskaplike diens
verskaffers kapasiteitsboudienste lewer, op die basiese vlak van ontwikkeling lê, word daar ver
volgens uitsluitlik verwys na die informele groepering van gemeenskapsgebaseerde organisasies,
tensy dit spesifiek anders aangedui word. Tans word geen onderskeid getref in die Staat se beleidstukke tussen groeperinge van IGO’s
nie en is die onderskeid tussen IGO’s en opkomende organisasies vaag (vergelyk Departement
van Maatskaplike Ontwikkeling 2012a). Daar is dus geen onderskeid tussen IGO’s wat informeel
funksioneer en dáárdie IGO’s wat effe meer gestruktureerd funksioneer nie. ’n Onderskeid tussen
verskillende groeperinge van IGO’s en ’n beter omskrywing van wat die Staat “opkomende
organisasies” noem, sou daartoe kon lei dat die Staat sou kon oorweeg om wél sekere tipe meer
gestruktureerde funksionerende IGO’s toegang tot staatsfinansiering te verleen, aangesien sodanige
tipe IGO’s tóg bewys sou kon lewer van ’n mate van verantwoordbare bestuur van sulke organi
sasies aan die publiek en befondsers. Omskrywing van gevestigde maatskaplike diensorganisasies Daar is egter genoegsame bewys dat ’n beduidende hoeveelheid
nasionale gevestigde diensorganisasies in ’n mindere of ’n meerdere mate betrokke is by
kapasiteitsbou van IGO’s en ’n waardevolle rol speel in die versterking van IGO’s se kapasiteit. Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016
doi.10.17159/2224-7912/2016/v56n2-2a5 600 Die beskikbare inligting kan dus as vertrekpunt dien en as bewyse aangevoer word in
onderhandeling met die Staat. Die beskikbare inligting kan dus as vertrekpunt dien en as bewyse aangevoer word in
onderhandeling met die Staat. Omskrywing van IGO’s IGO’s kom tot stand in kwesbare gemeenskappe om die nood van die gemeenskap en/of
individue wat betrokke is aan te spreek (Departement van Maatskaplike Ontwikkeling 2012a;
Ministerie van Welsyn en Bevolkingsontwikkeling 1997; Patel 1998). Die ligging van IGO’s is
dus nie bepalend in die behoefte aan kapasiteitsbou nie. Selfs IGO’s in informele gemeenskappe
aangrensend geleë tot groter stede, se kapasiteit is só beperk dat hulle sukkel om te bestaan,
alhoewel die hulpbronne geografies veronderstel is om nader aan die IGO’s te wees as op die
platteland. Die kapasiteitsbou van sommige IGO’s mag moontlik wél vanweë geografiese
afgesonderdheid en groter ontoeganklikheid tot hulpbronne benadeel word. IGO’s is in kwesbare gemeenskappe geleë. Vrywilligers betrokke by IGO’s is ook afkomstig
uit dieselfde kwesbare gemeenskappe (vergelyk Departement van Maatskaplike Ontwikkeling Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016
doi.10.17159/2224-7912/2016/v56n2-2a5 601 2006; Patel 1998). Armoede en bestaansbeveiliging noodsaak vrywilligers om nie gratis vanuit
’n blote diensmotief dienste te lewer nie, maar om geleenthede te ontgin wat tot armoedeverligting
en inkomstegenerering kan lei. Deelnemers se verwysing na spesifiek drie groepe uitdagings as
deel van die omskrywing van IGO’s, te wete beperkte kapasiteit, wisseling van bestuurslede en
die beduidende rol wat finansies speel, wys daarop dat hierdie genoemde aspekte as kenmerkend
van IGO’s beskou kan word. Kerke speel ’n belangrike ondersteuningsrol in die identifisering
van nood en die betrokke raak by gemeenskapslede in die aanpak daarvan – iets wat die
volhoubaarheid van IGO’s verhoog. Hierdie tipe ondersteuning blyk meer deurlopend van aard
te wees, omdat die kerke ook in die gemeenskappe geleë is waarin die IGO’s hulle bevind. Personeelbestuur verg bepaalde kennis en vaardighede. Aangesien weinige IGO’s oor
personeel beskik of sodanig saamgestel is, sal die ontwikkeling van dergelike bestuursvaardighede
tydens kapasiteitsbou van IGO’s ’n intensiewe uitdaging wees (vergelyk Departement van
Maatskaplike Ontwikkeling 2006; Ministerie vir Welsyn en Bevolkingsontwikkeling 1997). Wanneer die besture van IGO’s as werkgewers begin optree en ’n duideliker rolverdeling tussen
werkgewers en werknemers plaasvind, tree konflik dikwels in as gevolg van beperkte kapasiteit
om die verskillende rolle te vervul en die vreemdheid van die rolverdeling vir individue wat vroeër
almal op dieselfde vlak gewerk het. Die handhawing van gesagsverhoudinge tussen werkgewers
en kernpersoneel blyk ’n noodsaaklike fokusarea in kapasiteitsbou te wees. Benewens personeelbestuur blyk IGO’s se beperkte finansiële vermoëns oor die algemeen
’n beduidende kenmerk van IGO’s te wees (vergelyk Departement van Maatskaplike Ontwikkeling
2012a). Omskrywing van IGO’s Die gebrek aan finansies word nie pertinent deur deelnemers in die omskrywing van
IGO’s vermeld nie, maar wél beperkte finansiële vaardighede en stelsels. Dit gaan dus nie
noodwendig bloot oor die beperkte hoeveelheid fondse wat IGO’s het nie, maar pertinent oor
IGO’s se onvermoë om gesonde finansiële beginsels te handhaaf. Wanneer finansiering wel bekom
word, tree konflik in oor die nakoming van finansiële vereistes, waar daar dan dikwels op die
betrokke maatskaplike diensorganisasie gefokus word. Daar is nie by deelnemers ’n oortuiging dat alle IGO’s ’n georganiseerde struktuur wíl vorm
nie. Baie IGO’s blyk onkundig te wees oor die implikasies en inhoud van die stigting van ’n
organisasiestruktuur en die nakoming van staatsvereistes om aan staatsfinansiering te voldoen. Deelnemers vermeld ook hulle vertwyfeling of IGO’s oorwegend ’n behoefte het om gestruktureer
te word. Te oordeel aan die belewenis van deelnemers van gevestigde diensorganisasies is IGO’s
óf positief ingestel om meer gestruktureerd te werk, óf wil IGO’s in wese ongestruktureerd bly
in terme van wetlike vereistes en voortgaan om informeel ’n diens aan die gemeenskap te lewer. Daar behoort dus nie sonder meer aanvaar te word dat alle IGO’s oor die begeerte of vermoëns
beskik om aan staatsvereistes vir die verkryging van staatsfinansiering te voldoen nie. Suksesse behaal in kapasiteitsbou van IGO’s deur gevestigde maatskaplike diensverskaffers Gevestigde diensorganisasies se sienings en ervarings van suksesse wat in kapasiteitsbou van
IGO’s behaal is, gee ’n aanduiding van die ingesteldheid waarmee diensverskaffers kapasiteit van
IGO’s bou en tot welke mate hierdie ingesteldheid die maatskaplike ontwikkelingsgerigte
benadering weerspieël. Dit toon ook aan op welke wyse die suksesse aansluit by die geformuleerde
doel van kapasiteitsbou van IGO’s soos gestel deur gevestigde maatskaplike diensorganisasies. Die beginsel van volhoubaarheid van IGO’s blyk relatief te wees. Dit word grotendeels deur
die IGO’s se ingesteldheid bepaal en wanneer fondse vir oorlewing as genoegsaam geag word. Daaruit word afgelei dat IGO’s dikwels oorwegend in wese klein wil bly en slegs beperkte dienste
wil lewer. Hulle wil wel genoegsame fondse hê om daardie dienste te kan lewer. Waarskynlik Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016
doi.10.17159/2224-7912/2016/v56n2-2a5 602 veroorsaak eksterne finansiering spanning by IGO’s, omdat dit gepaardgaan met omvangryke
vereistes van die befondsers of finansierders wat addisionele sisteme en vaardighede van IGO’s
vereis as waaraan hulle gewoond is. g
Eie aan ʼn maatskaplike ontwikkelingsgerigte benadering, fokus maatskaplike werkers betrokke
by kapasiteitsbou van IGO’s op insigontwikkeling en die bereiking van individuele vaardighede3
en dui die deelnemers sodanige ontwikkeling as suksesse in kapasiteitsbou aan. Dit dien as verdere
bewys dat maatskaplike werkers wél in die praktyk die maatskaplike ontwikkelingsgerigte
benadering volg. Die meting van sukses aan die mate waartoe dienslewering aan kwesbare gemeenskappe
verbeter, bevestig dat gevestigde maatskaplike diensorganisasies die kapasiteit van IGO’s as
gemeenskapsbronne bou, eerder as om die belange van hul eie organisasies daardeur te wil uitbrei. Dit bevestig ook dat IGO’s ’n noodsaaklike rol in gemeenskappe speel om nood aan te durf,
ongeag die beperkte omvang daarvan. g
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Daar is ’n oënskynlike teenstrydigheid tussen enersyds die Staat se beskouing dat NPO-
registrasie as suksesbereiking in kapasiteitsbou van IGO’s gereken word (vergelyk Departement
van Maatskaplike Ontwikkeling 2012c), en andersyds gevestigde diensorganisasies se siening dat
NPO-registrasie bloot één van die stappe en suksesse in organisasie-ontwikkeling is. Daaruit word
afgelei dat NPO-registrasie wel ’n belangrike mylpaal in kapasiteitsbou is, maar dat dit nie
noodwendig as die bereiking van selfstandige funksionering van IGO’s beskou kan word nie. Een
van die groot redes hiervoor is dat NPO-registrasie oorhoofs bloot die samestelling van die bestuur
en konstitusie weergee, maar nie die bestuursvaardighede van die organisasie evalueer nie. 3
Vergelyk Homfeldt & Reutlinger (2012); Lombard (2011); Lombard, Kemp, Viljoen-Toet & Booyzen
(2012); Midgley (2014); Patel & Hochfeld (2008), (2012); Patel & Noyoo (2005). Suksesse behaal in kapasiteitsbou van IGO’s deur gevestigde maatskaplike diensverskaffers Dit
mag moontlik in ’n beperkte mate wel deur middel van die jaarverslag (wat jaarliks by die NPO-
Direktoraat ingedien moet word) geëvalueer word. Dit blyk dat die indien van die jaarverslae nie
noodwendig nagegaan word deur die NPO-Direktoraat om die aard en kategorie van vaardighede
vas te stel en NPO’s of niewinsgerigte organisasies (veral IGO’s) verder deur kommentaar daaroor
leiding te gee nie. Die afleiding oor die ontoereikendheid van NPO-registrasie as bewys van
verantwoordbare bestuur van ’n niewinsgerigte organisasie stem ooreen met die Departement van
Maatskaplike Ontwikkeling (2006) se eie kritiek op die Wet op Organisasies sonder Winsoogmerk
(1997), te wete dat registrasie as NPOs nie noodwendig lei tot selfstandige funksionering van die
meer informele organisasies nie, wat dié departement onder meer aan gebrekkige bestuurskapasiteit
toeskryf. Uitdagings vir gevestigde diensorganisasies se kapasiteitsbou van IGO’s In teenstelling met bestuursteorie en organisasie-ontwikkeling in die algemeen, word gebrekkige
organisasiestruktuur en menslike hulpbronne (veral vrywilligers as bestuurslede en personeel)
van IGO’s uitgesonder as die grootste uitdaging in effektiewe kapasiteitsbou van IGO’s deur
gevestigde diensorganisasies. Dit blyk dus dat die praktykervaring daarop dui dat daar ’n groepering
van IGO’s is waar die vaardighede en beskikbaarheid van persone wat op die besture dien op só
’n ontwikkelingsvlak lê dat kapasiteitsbou meer op hulpverlening en ontwikkeling van individue
neerkom, of op ’n meer vereenvoudigde vlak van organisasie-ontwikkeling of organisasiebou lê. Daaruit word afgelei dat IGO’s minder ontwikkeld is as wat bestuursteorie en organisasie-
ontwikkeling (vergelyk Yachakaschi 1995) oor die algemeen as vertrekpunt neem. Die vereistes
wat onder andere deur die Wet op Organisasies sonder Winsoogmerk (1997) en ander staatsbeleide Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016
doi.10.17159/2224-7912/2016/v56n2-2a5 603 gestel word, hou ook waarskynlik nie tred met die uitdagings wat – weens die aard van die
vrywilligers-besture en personeel as menslike hulpbronne, tesame met die vestiging van
organisasiestrukture deur diensverskaffers – deur IGO’s ervaar word nie.i Deelnemers se response oor uitdagings verwys spesifiek na daardie uitdagings wat kapasiteits
bou aan gevestigde maatskaplike diensorganisasies self stel, spesifiek verwysend na die finansiële
impak van kapasiteitsbou van IGO’s op gevestigde diensorganisasies. Die rede daarvoor is diens
organisasies se eie finansiële oorlewingstryd (vergelyk Davids 2012; NACOSS 2013; Swart 2012),
wye omvang en intensiteit van hul eie fokusse van dienslewering op kwesbare gemeenskappe en
groepe, asook die effek wat kapasiteitsbou op die tyd en omvang van werk van die maatskaplike
werkers in hul diens het. Die tydsaamheid van IGO’s se kapasiteitsbou hou direk verband met die
maatskaplike ontwikkelingsgerigte benadering wat gevolg word en wat as gegewe beskou behoort
te word. ʼn Verdere uitdaging vermeld deur beide die gevestigde diensorganisasies en IGO’s is die
finansiële beperkinge van laasgenoemde. Gevestigde organisasies beskou finansiële beperkinge
egter veel wyer as IGO’s, wat suiwer fokus op bestaansnood en basiese finansiering van dienste
en toerusting. Uitdagings vir gevestigde diensorganisasies se kapasiteitsbou van IGO’s Vir diensorganisasies sluit finansiële beperkinge die volgende aspekte in: gebrek
aan finansies om dienste te lewer; gebrekkige infrastruktuur; geen of weinig insig en kennis ten
opsigte van finansiële begrippe en stelsels; gebrekkige of baie beperkte finansiële bestuursvermoë;
kompetisie tussen IGO’s om finansiering en befondsing; asook die wantroue wat IGO’s van
donateurs en befondsers ervaar.i In aansluiting by die voorgaande uitdaging oor finansies blyk gevestigde maatskaplike
diensorganisasies, wat reeds uit eie oortuiging en oorwegend uit eie fondse IGO’s se kapasiteit
bou, in ’n onbenydenswaardige posisie geplaas te word wanneer IGO’s finansiering bekom. Konflik tussen informele en gevestigde diensorganisasies tree dikwels in wanneer IGO’s
finansiering ontvang. Alhoewel baie tyd vooraf bestee word aan die insigontwikkeling van
finansiële begrippe en die daarstel van finansiële stelsels, blyk die besteding van fondse wat nie
voorheen deel van die IGO’s se ervaringswêreld was nie, in te druis teen die aard of wese van
IGO’s se natuurlike ingesteldheid. Weens die verantwoordbaarheid vir terugvoer wat deur donateurs
of finansierders van die gevestigde maatskaplike diensorganisasies verwag word, asook die
gevestigde diensorganisasies se ingesteldheid op gestruktureerde finansiële bestuur, stel gevestigde
diensorganisasies streng vereistes oor finansiering aan IGO’s − ’n benadering wat tot konflik lei. Aangesien IGO’s nie regstegnies deel vorm van die gevestigde organisasies se strukture nie, word
die dilemma ten opsigte van afdwingbaarheid van vereistes verder verhoog. Hierdie dilemma
word vererger deurdat die ooreenkoms of kontrak, as die enigste verbintenis tussen die
diensorganisasie en die IGO, oorwegend informeel en mondelings van aard is juis om by die vlak
van ontwikkeling van die IGO’s aan te pas.i Soms lei vorige kapasiteitsbou-aksies deur die Staat of spesialiteit-organisasies, firmas of
individue tot IGO’s se wantroue teenoor gevestigde diensorganisasies. Dit word in verband gebring
met die deurlopende behoefte van IGO’s aan praktykgerigte ondersteuning soos reeds aangetoon,
wat waarskynlik nie deel vorm van tydbeperkende insette deur sommige kapasiteitsbouers nie. Alhoewel dit eerder dui op IGO’s se onrealistiese verwagting aan die Staat en ander diensver
skaffers, ervaar IGO’s waarskynlik dat sodanige instansies hulle in die steek laat. Te oordeel aan
die tydrowendheid van insette in die aanleer en oefening van vaardighede in kapasiteitsbou van
IGO’s deur gevestigde maatskaplike diensorganisasies, vorm praktykgerigte ondersteuning
onlosmaaklik deel van kapasiteitsbou. Organisasiekultuur blyk by IGO’s ’n ander rol te speel as by meer gevorderde organisasies. Uitdagings vir gevestigde diensorganisasies se kapasiteitsbou van IGO’s By IGO’s blyk organisasiekultuur nie te verwys na hul ingesteldheid teenoor prestasie of die etos
van die organisasie oor die manier hoe dinge in die organisasie gedoen word soos bevestig word Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016
doi.10.17159/2224-7912/2016/v56n2-2a5 604 in die McKinsey-model (2001) nie. Dit blyk uit deelnemers se terugvoer dat die organisasiekultuur
by IGO’s direk verband hou met die kultuur van die gemeenskap en manier van lewe, omdat
IGO’s in dié opsig integraal deel van die gemeenskap vorm en nie ’n eiesoortige identiteit aanneem
nie. Dit is waarskynlik daaraan toe te skryf dat IGO’s grotendeels eerder as individue in plaas van
as organisasies funksioneer. IGO’s se ervaring van kultuur het dus ʼn bepalende effek op die model
van kapasiteitsbou. p
IGO’s se kapasiteitsbou oor kultuurgrense heen blyk nie een van die grootste uitdagings vir
gevestigde diensorganisasies te wees nie, en word moontlik daaraan toegeskryf dat maatskaplike
werkers gewoond daaraan is om oor taal- en kultuurgrense te werk en hulpsisteme te vestig om
hierdie uitdaging die hoof te bied. Deelnemers beklemtoon egter die belangrikheid dat ’n
sensitiwiteit teenoor kultuur deurentyd in dienslewering teenwoordig behoort te wees. y
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Die IGO’s se geïntegreerdheid met hulle gemeenskappe lei tot verdere uitdagings vir
kapasiteitsbou. Die effek van die gemeenskappe se sienings, politieke beskouinge en groeperinge
op IGO’s lei dikwels tot konflik tussen gemeenskapslede. Dit blyk ook dat wanneer onderskeidende
vlakke in die organisasie gevorm word, soos met die aanstelling van personeel, of wanneer sekere
vereistes gestel word oor die besteding van fondse, verwydering tussen die IGO’s en die betrokke
gemeenskappe intree en konflik plaasvind. Vanweë IGO’s se ongestruktureerdheid en as gevolg
van die integrering in die gemeenskap blyk IGO’s nie normaalweg oor maniere en meganismes
te beskik om die konflik aan te spreek nie. Hierdie aspek vorm ’n spesifieke fokus in kapasiteitsbou
van IGO’s. Die integrering met die gemeenskap lei direk daartoe dat die gemeenskap se behoeftes
nét so belangrik geag word as die IGO’s se behoeftes. Die bepaling van die korrekte beginpunt
in die kapasiteitsbouproses deur ’n behoeftebepaling bied ook ’n uitdaging weens die
omvangrykheid en tydrowendheid van hierdie fase in kapasiteitsbou. Kapasiteitsbou het inherent verandering of transformasie ten doel, wat op sigself uitdagings
bied. Verteenwoordigers van IGO’s het pertinent aangedui dat die veranderingsproses die grootste
uitdaging in kapasiteitsbou bied, wat bevestig dat verandering binne normale organisasie-
ontwikkeling ’n veeleisende proses is. Uitdagings vir gevestigde diensorganisasies se kapasiteitsbou van IGO’s By IGO’s is hierdie verandering lewensingrypend en dus
uiters moeilik omdat beweeg word vanaf ’n informele groepering wat funksioneer soos en hoe
die inisieerder van die diens besluit tot ’n georganiseerde entiteit wat aan wetsvereistes voldoen. AANBEVELINGS In onderhandeling met die Staat behoort gevestigde maatskaplike diensorganisasies die omvangryke
implikasies van kapasiteitsbou vir gevestigde diensorganisasies te beklemtoon, sodat die Staat ’n
beter begrip kan ontwikkel wanneer vereistes gestel word vir verpligte kapasiteitsbou van IGO’s
deur gevestigde diensorganisasies. Die Staat behoort voorts erkenning te gee aan gevestigde
maatskaplike diensorganisasies vir die feit dat hulle toeganklik is vir kwesbare gemeenskappe en
behoort hul georganiseerdheid in terme van geografiese dienslewering as bate te benut. g
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Gevestigde diensorganisasies behoort statistiek te bekom en by te hou oor die aantal IGO’s
waarvan kapasiteit gebou word om die saak van gevestigde diensorganisasies in onderhandelinge
met die Staat en ander befondsers te versterk. Desnieteenstaande behoort die Staat oorweging
daaraan te verleen om die beste praktyke uit die bestaande voorbeelde van kapasiteitsbou deur
gevestigde diensorganisasies te ondersoek en te identifiseer, sodat toepaslike beleide en vereistes
gestel kan word en ook om ’n instaatstellende omgewing vir gevestigde maatskaplike diens
organisasies te skep. g
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Tans blyk IGO’s die Suid-Afrikaanse regering se fokus te wees in die herstrukturering van
die maatskaplike diensleweringsisteem (vergelyk Departement van Maatskaplike Ontwikkeling
2012a; Ministerie van Welsyn en Bevolkingsontwikkeling 1997). In hierdie fokus behoort Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016
doi.10.17159/2224-7912/2016/v56n2-2a5 605 kategorisering van IGO’s in onderskeie vlakke van gestruktureerdheid, bestuur en dienslewering
mee te werk om prioriteite ten opsigte van strategiese doelwitte te bepaal en skaars hulpbronne
meer effektief as tans te kan aanwend. Gevestigde maatskaplike diensorganisasies kan as een van
die skaars hulpbronne in die proses van kapasiteitsbou van IGO’s in Suid-Afrika beskou word,
maar sal beslis staatsfinansiering benodig om wel die kapasiteitsbou van IGO’s te kan implementeer. i
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Diensorganisasies behoort ernstig te oorweeg om ’n bondige geskrewe beleid neer te lê vir
kapasiteitsbou van IGO’s vir besture en veral personeel in daardie spesifieke organisasies. Sodanige
beleid sal rigting verskaf oor die tyd en aandag wat aan IGO’s se kapasiteitsbou bestee kan word. Vanweë die reeds bestaande uitdagings wat aan gevestigde maatskaplike diensorganisasies gestel
word, word aanvaar dat hierdie beleid gereeld hersien sal word ten einde die interne kapasiteit
van die diensorganisasies ten beste strategies en operasioneel te kan bestuur. AANBEVELINGS Indringende samesprekings tussen die gevestigde maatskaplike diensorganisasies en die
Departement van Maatskaplike Ontwikkeling behoort plaas te vind, sodat die stel van verwagtinge
aan hierdie groep diensverskaffers realisties kan wees, gedagtig daaraan dat kapasiteitsbou van
IGO’s nie noodwendig deel van hulle kernfokus van dienslewering vorm nie. Die doel van die
gesprekke in hierdie verband sou wees om insig by die Staat te ontwikkel oor die effek wat veral
die implementering van die Kinderwet op kinderbeskermingsorganisasies blyk te hê, die effek
van gebrekkige finansiering selfs op gevestigde diensorganisasies, en ten slotte wat die aard van
kapasiteitsbou van IGO’s in die praktyk behels. ii Die groot uitdaging wat beperkte finansiële vaardighede op die hantering van finansies deur
IGO’s het, moet onder die Departement van Maatskaplike Ontwikkeling se aandag gebring word,
sodat beleidstukke spesifiek voorsiening kan maak vir die skep van kanale vir IGO’s vir toegang
tot staatsfinansiering aan die een kant; maar aan die ander kant ook sodat ’n sekere vlak van
finansiële vaardighede ontwikkel kan word alvorens staatsfinansiering toegeken word. Veral waar
die Departement van Maatskaplike Ontwikkeling geld bewillig vir die opleiding van IGO’s in
bestuur, moet aandag geskenk word aan die wyse waarop meer deurlopende ondersteuning aan
sodanige IGO’s gebied kan word. Aangesien IGO’s se geïntegreerdheid met die gemeenskappe waarbinne hulle funksioneer
een van hul wesenskenmerke is, moet hierdie aspek deurlopend op beleids- en diensleweringsvlak
in ag geneem word in die kapasiteitsbou van IGO’s, ongeag die velerlei uitdagings wat die
geïntegreerdheid tot gevolg het vir kapasiteitsbou. Veranderingsbestuur blyk inherent deel van organisatoriese kapasiteitsbou te wees en veral
vanweë die impak daarvan op die kapasiteitsbouproses van IGO’s behoort maatskaplike werkers
wat hierdie tipe maatskaplikewerk-intervensie doen, opleiding in veranderingsteorie te ontvang. SAMEVATTING Uit hierdie navorsing is dit duidelik dat gevestigde maatskaplike diensorganisasies reeds wel ʼn
waardevolle bydrae tot die kapasiteitsbou van informele gemeenskapsgebaseerde organisasies
lewer. In die stel van vereistes aan gevestigde diensorganisasies oor die kapasiteitsbou van IGO’s
behoort daar allereers deur die Staat in ag geneem te word dat gevestigde organisasies rééds ’n
finansiële oorlewingstryd en vele uitdagings in die aanspreek van nood in hul kerndienslewering
ervaar. Die ernstige finansiële uitdaging aan gevestigde maatskaplike diensorganisasies word deur
verskeie navorsers beklemtoon (Gray 2006; Lombard 2011; Patel & Hochfeld 2008; 2012). Erkenning behoort dus gegee te word vir die feit dat kapasiteitsbou van IGO’s nodig is vir die
herstrukturering van die Suid-Afrikaanse diensleweringsisteem, maar dat eiesoortige uitdagings
tans aan gevestigde maatskaplike diensverskaffers gestel word, wat hulle waardevolle diensle
wering aan kwesbare gemeenskappe in gevaar stel. Tydskrif vir Geesteswetenskappe, Jaargang 56 No. 2-2: Junie 2016
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Potent Anticancer Effects of Epidithiodiketopiperazine NT1721 in Cutaneous T Cell Lymphoma
|
Cancers
| 2,021
|
cc-by
| 11,689
|
UC Irvine Journal
Cancers, 13(13)
ISSN
2072-6694
Authors
Lin, Min
Kowolik, Claudia M
Xie, Jun
et al. Publication Date
2021
DOI
10.3390/cancers13133367
Peer reviewed UC Irvine
UC Irvine Previously Published Works
Title
Potent Anticancer Effects of Epidithiodiketopiperazine NT1721 in Cutaneous T Cel
Lymphoma
Permalink
https://escholarship.org/uc/item/7t15q0p6
Journal
Cancers, 13(13)
ISSN
2072-6694
Authors
Lin, Min
Kowolik, Claudia M
Xie, Jun
et al.
Publication Date
2021
DOI
10.3390/cancers13133367
Peer reviewed UC Irvine
UC Irvine Previously Published Works
Title
Potent Anticancer Effects of Epidithiodiketopiperazine NT1721 in Cutaneous T Cel
Lymphoma
Permalink
https://escholarship.org/uc/item/7t15q0p6
Journal
Cancers, 13(13)
ISSN
2072-6694
Authors
Lin, Min
Kowolik, Claudia M
Xie, Jun
et al. Publication Date
2021
DOI
10.3390/cancers13133367
Peer reviewed
Abstract: Cutaneous T cell lymphomas (CTCLs) are a heterogeneous group of debilitating, incurable
malignancies. Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common subtypes,
accounting for ~65% of CTCL cases. Patients with advanced disease have a poor prognosis and low
median survival rates of four years. CTCLs develop from malignant skin-homing CD4+ T cells that
spread to lymph nodes, blood, bone marrow and viscera in advanced stages. Current treatments
options for refractory or advanced CTCL, including chemotherapeutic and biological approaches,
rarely lead to durable responses. The exact molecular mechanisms of CTCL pathology remain unclear
despite numerous genomic and gene expression profile studies. However, apoptosis resistance is
thought to play a major role in the accumulation of malignant T cells. Here we show that NT1721, a
synthetic epidithiodiketopiperazine based on a natural product, reduced cell viability at nanomolar
concentrations in CTCL cell lines, while largely sparing normal CD4+ cells. Treatment of CTCL
cells with NT1721 reduced proliferation and potently induced apoptosis. NT1721 mediated the
downregulation of GLI1 transcription factor, which was associated with decreased STAT3 activation
and the reduced expression of downstream antiapoptotic proteins (BCL2 and BCL-xL). Importantly,
NT1721, which is orally available, reduced tumor growth in two CTCL mouse models significantly
better than two clinically used drugs (romidepsin, gemcitabine). Moreover, a combination of NT1721
with gemcitabine reduced the tumor growth significantly better than the single drugs. Taken together,
these results suggest that NT1721 may be a promising new agent for the treatment of CTCLs. Citation: Lin, M.; Kowolik, C.M.; Xie,
J.; Yadav, S.; Overman, L.E.; Horne,
D.A. Potent Anticancer Effects of
Epidithiodiketopiperazine NT1721 in
Cutaneous T Cell Lymphoma. Cancers
2021, 13, 3367. https://doi.org/
10.3390/cancers13133367 Received: 18 May 2021
Accepted: 1 July 2021
Published: 5 July 2021 Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Keywords: cutaneous T cell lymphoma (CTCL); epidithiodiketopiperazine; NT1721; GLI1; STAT3 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/). Article
Potent Anticancer Effects of Epidithiodiketopiperazine NT1721
in Cutaneous T Cell Lymphoma a M. Kowolik 1,*,†, Jun Xie 1, Sushma Yadav 1,2
, Larry E. Overman 3 and David A. Horne 1,* Min Lin 1,†, Claudia M. Kowolik 1,*,†, Jun Xie 1, Sushma Yadav 1,2
, Larry E. Overman 3 and 1
Department of Molecular Medicine, City of Hope National Medical Center, 1500 E. Duarte Road, Duarte, CA 91010, USA; mlin@coh.org (M.L.); jxie@coh.org (J.X.); syadav@coh.org (S.Y.)
2
Department of Translational Research and Cellular Therapeutics, City of Hope National Medical Center
1500 E. Duarte Road, Duarte, CA 91010, USA
3
Department of Chemistry, University of California, Irvine, CA 92697-2025, USA; leoverma@uci.edu
*
Correspondence: ckowolik@coh.org (C.M.K.); dhorne@coh.org (D.A.H.) p
y
y
*
Correspondence: ckowolik@coh.org (C.M.K.); dhorne@coh.org (D.A.H.) p
g
†
These authors contributed equally to this work. †
These authors contributed equally to this work. †
These authors contributed equally to this work. Simple Summary: Cutaneous T cell lymphomas (CTCLs) are a group of blood cancers that cannot
be cured with current chemotherapeutical or biological drugs. Patients with advanced disease are
severely immunocompromised due to the unchecked expansion of malignant T cells and have low
survival rates of less than four years. Hence, new treatment options for CTCLs are urgently needed. In this study the anti-CTCL activity of a new compound, NT1721, was determined in vitro and in
two CTCL mouse models. We found that NT1721 increased apoptosis (programmed cell death) in
the malignant T cells and reduced tumor growth better than two drugs that are currently clinically
used for CTCL treatment (i.e., gemcitabine, romidepsin). These results suggest that NT1721 may
represent a potent new agent for the treatment of advanced CTCL.
Citation: Lin, M.; Kowolik, C.M.; Xie,
J.; Yadav, S.; Overman, L.E.; Horne,
D.A. Potent Anticancer Effects of
Epidithiodiketopiperazine NT1721 in
Cutaneous T Cell Lymphoma. Cancers
2021, 13, 3367. https://doi.org/
10.3390/cancers13133367
Academic Editor: Daruka Mahadevan
Received: 18 May 2021
Accepted: 1 July 2021
Published: 5 July 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. 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/). Powered by the California Digital Library
University of California Powered by the California Digital Library
University of California eScholarship.org eScholarship.org cancers cancers cancers Citation: Lin, M.; Kowolik, C.M.; Xie,
J.; Yadav, S.; Overman, L.E.; Horne,
D.A. Potent Anticancer Effects of
Epidithiodiketopiperazine NT1721 in
Cutaneous T Cell Lymphoma. Cancers
2021, 13, 3367. https://doi.org/
10.3390/cancers13133367 1. Introduction Cutaneous T cell lymphomas (CTCLs) are a biologically and clinically heterogeneous
group of rare, incurable non-Hodgkin lymphomas that typically affect adults with a median
age of 55 to 60 years [1–3]. The annual age-adjusted incidence rate in the United States is
6.4 per million persons [3]. CTCLs develop from malignant skin-homing CD4+ T cells that https://www.mdpi.com/journal/cancers Cancers 2021, 13, 3367. https://doi.org/10.3390/cancers13133367 Cancers 2021, 13, 3367 2 of 15 spread to lymph nodes, blood, bone marrow and viscera in advanced stages [4]. The most
common subtypes, mycosis fungoides (MF) and Sézary syndrome (SS), account for 65%
of CTCL cases [5,6]: MF is an indolent disease with only 10% of patients progressing to
advanced stages, while SS represents the aggressive leukemic variant of CTCL that can
develop de novo or progress from MF [7]. Differences in molecular profiles and responses
to therapies suggest that MF and SS are distinct diseases [8,9]. Patients with SS present
with generalized erythroderma and lymphadenopathy and have a poor prognosis with
low median survival rates of 2–4 years [10]. SS is characterized by the clonal expansion
of circulating malignant T cells with cerebriform nuclei (Sézary cells), which leads to the
loss of a normal T cell receptor repertoire and thus immunosuppression [10–14]. Hence,
patients with advanced disease are highly susceptible for opportunistic infections, which
represent the most common CTCL-related cause of death [5,14]. Current treatment options
for CTCL depend on the tumor stage. Unlike early stage CTCL, which is treated with skin-
directed therapies, refractory early stage and advanced stage CTCL requires systemic mono-
or combination therapy such as photopheresis, interferon alpha, bexarotene, monoclonal
antibodies (e.g., mogamulizumab), histone deacetylase inhibitors (e.g., romidepsin (FK228))
and chemotherapeutics (e.g., gemcitabine) [4,15]. Response rates for most treatments vary
between 30 and 50% and are rarely durable (7.5 to 22.4 months) [15,16]. Relapses are
common and treatment for the aggressive forms remains palliative, not curative despite
recent advances with chemotherapeutic and biological approaches [4]. Moreover, current
therapies often lead to progressive drug resistance [17]. Thus, new treatment options for
advanced CTCL are urgently needed. Numerous genomic and gene expression profile studies have been performed over
the past decade to discover genes that play a role in CTCL pathogenesis and to find
potential new targets for therapy [11,14,18,19]. 2.1. Reagents NT1721 was synthesized as previously described [32]. Gemcitabine, romidepsin
(FK228) and the ERK inhibitor SCH772984 were purchased from Selleckchem (Houston,
TX, USA). 2.5. Cell Cycle Analysis Cells were treated with NT1721 as indicated and harvested after 48 h, stained with
propidium iodide (PI) (ThermoFisher) as previously described and subjected to FACS
analysis [27]. 2.4. Proliferation Assay Cells were stained with CFSE ((5(6)-carboxyfluorescein N-hydroxysuccinimidylester,
CellTrace, ThermoFisher) according to the manufacturer’s instructions, and seeded at a
concentration of 10,000 cells/well. The cells were treated with NT1721 or DMSO the next
day, harvested after 48 h or 72 h, stained with 0.2 µg/mL DAPI and subjected to FACS
analysis. Fluorescence data were collected on a CyAN flow cytometer (Beckman Coulter,
Brea, CA, USA) and analyzed with FlowJo software (TreeStar, Ashland, OR, USA). 2.3. Determination of IC50 Values The CellTiter-GLO viability assay (Promega, Madison, WI, USA) was used according
to the manufacturer’s instructions to determine cell viability: Briefly, 10,000 cells/well were
seeded in 96-well plates, cultured overnight and then treated with various concentrations
of NT1721 for 48 h or 72 h. Data from the assay were expressed as percent of viable cells
compared to the vehicle control (0.1% DMSO). 2.2. Cell Culture HUT78 and HH cells were obtained from ATCC (Manassas, VA, USA), authenticated
by STR-profiling at the source and passaged for less than 6 months after receipt or re-
suscitation. Peripheral blood mononuclear cells (PBMCs) were obtained from ReachBio
(Spokane, WA, USA). All cells were grown in RPMI supplemented with 10% FBS (Atlas
Biologicals, Fort Collins, CO, USA). CD4+ cells were isolated from PBMCs using Dynabeads
(ThermoFisher, Waltham, MA, USA). 1. Introduction However, the molecular mechanisms of
CTCL pathogenesis remain obscure as there is little overlap among the study results, which
highlights the genetic heterogeneity of CTCL [11,19,20]. Genes with the most frequently
altered expression profiles in CTCL are associated with the constitutive production of
cytokines characteristic for T helper type 2 cells, resistance to growth inhibition via TGFβ
and apoptosis induction [11]. Importantly, apoptosis resistance rather than increased
proliferation is thought to be the cause for the accumulation of malignant T cells in the early
stages of CTCL [21,22]. Aberrantly activated STAT3 has been identified as one important
factor in CTCL progression and apoptosis resistance [18,23–25]. Activated STAT3 has
been associated with increased expression of antiapoptotic proteins (BCL2, BCL-xL) and
decreased overall and progression-free survival in CTCL [23,26]. p
g
In this study, the anti-CTCL efficacy of an epidithiodiketopiperazine, NT1721, was
investigated. Epidithiodiketopiperazines (ETPs) are a broad class of fungal metabolites
with potent antitumor activity in multiple solid and non-solid tumors. We have shown
in a previous study that NT1721, an ETP chosen from a library of ETPs because of its
potent antitumor activity against various solid and non-solid tumors, attenuated hedge-
hog/GLI signaling through downregulation of GLI transcription factors [27]. Aberrant
hedgehog/GLI signaling promotes tumorigenesis, tumor progression, metastasis and drug
resistance in various types of cancers [28,29]. Here we show that treatment with NT1721
mediated GLI1 downregulation in CTCL cells, which was associated with decreased STAT3
activation and reduced expression of downstream antiapoptotic proteins (BCL2 and BCL-
xL). NT1721 led to potent apoptosis induction in vitro and significantly reduced tumor
growth in two CTCL xenograft mouse models. Importantly, NT1721 impaired tumor
growth significantly better than romidepsin or gemcitabine, two clinically used drugs
for the treatment of refractory or relapsed CTCL [30,31]. Drug combinations of NT1721
with gemcitabine showed significantly improved antitumor effects in vitro and in vivo
compared to the single agents. Our data suggest that NT1721 is, potentially, a promising
new agent for the treatment of CTCL either by itself or in combination with gemcitabine. 3 of 15 Cancers 2021, 13, 3367 2.6. QPCR Total RNA was isolated using the Direct-zol kit (Zymo Research, Irvine, CA, USA)
and reverse transcribed using the Tetro cDNA synthesis kit (Bioline, Taunton, MA, USA). The following qPCR primers were used: The following qPCR primers were used:
BCL2: 5′AGTACCTGAACCGGCACCT/5′GCCGTACAGTTCCACAAAGG
BCL-xL: 5′CTGAGTTACCGGCATCCC/5′TTCTGAAGGGAGAGAAAGAGATTC
BMI1: 5′CCATTGAATTCTTTGACCAGAA/5′CTGCTGGGCATCGTAAGTATC
CCNE1: 5′GGCCAAAATCGACAGGAC/5′GGGTCTGCACAGACTGCAT;
GAPDH: 5′AGCCACATCGCTCAGACAC/5′GCCCAATACGACCAAATCC;
GLI1: 5′ACCCGGGGTCTCAAACTG/5′GGCTGACAGTATAGGCAGAGC;
Quantitative PCR was performed using a CFX96 Touch Real-Time PCR detection
system (Bio-Rad, Hercules, CA, USA). Relative expression levels were calculated using the
2−∆∆Ct method and GAPDH as reference gene. g q
p
BCL2: 5′AGTACCTGAACCGGCACCT/5′GCCGTACAGTTCCACAAAGG
BCL
L 5′CTGAGTTACCGGCATCCC/5′TTCTGAAGGGAGAGAAAGAGATTC g q
p
BCL2: 5′AGTACCTGAACCGGCACCT/5′GCCGTACAGTTCCACAAAGG
BCL-xL: 5′CTGAGTTACCGGCATCCC/5′TTCTGAAGGGAGAGAAAGAGATTC BMI1: 5′CCATTGAATTCTTTGACCAGAA/5′CTGCTGGGCATCGTAAGTATC CCNE1: 5′GGCCAAAATCGACAGGAC/5′GGGTCTGCACAGACTGCAT; GAPDH: 5′AGCCACATCGCTCAGACAC/5′GCCCAATACGACCAAATCC; GLI1: 5′ACCCGGGGTCTCAAACTG/5′GGCTGACAGTATAGGCAGAGC Quantitative PCR was performed using a CFX96 Touch Real-Time PCR detection
system (Bio-Rad, Hercules, CA, USA). Relative expression levels were calculated using the
2−∆∆Ct method and GAPDH as reference gene. 2.7. Western Blots SDS-PAGE and Western blots were carried out as previously described [27]. Briefly,
cells were lyzed using RIPA buffer (Cell signaling, Danvers, MA, USA) supplemented
with Halt Protease inhibitor cocktail (ThermoFisher). The protein concentration was
quantified using the Pierce BCA Protein Assay Kit (ThermoFisher). Equal amounts of
protein were loaded on precast gels (Bio-Rad, Hercules, CA, USA) and transferred to PVDF Cancers 2021, 13, 3367 4 of 15 membranes (Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes
were blocked for 1 h at RT in blocking buffer (5% w/v nonfat dry milk, 0.1% Tween-20 in
TBS), then incubated with primary antibodies overnight at 4 ◦C. The NE-PER Nuclear and
Cytoplasmic Extraction Kit (ThermoFisher) was used for the preparation of cytoplasmic
and nuclear extracts. Primary antibodies were purchased from Cell Signaling Technology
(Danvers, MA, USA): β-actin (#4970), BCL2 (#4223), BCL-xL (#2764), BMI1 (#6964), cleaved
PARP (#5625), cleaved CASP3 (#9664), ERK (#4695), pERK1/2 (#4370), GAPDH (#5174),
GLI1 (#3538), H2AX (#2595), γH2AX (#9718), H3 (#4499), p21 (#2947), p27 (#3686), STAT3
(#9139), pSTAT3 (Y705) (#9145), TBP (#44059); additionally, tubulin (05-829) was purchased
from Sigma (St. Louis, MO, USA). The membranes were washed three times with TBS and
then incubated 1 h at RT with the appropriate secondary antibodies. Bands were visualized
using X-ray film or the ChemiDoc Imaging System (Bio-Rad). 2.8. In Vivo Studies Mouse care and experimental procedures were performed under pathogen-free condi-
tions in accordance with approved protocols from the institutional animal care and use
committee of City of Hope National Medical Center. For the xenograft mouse models,
5 million HuT78 or HH cells were injected subcutaneously into the flank of 6- to 8-week old
NSG mice (Jackson Laboratory, Bar Harbor, ME, USA). The tumor volume was calculated
using the equation: V = 1
2 × W2 × L with W and L representing the width and length. Treat-
ment started when the tumor volume reached ~200 mm3. The mice were then distributed
into groups bearing equal tumor burdens and treated with NT1721 by gavage, gemcitabine
(i.p. injection), romidepsin (i.p. injection) or the vehicle control as indicated. PBS was
used as vehicle control for treatments by i.p. injections; 30% solutol/3.3% DMSO in PBS or
Ora-Blend (ThermoFisher)/10% saline were used as vehicle controls for gavage treatments. 2.9. Statistical Analysis The mean ± standard deviation (SD) was calculated for each treatment group. The
two-tailed t-test (GraphPad Software Inc., La Jolla, CA, USA) was used to determine
statistical significance between two treatment groups in the in vitro studies. The Mann–
Whitney U test was used to determine statistical significance between treatment groups in
mouse models. p values < 0.05 were considered significant. 3.1. NT1721 Led to Reduced CTCL Cell Viability, Proliferation and G2 Cell Cycle Arrest at
Nanomolar Concentrations (B) Effect of NT1721 on normal cells. HuT78 cells, normal PBMCs and normal CD4+ cells were treated with
300 nM NT1721 or 0.3% DMSO. (C) Proliferation. CTCL cells were stained with CFSE, treated with NT1721 and subjected to
FACS analysis after 48 or 72 h to determine the mean fluorescence intensity. The data were normalized to the controls. (D)
Cell cycle analysis. HuT78 cells were treated with NT1721, stained with PI after 24 h and 48 h and analyzed by FACS. The
graphs represent the mean ± SD from triplicate values. Figure 1. NT1721 decreased CTCL cell viability, proliferation and led to G2 cell cycle arrest. (A) Cell viability. HuT78 and
HH cells were treated with increasing concentrations of NT1721. Cell viability and IC50 values were determined after 48
and 72 h. (B) Effect of NT1721 on normal cells. HuT78 cells, normal PBMCs and normal CD4+ cells were treated with
300 nM NT1721 or 0.3% DMSO. (C) Proliferation. CTCL cells were stained with CFSE, treated with NT1721 and subjected to
FACS analysis after 48 or 72 h to determine the mean fluorescence intensity. The data were normalized to the controls. (D)
Cell cycle analysis. HuT78 cells were treated with NT1721, stained with PI after 24 h and 48 h and analyzed by FACS. The
graphs represent the mean ± SD from triplicate values. We then determined the effect of NT1721 on the cell cycle by staining NT1721-treated
cells with PI. As shown in Figure 1D, NT1721 led to cell cycle arrest in CTCL cells. Treat-
ment with NT1721 led to G2 cell cycle arrest as indicated by the dose- and time-dependent
increase in the percentage of cells in the G2 phase and concomitant decrease in the percent-
age of cells in the G1 phase. Treatment with 500 nM NT1721 decreased the number of cells
in the G1 phase from ~70% to 30% and 22% after 24 h and 48 h, respectively; the number of
cells in the G2 phase increased from ~12% to 35 and 38% in NT1721-treated cells after 24 h
and 48 h, respectively. Taken together, the results indicate that NT1721-mediated preferen-
tial reduction in CTCL cell viability (compared to normal CD4+ cells) was associated with
decreased proliferation and G2 cell cycle arrest. 3.1. NT1721 Led to Reduced CTCL Cell Viability, Proliferation and G2 Cell Cycle Arrest at
Nanomolar Concentrations To assess the potency of NT1721 against CTCL, we treated HuT78 and HH cells with
increasing concentrations of NT1721 and determined viability after 48 and 72 h. The IC50
values were in the nanomolar range for both cell lines and time points: 125 nM and 6 nM in
HuT78 cells after 48 and 72 h, respectively; 542 nM and 70 nM in HH cells after 48 and 72 h,
respectively (Figure 1A). To assess the effect of NT1721 on normal cells we also treated
Hut78 cells, normal peripheral blood mononuclear cells (PBMCs) and CD4+ cells from two
healthy donors with NT1721. As shown in Figure 1B, 300 nM NT1721 reduced the viability
of HuT78 by 83%, while only decreasing the viability of normal PBMCs and normal CD4+
by ~32%. The result suggests that NT1721 preferentially decreased the viability of CTCL
cells. To investigate the effect of NT1721 on CTCL proliferation we stained HuT78 and HH
cells with ((5(6)-Carboxyfluorescein N-hydroxysuccinimidylester (CFSE) and treated them
with NT1721 (up to 300 nM) for 48 or 72 h. FACS analysis of live cells showed that NT1721
reduced proliferation faster and at lower concentrations in Hut78 compared to HH cells
(Figure 1C). However, treatment with 100 nM NT1721 dramatically reduced proliferation
in both cell lines after 72 h by 50–75%. 5 of 15 Cancers 2021, 13, 3367 Figure 1. NT1721 decreased CTCL cell viability, proliferation and led to G2 cell cycle arrest. (A) Cell viability. HuT78 and
HH cells were treated with increasing concentrations of NT1721. Cell viability and IC50 values were determined after 48
and 72 h. (B) Effect of NT1721 on normal cells. HuT78 cells, normal PBMCs and normal CD4+ cells were treated with
300 nM NT1721 or 0.3% DMSO. (C) Proliferation. CTCL cells were stained with CFSE, treated with NT1721 and subjected to
FACS analysis after 48 or 72 h to determine the mean fluorescence intensity. The data were normalized to the controls. (D)
Cell cycle analysis. HuT78 cells were treated with NT1721, stained with PI after 24 h and 48 h and analyzed by FACS. The
graphs represent the mean ± SD from triplicate values. Figure 1. NT1721 decreased CTCL cell viability, proliferation and led to G2 cell cycle arrest. (A) Cell viability. HuT78 and
HH cells were treated with increasing concentrations of NT1721. Cell viability and IC50 values were determined after 48
and 72 h. 3.2. NT1721 Downregulated GLI1 Transcription Factor in CTCL Cells Since we previously found that NT1721 potently downregulated GLI transcription
factors [27], we first determined the effect of NT1721 on GLI1 expression in CTCL cells. As shown in (Figure 2A,B), NT1721 dramatically reduced GLI1 expression on the
mRNA and protein level in both HuT78 and HH cells after 24 h at concentrations of
≤100 nM. A comparison of GLI1 expression levels in the CTCL cell lines revealed ~3-
fold higher GLI1 mRNA expression in Hut78 as well as higher GLI1 protein expression Cancers 2021, 13, 3367 6 of 15 compared to HH cells (Figure 2B,C). In contrast, no GLI1 protein expression was detected
in two samples of normal CD4+ cells (Figure 2C), suggesting that GLI1 might be aberrantly
expressed in the CTCL cell lines. Moreover, this result is in agreement with a previous
report showing strong GLI1 expression in T cell lymphomas including CTCL while no
GLI1 expression was observed in healthy peripheral blood T cells [33]. compared to HH cells (Figure 2B,C). In contrast, no GLI1 protein expression was detected
in two samples of normal CD4+ cells (Figure 2C), suggesting that GLI1 might be aberrantly
expressed in the CTCL cell lines. Moreover, this result is in agreement with a previous
report showing strong GLI1 expression in T cell lymphomas including CTCL while no
GLI1 expression was observed in healthy peripheral blood T cells [33]. Figure 2. NT1721 downregulated GLI and GLI target genes in CTCL cells. (A) Protein expression levels were assessed
by Western blot after 24 h treatment with NT1721. (B) QPCR analysis of gene expression. The data were analyzed using
GAPDH as the reference gene and represent the mean ± SD from 3 independent experiments. (* indicates p ≤0.05;
** indicates p ≤0.01) (C) GLI1, pSTAT3, STAT3 and BCL-xL protein expression in normal CD4+ cells, CTCL cells and
NT1721-treated CTCL cells. Original Western Blots of Figure 2A,C available in Figure S4. Figure 2. NT1721 downregulated GLI and GLI target genes in CTCL cells. (A) Protein expression levels were assessed
by Western blot after 24 h treatment with NT1721. (B) QPCR analysis of gene expression. The data were analyzed using
GAPDH as the reference gene and represent the mean ± SD from 3 independent experiments. (* indicates p ≤0.05;
** indicates p ≤0.01) (C) GLI1, pSTAT3, STAT3 and BCL-xL protein expression in normal CD4+ cells, CTCL cells and
NT1721-treated CTCL cells. 3.3. NT1721 Induced Apoptosis in CTCL In Vitro and In Vivo Since NT1721 mediated the downregulation of antiapoptotic BCL2 and BCL-xL, we
decided to further investigate the effect of NT1721 on apoptosis. HuT78 cells were treated
with NT1721 (up to 500 nM) and stained with annexin V after 24 h and 48 h. As shown
in Figure 3A, low concentrations of NT1721 (10–30 nM) significantly increased the per-
centage of apoptotic cells to 20–40% after 48 h. Treatment with a higher dose of NT1721
(500 nM) resulted in 35% and 62% apoptotic cells after 24 h and 48 h, respectively. To
further investigate apoptosis induction by NT1721 on the molecular level, NT1721-treated
HuT78 and HH samples were analyzed by Western blots. As shown in Figure 3B, NT1721
increased the expression of proteins related to apoptosis induction (i.e., cleaved CASP3 and
cleaved PARP) in a concentration- and time-dependent manner. Moreover, this increase
in apoptotic markers was accompanied by H2AX upregulation and increased levels of γ
H2AX. Subcellular fractionation revealed that in NT1721-treated Hut78 cells the cytoplas-
mic fractions contained the majority of γH2AX and total H2AX; only a small amount of
these proteins was detected in the nuclear fractions (Figure S1). Also observed was a dra-
matic increase in γH2AX and total H2AX levels in NT1721-treated HH cells; a significant
portion of these proteins was also found in the cytoplasmic fractions. This H2AX upreg-
ulation and cytoplasmic localization provided additional support that NT1721 induced
apoptosis in CTCL. Increased H2AX levels have been identified as a cause of apoptosis in
previous studies [35,36]. Since ERK activation has been linked to both apoptosis induction and G2 cell cycle
arrest [37,38], the effect of NT1721 on ERK activation and p21 (CDKN1A) levels was
investigated. As shown in Figure 3B, low concentrations of NT1721 (10–30 nM) led to
concentration-dependent activation of ERK in Hut78 starting after 24 h. In HH cells,
ERK activation was detected after 48 h of treatment with NT1721 and at higher NT1721
concentrations. Dramatic upregulation of p21 by NT1721 was detected in both cell lines
(Figure 3B). To explore the importance of ERK phosphorylation on apoptosis induction,
HuT78 cells were treated with an ERK inhibitor SCH772984, NT1721, or a combination
of both agents. Western blot analyses showed that NT1721 alone increased phospho-
ERK (pERK), p21, cleaved CASP3, cleaved PARP and γH2AX levels in a concentration-
dependent manner (Figure S2A). In contrast, SCH772984 alone had no effect on these
protein levels. 3.2. NT1721 Downregulated GLI1 Transcription Factor in CTCL Cells Original Western Blots of Figure 2A,C available in Figure S4. We next investigated the effect of GLI1 downregulation on the expression of GLI target Figure 2. NT1721 downregulated GLI and GLI target genes in CTCL cells. (A) Protein expression levels were assessed
by Western blot after 24 h treatment with NT1721. (B) QPCR analysis of gene expression. The data were analyzed using
GAPDH as the reference gene and represent the mean ± SD from 3 independent experiments. (* indicates p ≤0.05;
** indicates p ≤0.01) (C) GLI1, pSTAT3, STAT3 and BCL-xL protein expression in normal CD4+ cells, CTCL cells and
NT1721-treated CTCL cells. Original Western Blots of Figure 2A,C available in Figure S4. Figure 2. NT1721 downregulated GLI and GLI target genes in CTCL cells. (A) Protein expression levels were assessed
by Western blot after 24 h treatment with NT1721. (B) QPCR analysis of gene expression. The data were analyzed using
GAPDH as the reference gene and represent the mean ± SD from 3 independent experiments. (* indicates p ≤0.05;
** indicates p ≤0.01) (C) GLI1, pSTAT3, STAT3 and BCL-xL protein expression in normal CD4+ cells, CTCL cells and
NT1721-treated CTCL cells. Original Western Blots of Figure 2A,C available in Figure S4. We next investigated the effect of GLI1 downregulation on the expression of GLI target
genes that are involved in apoptosis resistance and cell cycle progression (i.e., BCL2, BMI1,
CCNE1) and that were also downregulated by NT1721 in our previous study [27]: NT1721
concentrations of 100 nM significantly reduced BMI1 and CCNE1 expression in HuT78 and
HH cells (Figure 2A,B). NT1721 also significantly decreased BCL2 expression in HuT78
(Figure 2A,B). However, in line with previous reports [17,34], no BCL2 mRNA or protein
expression could be detected in HH cells. We decided to determine the effect of NT1721 on STAT3 activation since several reports
show that GLI1 inhibition or knockdown decreased phosho-STAT3 (pSTAT3) levels in T cell
lymphomas and that pSTAT3 may be an important driver of CTCL, especially in advanced
disease [26]. As shown in Figure 2C, strong pSTAT3 was only detected in the CTCL cells Cancers 2021, 13, 3367 7 of 15 and not in the normal CD4+ samples. Treatment of CTCL cells with NT1721 resulted in
reduced pSTAT3 levels (Figure 2C). The reduced STAT3 activation was accompanied by
a significant reduction in mRNA and protein levels of the STAT3 target gene BCL-xL in
Hut78 (Figure 2B,C). 3.2. NT1721 Downregulated GLI1 Transcription Factor in CTCL Cells In contrast, very low BCL-xL expression was detected in HH and
normal CD4+ cells. 3.4. NT1721 Suppressed Tumor Growth Significantly Better Than Romidepsin in CTCL
Mouse Models 3.4. NT1721 Suppressed Tumor Growth Significantly Better Than Romidepsin in CTCL
Mouse Models
To assess the antitumor efficacy of NT1721 in vivo, NSG mice were injected subcu-
taneously with HuT78 cells. When the tumor volumes reached ~200 mm3 the mice were
treated with 20 mg/kg NT1721 by gavage three times per week on consecutive days. The
control group received the vehicle control (30% solutol/3.3% DMSO in PBS). As shown
in Figure 4A,B, NT1721 reduced the tumor volume and tumor weight significantly by
90%. To compare the efficacy of NT1721 to romidepsin, a clinically used drug for CTCL
treatment [39], mice were treated with romidepsin (2 mg/kg) twice per week. Romidepsin
did not significantly reduce the tumor weight (Figure 4A,B). Moreover, romidepsin-treated
mice showed signs of toxicity, i.e., ascites and diarrhea, while no obvious signs of toxicity
were observed in NT1721-treated mice. We next investigated the in vivo efficacy of NT1721
against CTCL in another mouse model. NT1721 also reduced the tumor volume and tumor
weight of HH tumors by ~50% (Figure 4D–F). Treatment of HH tumors with romidepsin
did not result in smaller tumor volumes (data not shown). These results indicate that
NT1721 reduced CTCL tumor growth better than romidepsin and that NT1721 may be less
toxic than romidepsin. To assess the antitumor efficacy of NT1721 in vivo, NSG mice were injected subcu-
taneously with HuT78 cells. When the tumor volumes reached ~200 mm3 the mice were
treated with 20 mg/kg NT1721 by gavage three times per week on consecutive days. The
control group received the vehicle control (30% solutol/3.3% DMSO in PBS). As shown
in Figure 4A,B, NT1721 reduced the tumor volume and tumor weight significantly by
90%. To compare the efficacy of NT1721 to romidepsin, a clinically used drug for CTCL
treatment [39], mice were treated with romidepsin (2 mg/kg) twice per week. Romidepsin
did not significantly reduce the tumor weight (Figure 4A,B). Moreover, romidepsin-treated
mice showed signs of toxicity, i.e., ascites and diarrhea, while no obvious signs of toxicity
were observed in NT1721-treated mice. We next investigated the in vivo efficacy of NT1721
against CTCL in another mouse model. NT1721 also reduced the tumor volume and tumor
weight of HH tumors by ~50% (Figure 4D–F). Treatment of HH tumors with romidepsin
did not result in smaller tumor volumes (data not shown). 3.3. NT1721 Induced Apoptosis in CTCL In Vitro and In Vivo Samples treated with combinations of NT1721 and SCH772984 displayed
reduced ERK phosphorylation, cleaved PARP, cleaved CASP3, p21 and γH2AX induction
compared to samples treated with NT1721 alone. To further investigate the effect of the
drug combination on apoptosis induction we stained Hut78 cells that were treated with the
drug combination with annexin V. As shown in Figure S2B, treatment with both NT1721
and SCH772984 reduced the percentage of apoptotic HuT78 cells significantly compared to
cells treated with NT1721 alone. Taken together, these results suggest that increased ERK
phosphorylation may be critical for efficient apoptosis induction by NT1721. To evaluate
whether treatment with NT1721 could also induce apoptosis in vivo, NSG mice were
injected with HuT78 cells subcutaneously. When the tumor volumes reached ~200 mm3 the
mice were treated with a single oral dose of NT1721 or the vehicle control. The mice were
euthanized 15, 24, 48 or 72 h later, and tumor tissue from individual mice was used for
Western blot analysis. As shown in Figure 3C, NT1721 increased cleaved CASP3, cleaved
PARP and p21 expression while decreasing GLI1 and BCL2 expression, thus recapitulating
the changes in protein expression that were observed in vitro. These results suggest that
NT1721 induces apoptosis in CTCL in vivo. 8 of 15 Cancers 2021, 13, 3367 Figure 3. NT1721 induced apoptosis in CTCL cells in vitro and in vivo. (A) HuT78 cells were treated with NT1721 for 24 h
or 48 h, then stained with annexin V and subjected to FACS analysis. (B) HuT78 and HH cells were treated with NT1721
as indicated. Western blots were used to analyze the expression of genes related to apoptosis induction. (C) Apoptosis
induction in vivo. NSG mice harboring HuT78 tumors (n = 4 per group) were treated by gavage with a single dose of
NT1721 (20 mg/kg) or the vehicle (3.3% DMSO/30% solutol in PBS). The tumors were then harvested at the indicated time
points and subjected to Western blot analysis. Original Western Blots of Figure 3B,C available in Figure S5. Figure 3. NT1721 induced apoptosis in CTCL cells in vitro and in vivo. (A) HuT78 cells were treated with NT1721 for 24 h
or 48 h, then stained with annexin V and subjected to FACS analysis. (B) HuT78 and HH cells were treated with NT1721
as indicated. Western blots were used to analyze the expression of genes related to apoptosis induction. (C) Apoptosis
induction in vivo. 3.3. NT1721 Induced Apoptosis in CTCL In Vitro and In Vivo NSG mice harboring HuT78 tumors (n = 4 per group) were treated by gavage with a single dose of
NT1721 (20 mg/kg) or the vehicle (3.3% DMSO/30% solutol in PBS). The tumors were then harvested at the indicated time
points and subjected to Western blot analysis. Original Western Blots of Figure 3B,C available in Figure S5. 3.4. NT1721 Suppressed Tumor Growth Significantly Better Than Romidepsin in CTCL
Mouse Models 3.4. NT1721 Suppressed Tumor Growth Significantly Better Than Romidepsin in CTCL
Mouse Models 3.4. NT1721 Suppressed Tumor Growth Significantly Better Than Romidepsin in CTCL
Mouse Models These results indicate that
NT1721 reduced CTCL tumor growth better than romidepsin and that NT1721 may be less
toxic than romidepsin. 9 of 15 Cancers 2021, 13, 3367 Figure 4. NT1721 suppressed tumor growth significantly better than romidepsin in a CTCL mouse model. (A) NSG mice
(n = 7/group) were injected s.c. with HuT78 cells and treated by gavage three times/week (on consecutive days) with
the vehicle control (30% solutol/3.3% DMSO in PBS), 20 mg/kg NT1721 or with 2 mg/kg romidepsin by I.P. injection
twice per week. Treatment with NT1721 reduced tumor volumes significantly better than romidepsin on Day 4, 7 and 11
(p values < 0.01). (B) Weight of HuT78 tumors from individual mice (p = 0.0003 for control vs. NT1721 and for romidepsin
vs. NT1721; no significant difference for control vs. romidepsin: p = 0.317). (C) Picture of HuT78 tumors from the treated
mice. (D) NSG mice were injected s.c. with HH cells and treated by gavage on three consecutive days per week with
20 mg/kg NT1721 or the vehicle control. The differences in tumor volume were significant from day 14 through day 25
(p values < 0.0012). (E) Weight of HH tumors from individual mice (p = 0.0003). (F) Picture of representative HH tumors
from the treated mice. Figure 4. NT1721 suppressed tumor growth significantly better than romidepsin in a CTCL mouse model. (A) NSG mice
(n = 7/group) were injected s.c. with HuT78 cells and treated by gavage three times/week (on consecutive days) with
the vehicle control (30% solutol/3.3% DMSO in PBS), 20 mg/kg NT1721 or with 2 mg/kg romidepsin by I.P. injection
twice per week. Treatment with NT1721 reduced tumor volumes significantly better than romidepsin on Day 4, 7 and 11
(p values < 0.01). (B) Weight of HuT78 tumors from individual mice (p = 0.0003 for control vs. NT1721 and for romidepsin
vs. NT1721; no significant difference for control vs. romidepsin: p = 0.317). (C) Picture of HuT78 tumors from the treated
mice. (D) NSG mice were injected s.c. with HH cells and treated by gavage on three consecutive days per week with
20 mg/kg NT1721 or the vehicle control. The differences in tumor volume were significant from day 14 through day 25
(p values < 0.0012). (E) Weight of HH tumors from individual mice (p = 0.0003). (F) Picture of representative HH tumors
from the treated mice. 3.5. 3.4. NT1721 Suppressed Tumor Growth Significantly Better Than Romidepsin in CTCL
Mouse Models DMSO-Free Formulation of NT1721 Potently Decreased CTCL Tumor Growth To assess the efficacy of NT1721 under more clinically relevant conditions we used a
DMSO-free formulation of NT1721 for the treatment of mice bearing CTCL tumors: NT1721
was suspended in Ora-Blend containing 10% saline. Mice bearing HuT78 tumors were
treated with the vehicle control, 30 mg/kg five times per week or 100 mg/kg NT1721 twice
per week. As shown in Figure 5A–C, treatment with 30 mg/kg or 100 mg/kg NT1721
reduced the tumor volume and tumor weight significantly by 70% and 91%, respectively
(p < 0.0001). No weight loss or other obvious signs of toxicity were observed with this
formulation and drug schedule. In addition, mice bearing HH tumors were treated once
or twice per week with 100 mg/kg NT1721 (in Ora-Blend). As shown in Figure 5D–F, the
treatment led to a significant reduction in tumor growth by 35% and 55%, respectively. Taken together, the results show that treatment with NT1721 administered in a DMSO-free,
more clinically relevant formulation potently suppressed CTCL tumor growth in mice. 10 of 15 Cancers 2021, 13, 3367 Figure 5. In vivo efficacy of a DMSO-free formulation of NT1721. (A–C) NSG mice bearing HuT78 tumors were treated
with 30 mg/kg (five times per week), 100 mg/kg NT1721 (twice per week) or the vehicle control (Ora-Blend containing
10% saline). (A) Tumor volume. (B) Representative mice and tumors from the treatment and control groups. (C) Tumor
weight in treated and control mice. The differences between control and treatment groups as well as between the treatment
groups were statistically significant (p < 0.0001). (D–F) NSG mice bearing HH tumors were treated with 100 mg/kg NT1721
once a week, 100 mg/kg NT1721 twice per week or the vehicle control (Ora-Blend containing 10% saline). (D) Tumor
volume. (E) Representative mice from the treatment and control groups. (F) Tumor weight in treated and control mice. The
differences between control and treatment groups were statistically significant (control compared to 100 mg/kg 1×/week:
p = 0.021 and control compared to 100 mg/kg 2×/week: p = 0.0002). o efficacy of a DMSO-free formulation of NT1721. (A–C) NSG mice bearing HuT78 tumors were treated Figure 5. In vivo efficacy of a DMSO-free formulation of NT1721. (A–C) NSG mice bearing HuT78 tumors were treated
with 30 mg/kg (five times per week), 100 mg/kg NT1721 (twice per week) or the vehicle control (Ora-Blend containing
10% saline). 3.4. NT1721 Suppressed Tumor Growth Significantly Better Than Romidepsin in CTCL
Mouse Models (A) Tumor volume. (B) Representative mice and tumors from the treatment and control groups. (C) Tumor
weight in treated and control mice. The differences between control and treatment groups as well as between the treatment
groups were statistically significant (p < 0.0001). (D–F) NSG mice bearing HH tumors were treated with 100 mg/kg NT1721
once a week, 100 mg/kg NT1721 twice per week or the vehicle control (Ora-Blend containing 10% saline). (D) Tumor
volume. (E) Representative mice from the treatment and control groups. (F) Tumor weight in treated and control mice. The
differences between control and treatment groups were statistically significant (control compared to 100 mg/kg 1×/week:
p = 0.021 and control compared to 100 mg/kg 2×/week: p = 0.0002). Figure 5. In vivo efficacy of a DMSO-free formulation of NT1721. (A–C) NSG mice bearing HuT78 tumors were treated
with 30 mg/kg (five times per week), 100 mg/kg NT1721 (twice per week) or the vehicle control (Ora-Blend containing
10% saline). (A) Tumor volume. (B) Representative mice and tumors from the treatment and control groups. (C) Tumor
weight in treated and control mice. The differences between control and treatment groups as well as between the treatment
groups were statistically significant (p < 0.0001). (D–F) NSG mice bearing HH tumors were treated with 100 mg/kg NT1721
once a week, 100 mg/kg NT1721 twice per week or the vehicle control (Ora-Blend containing 10% saline). (D) Tumor
volume. (E) Representative mice from the treatment and control groups. (F) Tumor weight in treated and control mice. The
differences between control and treatment groups were statistically significant (control compared to 100 mg/kg 1×/week:
p = 0.021 and control compared to 100 mg/kg 2×/week: p = 0.0002). 3.6. In Vivo Combinations of NT1721 and Gemcitabine Suppressed CTCL Tumor Growth Better
Than the Single Agents 3.6. In Vivo Combinations of NT1721 and Gemcitabine Suppressed CTCL Tumor Growth Better
Than the Single Agents To determine if combinations of NT1721 with gemcitabine, a clinically used drug for
CTCL treatment [39], could lead to improved anticancer responses, an in vitro viability
assay was employed. As shown in Figure 6A, a combination of NT1721 with gemcitabine
decreased the cell viability of HuT78 cells significantly better than the single drugs alone. To investigate what might cause this effect we assessed the expression of proteins
related to apoptosis induction as well as GLI1 in HuT78 samples that were treated with
NT1721, gemcitabine, or the drug combination. As shown in Figure 6B, the expression
of cleaved PARP, pERK, and γH2AX was increased in samples treated with the drug
combination compared to samples treated with the single agents. This result suggests that
the drug combination may increase apoptosis induction and hence decrease cell viability. Treatment with the drug combination also seemed to slightly enhance GLI1 downregulation
while treatment with gemcitabine alone did not affect GLI1 expression. To determine if combinations of NT1721 with gemcitabine, a clinically used drug for
CTCL treatment [39], could lead to improved anticancer responses, an in vitro viability
assay was employed. As shown in Figure 6A, a combination of NT1721 with gemcitabine
decreased the cell viability of HuT78 cells significantly better than the single drugs alone. To investigate what might cause this effect we assessed the expression of proteins
related to apoptosis induction as well as GLI1 in HuT78 samples that were treated with
NT1721, gemcitabine, or the drug combination. As shown in Figure 6B, the expression
of cleaved PARP, pERK, and γH2AX was increased in samples treated with the drug
combination compared to samples treated with the single agents. This result suggests that
the drug combination may increase apoptosis induction and hence decrease cell viability. Treatment with the drug combination also seemed to slightly enhance GLI1 downregulation
while treatment with gemcitabine alone did not affect GLI1 expression. 11 of 15 Cancers 2021, 13, 3367 Figure 6. In vivo combination of NT1721 with gemcitabine. (A) Cell viability of HuT78 cells treated with NT1721,
gemcitabine or a combination of the two drugs. (B) Protein expression of GLI1 and proteins related to apoptosis iHut78
H. Induction (cleaved PARP, pERK, γH2A.x) in HuT78 cells treated with NT1721, gemcitabine or a combination of the
two drugs for 48 h. 3.6. In Vivo Combinations of NT1721 and Gemcitabine Suppressed CTCL Tumor Growth Better
Than the Single Agents (C) Tumor weight in mice treated with NT1721, gemcitabine, a combination of the two drugs with the
vehicle controls (Ora-Blend containing 10% saline or PBS, respectively) for 3 weeks. Representative HuT78 tumors treated
with the single drugs or drug combinations are shown on the right side. (D) Expression of GLI1 and proteins related to
apoptosis iHut78 H. Induction (BCL2, p21, pERK, γH2AX) in individual mice treated with 10 mg/kg NT1721, 20 mg/kg
gemcitabine or the drug combination for 3 weeks. Original Western Blots of Figure 6B,D available in Figure S6. vo combination of NT1721 with gemcitabine. (A) Cell viability of HuT78 cells treated with NT1721, Figure 6. In vivo combination of NT1721 with gemcitabine. (A) Cell viability of HuT78 cells tre Figure 6. In vivo combination of NT1721 with gemcitabine. (A) Cell viability of HuT78 cells treated with NT1721,
gemcitabine or a combination of the two drugs. (B) Protein expression of GLI1 and proteins related to apoptosis iHut78
H. Induction (cleaved PARP, pERK, γH2A.x) in HuT78 cells treated with NT1721, gemcitabine or a combination of the
two drugs for 48 h. (C) Tumor weight in mice treated with NT1721, gemcitabine, a combination of the two drugs with the
vehicle controls (Ora-Blend containing 10% saline or PBS, respectively) for 3 weeks. Representative HuT78 tumors treated
with the single drugs or drug combinations are shown on the right side. (D) Expression of GLI1 and proteins related to
apoptosis iHut78 H. Induction (BCL2, p21, pERK, γH2AX) in individual mice treated with 10 mg/kg NT1721, 20 mg/kg
gemcitabine or the drug combination for 3 weeks. Original Western Blots of Figure 6B,D available in Figure S6. Figure 6. In vivo combination of NT1721 with gemcitabine. (A) Cell viability of HuT78 cells treated with NT1721,
gemcitabine or a combination of the two drugs. (B) Protein expression of GLI1 and proteins related to apoptosis iHut78
H. Induction (cleaved PARP, pERK, γH2A.x) in HuT78 cells treated with NT1721, gemcitabine or a combination of the
two drugs for 48 h. (C) Tumor weight in mice treated with NT1721, gemcitabine, a combination of the two drugs with the
vehicle controls (Ora-Blend containing 10% saline or PBS, respectively) for 3 weeks. Representative HuT78 tumors treated
with the single drugs or drug combinations are shown on the right side. (D) Expression of GLI1 and proteins related to
apoptosis iHut78 H. 3.6. In Vivo Combinations of NT1721 and Gemcitabine Suppressed CTCL Tumor Growth Better
Than the Single Agents Induction (BCL2, p21, pERK, γH2AX) in individual mice treated with 10 mg/kg NT1721, 20 mg/kg
gemcitabine or the drug combination for 3 weeks. Original Western Blots of Figure 6B,D available in Figure S6. To evaluate the efficacy of NT1721, gemcitabine, and the drug combination in vivo,
mice bearing HuT78 tumors were treated with 10 mg/kg NT1721 (in Ora-Blend, twice
per week), 10 mg/kg gemcitabine (twice per week), 20 mg/kg gemcitabine (twice per
week) or the respective combinations. A comparison of the in vivo efficacy of NT1721
and gemcitabine showed that NT1721 significantly decreased tumor growth by 55%. Both
10 and 20 mg/kg gemcitabine only non-significantly decreased tumor growth by ~15%
(Figure 6C). Better suppression of tumor growth by NT1721 was also observed in the other
CTCL mouse model (HH tumors). As shown in Figure S3, NT1721 (100 mg/kg, 1×/week)
decreased HH tumor growth by 52%, while gemcitabine (100 mg/kg, 2×/week) led to a
45% reduction. However, the difference between the two drugs did not reach statistical
significance in this tumor model. Taken together, these results show that NT1721 led to a
better suppression of CTCL tumor growth at a lower dose compared to gemcitabine. pp
g
p
g
Next, efficacies of the drug combinations (10 mg/kg NT1721 plus either 10 mg/kg
or 20 mg/kg gemcitabine) against Hut78 tumors were compared. Mice treated with the
drug combinations displayed 65% and 76% smaller tumors, respectively, while NT1721
alone only led to a 45% reduction (Figure 6C). The differences were significant: p = 0.0012
for NT1721 vs. NT1721 plus 10 mg/kg gemcitabine; p = 0.0003 for NT1721 vs. NT1721 plus Cancers 2021, 13, 3367 12 of 15 12 of 15 20 mg/kg gemcitabine. Thus, these results indicate that the drug combinations reduced
tumor growth significantly better than the single drugs alone. 20 mg/kg gemcitabine. Thus, these results indicate that the drug combinations reduced
tumor growth significantly better than the single drugs alone. g
g
y
g
g
We then determined the expression of proteins related to the hedgehog pathway
(GLI1, BCL2) and to apoptosis induction in individual mice treated with 10 mg/kg NT1721,
20 mg/kg gemcitabine or the drug combination. As shown in Figure 6D, NT1721 decreased
the expression of GLI1 and antiapoptotic BCL2 and increased the expression of apoptosis
induction-related proteins (p21, pERK, γH2AX), thus recapitulating what was observed
in vitro (Figures 3B and 6B). 4. Discussion Apoptosis resistance has been identified as a major cause for the accumulation of
malignant T cells in CTCL [22]. Patients with advanced disease have a poor prognosis with
low survivals rates of less than 4 years [40]. Since current treatment options for advanced
CTCL stages are rarely durable and remain largely palliative [4], new strategies for the
treatment of advanced CTCL are urgently needed. g
y
Here we investigated the anti-CTCL activity of an ETP, NT1721. Treatment of CTCL
cells derived from the blood of patients with either Sézary syndrome (Hut78) or aggres-
sive, extensively pretreated CTCL (HH) (ATCC.org) with NT1721 downregulated GLI1
transcription factor, which was associated with decreased viability, reduced proliferation
and increased apoptosis. This result aligns with previous reports showing that inhibition
of aberrantly activated hedgehog/GLI in various types of T cell lymphomas by hedgehog
inhibitors or GLI1 knockdown led to increased apoptosis and reduced proliferation and
viability [33,41–43]. Decreased pSTAT3 levels as well as reduced expression of downstream
targets were also observed in NT1721-treated cells. This agrees with a previous study show-
ing that GLI1 knockdown or inhibition leads to a reduction in pSTAT3 levels in T cell lym-
phomas including CTCL [33]. Aberrant STAT3 activation plays a major role in CTCL since
it has been linked to apoptosis resistance, progression, and inferior clinical outcome [23–25]. Both GLI1 expression and STAT3 activation are absent from healthy peripheral T cells
according to previous reports [24,33,43,44] and our results. This might partially explain
why NT1721 preferentially reduces the viability of CTCL cells. The NT1721-mediated
decreased GLI1 expression and STAT3 activation led to concentration-dependent downreg-
ulation of downstream antiapoptotic proteins, i.e., BCL2 and BCL-xL, which contributed to
apoptosis induction. Several recent studies have highlighted the importance of BCL2/BCL-
xL inhibition for efficient apoptosis induction in CTCL. Adding BCL2 or BCL2/BCL-xL
inhibitors to the treatment of CTCL cells with other agents (e.g., histone deacetylase or
bromodomain inhibitors) synergistically increased apoptosis induction [7,17,34,45,46]. In
contrast, no synergy was observed when BCL2-negative HH cells were treated with the
drug combinations [17,34]. Moreover, it has been shown that antiapoptotic BCL2 proteins
have a role in promoting hedgehog/GLI signaling by enhancing the turnover of a GLI
antagonist, SUFU tumor suppressor. This inhibits GLI-SUFU interaction and thus increases
the expression of GLI target genes such as BCL2 itself [47]. 3.6. In Vivo Combinations of NT1721 and Gemcitabine Suppressed CTCL Tumor Growth Better
Than the Single Agents BCL2 expression appeared to be decreased to a greater extend
in mice treated with the drug combination compared to mice treated with the single agents. Upregulation of pERK, which is essential for NT1721-mediated increased expression of
p21, yH2AX, cleaved PARP and cleaved CASP3 (Figure S2B), was elevated to a greater
extent in mice treated with the drug combination compared to mice treated with NT1721
alone (Figure 6D). Taken together these results indicate that combining gemcitabine with
NT1721 resulted in better antitumor effects compared to treatment with the single agents. This improved anticancer effect appears to be mediated by increased apoptosis. 4. Discussion Our in vivo results suggested that orally available, well-tolerated NT1721 inhibits
CTCL tumor growth significantly better than two clinically used drugs, i.e., romidepsin or
gemcitabine, in two CTCL mouse models. However, NT1721 reduced tumor growth in mice
bearing Hut78 tumors significantly better than in mice bearing HH tumors (85% and 60%
inhibition, respectively). Lower GLI1 and pSTAT3 levels as well as the lack of BCL2/BLC-
xl expression in HH cells might at least partially explain the lower in vitro sensitivity Cancers 2021, 13, 3367 13 of 15 13 of 15 and relatively lower in vivo response to NT1721 compared to Hut78 cells. In summary,
NT1721 may be a promising new agent for the treatment of CTCLs overexpressing GLI1
and activated STAT3 either as a single agent or in combination with gemcitabine. and relatively lower in vivo response to NT1721 compared to Hut78 cells. In summary,
NT1721 may be a promising new agent for the treatment of CTCLs overexpressing GLI1
and activated STAT3 either as a single agent or in combination with gemcitabine. 5. Conclusions NT1721 potently induces apoptosis in CTCL in vitro and in vivo. Orally available,
well-tolerated NT1721 reduces CTCL tumor growth better than two drugs that are currently
clinically used for CTCL treatment (romidepsin and gemcitabine). Moreover, combinations
of NT1721 with gemcitabine suppress CTCL growth better than the single agents. Thus,
our results provide a rationale for testing the anti-CTCL efficacy of NT1721 in the clinic
either as a single agent or in combination with gemcitabine. A patent related to this work was filed by City of Hope. A patent related to this work was filed by City of Hope. Supplementary Materials: The following are available online at https://www.mdpi.com/article/
10.3390/cancers13133367/s1. Figure S1: γH2AX expression. CTCL cells were treated with NT1721 as
indicated for 24 h. γH2AX expression was detected in the cytoplasmic and nuclear extracts. Tubulin
and TBP expression were used as loading controls; Figure S2: Combining NT1721 with an ERK
inhibitor reduced apoptosis induction in HuT78 cells. (A) Protein expression in HuT78 cells treated
with NT1721, the ERK inhibitor (SCH772984) or a combination. (B) Annexin V staining of HuT78
cells treated with NT1721, SCH772984 or combinations of both agents; Figure S3: In vivo efficacies
of NT171 (in DMSO-free formulation) and gemcitabine against HH tumors. The mice were treated
with NT1721, gemcitabine or the vehicle controls as indicated. (A) Tumor volume. (B) Tumor weight. (C) Picture of representative tumors from treatment and control groups; Figure S4: Original Western
Blots of Figure 2A,C; Figure S5: Original Western Blots of Figure 3B,C; Figure S6: Original Western
Blots of Figure 6B,D. Author Contributions: Conceptualization, C.M.K., L.E.O. and D.A.H.; methodology, formal analysis,
investigation, M.L., C.M.K., S.Y.; resources: J.X.; writing—original draft preparation, C.M.K.; writing—
review and editing, all authors; supervision, D.A.H.; funding acquisition, L.E.O. and D.A.H. All
authors have read and agreed to the published version of the manuscript. Funding: The work was supported by the A. Gary Anderson Family Foundation (to D.A.H.), the
Panda Charitable Foundation (to D.A.H.), and NIH grant R01GM098601 (to L.E.O.). Research
reported in this publication included work performed in the Drug Discovery and Structural Bi-
ology, Analytical Flow Cytometry, Pathology, and Light Microscopy Cores that are supported by
the National Cancer Institute of the National Institutes of Health Cancer Center Support Grant
(P30CA33572). Institutional Review Board Statement: Mouse care and experimental procedures were performed
under pathogen-free conditions in accordance with approved protocols from the institutional animal
care and use committee of City of Hope National Medical Center. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in this article. 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
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30. Duvic, M.; Talpur, R.; Wen, S.; Kurzrock, R.; David, C.L.; Apisarnthanarax, N. Phase II evaluation of gemcitabine monotherapy
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32. Baumann, M.; Dieskau, A.P.; Loertscher, B.M.; Walton, M.C.; Nam, S.; Xie, J.; Horne, D.; Overman
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Epidithiodioxopiperazine Alkaloids with Promising and Antitumor Activity. Chem. Sci. 2015, 6, 4451–4457. [CrossRef]
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Geng L ; Lu K ; Li P; Li X ; Zhou X ; Li Y; Wang X GLI1 inhibitor GANT61 exhibits antitumor efficacy in T cell lymphoma Epidithiodioxopiperazine Alkaloids with Promising and Antitumor Activity. Chem. Sci. 2015, 6, 4451–4457. [CrossRef]
33. Geng, L.; Lu, K.; Li, P.; Li, X.; Zhou, X.; Li, Y.; Wang, X. GLI1 inhibitor GANT61 exhibits antitumor efficacy in T-cell lymphoma
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33. Geng, L.; Lu, K.; Li, P.; Li, X.; Zhou, X.; Li, Y.; Wang, X. GLI1 inhibitor GANT61 exhibits antitumor efficacy in T-cell lymphoma
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34. Chen, J.; Fiskus, W.; Eaton, K.; Fernandez, P.; Wang, Y.; Rao, R.; Lee, P.; Joshi, R.; Yang, Y.; Kolhe, R.; et al. References Med Sci. Monit. Int. Med J. Exp. Clin. Res. 2019, 25, 8579–8586. [CrossRef] 44. Van Kester, M.S.; Out-Luiting, J.J.; von dem Borne, P.A.; Willemze, R.; Tensen, C.P.; Vermeer, M.H. Cucurbitacin I inhibits Stat3
and induces apoptosis in Sezary cells. J. Investig. Dermatol. 2008, 128, 1691–1695. [CrossRef] [PubMed] 45. Cyrenne, B.M.; Lewis, J.M.; Weed, J.G.; Carlson, K.R.; Mirza, F.N.; Foss, F.M.; Girardi, M. Synergy of BCL2 and histone deacetylase
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46. Yumeen, S.; Mirza, F.N.; Lewis, J.M.; King, A.L.O.; Kim, S.R.; Carlson, K.R.; Umlauf, S.R.; Surovtseva, Y.V.; Foss, F.M.; Girardi, M. JAK inhibition synergistically potentiates BCL2, BET, HDAC, and proteasome inhibition in advanced CTCL. Blood Adv. 2020, 4,
2213–2226. [CrossRef] [PubMed] 45. Cyrenne, B.M.; Lewis, J.M.; Weed, J.G.; Carlson, K.R.; Mirza, F.N.; Foss, F.M.; Girardi, M. Synergy of BCL2 and histone deacetylase
inhibition against leukemic cells from cutaneous T-cell lymphoma patients. Blood 2017, 130, 2073–2083. [CrossRef] [PubMed]
46. Yumeen, S.; Mirza, F.N.; Lewis, J.M.; King, A.L.O.; Kim, S.R.; Carlson, K.R.; Umlauf, S.R.; Surovtseva, Y.V.; Foss, F.M.; Girardi, M. inhibition against leukemic cells from cutaneous T cell lymphoma patients. Blood 2017, 130, 2073 2083. [CrossRef] [PubMed]
46. Yumeen, S.; Mirza, F.N.; Lewis, J.M.; King, A.L.O.; Kim, S.R.; Carlson, K.R.; Umlauf, S.R.; Surovtseva, Y.V.; Foss, F.M.; Girardi, M. JAK inhibition synergistically potentiates BCL2, BET, HDAC, and proteasome inhibition in advanced CTCL. Blood Adv. 2020, 4,
2213–2226. [CrossRef] [PubMed] 47. Wu, X.; Zhang, L.S.; Toombs, J.; Kuo, Y.C.; Piazza, J.T.; Tuladhar, R.; Barrett, Q.; Fan, C.W.; Zhang, X.; Walensky, L.D.; et al. Extra-mitochondrial prosurvival BCL-2 proteins regulate gene transcription by inhibiting the SUFU tumour suppressor. Nat. Cell
Biol. 2017, 19, 1226–1236. [CrossRef]
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https://openalex.org/W4392570306
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https://bop.unibe.ch/linguistik-online/article/download/10789/13893
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Italian
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La fraseologia nei dizionari istrioto-italiano
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Linguistik online
| 2,024
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cc-by
| 10,506
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Abstract In this paper, the treatment of phraseological elements in four bilingual dictionaries of Istriot
varieties – Gallesanese, Vallese, and Rovignese – and Italian is analyzed. First, an overview of
the sociolinguistic situation of Istriot in the complex linguistic reality of Croatian Istria is
provided. This is followed by a concise outline of Istriot lexicography. In the empirical part,
after describing the analyzed dictionaries, the research results of the treatment of phraseology
in the latter are presented and discussed. Finally, some concluding remarks are made. 1 Per una discussione sul concetto di “esperto” in lessicografia, cf. Melchior (2024: 233–235), più in generale sul
rapporto esperti/non esperti con riferimento al sapere e al discorso sulla lingua, cf. i contributi raccolti in Hoffmei-
ster/Hundt/Naths (2021). 1
Introduzione Nel presente articolo si analizzerà il trattamento degli elementi fraseologici in quattro dizionari
bilingui di varietà istriote – gallesanese, vallese e rovignese – e italiano. Innanzitutto, si illu-
strerà a grandi linee la situazione sociolinguistica dell’istrioto nella complessa realtà linguistica
dell’Istria croata. Si delineerà poi brevemente un profilo della lessicografia istriota, spesso
opera di non esperti,1 per passare quindi all’analisi empirica. Dopo una descrizione dei voca-
bolari in oggetto si procederà alla presentazione e discussione dei risultati della ricerca sul
trattamento della fraseologia negli stessi e, infine, ad alcune considerazioni conclusive. Luca Melchior (Klagenfurt a. W./Graz) Luca Melchior (Klagenfurt a. W./Graz) 2 Per quanto assai ridotta, la diffusione territoriale dell’istrioto è dunque più ampia di quanto indicato ad es. da
Banfi (2003: 629), che lo vedeva parlato solamente nelle località di Dignano e Rovigno. L’istrioto Parlando di istrioto ci si riferisce all’unico gruppo di idiomi autoctoni preveneti della penisola
istriana, la cui estensione territoriale, un tempo verosimilmente estesa a un’area più ampia
dell’Istria meridionale, al di sotto del Canale di Leme, si è ridotta ormai a sei – secondo alcune
stime appena cinque – località dell’Istria meridionale, come visualizzato nella cartina rappre-
sentata nella figura 1.2 Sia il termine istrioto, introdotto da Graziadio Isaia Ascoli (cf. Ascoli 1873: 436), cui si deve
“il primo inquadramento scientifico, nel quale si colgono in nuce tutti gli elementi sviluppati
nelle analisi successive” (Ursini 1989: 537), sia il più recente termine istroromanzo proposto
da Petar Skok (1943) sono termini dotti, che non corrispondono alle denominazioni in uso in
loco. Tradizionalmente i sei idiomi vengono infatti indicati ognuno con un suo nome, in genere 1 Per una discussione sul concetto di “esperto” in lessicografia, cf. Melchior (2024: 233–235), più in generale sul
rapporto esperti/non esperti con riferimento al sapere e al discorso sulla lingua, cf. i contributi raccolti in Hoffmei-
ster/Hundt/Naths (2021). 2 Per quanto assai ridotta, la diffusione territoriale dell’istrioto è dunque più ampia di quanto indicato ad es. da
Banfi (2003: 629), che lo vedeva parlato solamente nelle località di Dignano e Rovigno. Linguistik online 125, 1/24 − http://dx.doi.org/10.13092/lo.125.10789
CC by 3.0 118 Linguistik online 125, 1/24 Figura 1: Località istriotofone3 Figura 1: Località istriotofone3 deonomastico, derivante dal nome della località in cui lo si parla: si ha dunque, procedendo da
nord a sud, il ruvigniʃ ‘rovignese’ a Rovinj-Rovigno, il dignanìʃ ‘dignanese’ o boumbaro ‘bum-
baro’ a Vodnjan-Dignano, il valeʃ ‘vallese’ a Bale-Valle, il galiʃaneʃ ‘gallesanese’ a Galižana-
Gallesano, il faʃaneʃ ‘fasanese’ a Fažana-Fasana e il sisaneʃ ‘sissanese’ a Šišan-Sissano. Più in
generale, i parlanti sembrano riferirsi alla propria varietà semplicemente con il termine dialetto. I sei idiomi sono sostanzialmente privi di codificazione sia a livello locale sia, e ancor di più, a
livello di una norma di riferimento sovralocale. Considerato lingua “severly endangered”
dall’UNESCO (cf. Moseley 32010: 40), l’istrioto nel suo complesso conta attualmente, secondo
le stime di Cergna (2014: 317), “non più di circa 1070 individui” parlanti attivi residenti in
Istria,4 a fronte di stime di circa 20.000 parlanti a inizio 1800 (cf. Buršić Giudici 2017: 59). 4 Per un totale di circa duemila potenziali parlanti, considerando anche gli istriotofoni emigrati fuori Istria e fuori
Croazia (cf. Cergna 2014: 317). 3 L’immagine 1 è stata collocata sulla presente pagina per motivi di spazio e su richiesta delle curatrici E. Autelli
e C. Konecny. 3 L’immagine 1 è stata collocata sulla presente pagina per motivi di spazio e su richiesta delle curatrici E. Autelli
e C. Konecny.
4 Per un totale di circa duemila potenziali parlanti, considerando anche gli istriotofoni emigrati fuori Istria e fuori
Croazia (cf. Cergna 2014: 317). L’istrioto In
realtà, lo “stato di salute” nelle sei località in cui si parla(va)no idiomi istrioti è piuttosto di-
verso: mentre per Valle e Gallesano la trasmissione intergenerazionale pare resistere (come deonomastico, derivante dal nome della località in cui lo si parla: si ha dunque, procedendo da
nord a sud, il ruvigniʃ ‘rovignese’ a Rovinj-Rovigno, il dignanìʃ ‘dignanese’ o boumbaro ‘bum-
baro’ a Vodnjan-Dignano, il valeʃ ‘vallese’ a Bale-Valle, il galiʃaneʃ ‘gallesanese’ a Galižana-
Gallesano, il faʃaneʃ ‘fasanese’ a Fažana-Fasana e il sisaneʃ ‘sissanese’ a Šišan-Sissano. Più in
generale, i parlanti sembrano riferirsi alla propria varietà semplicemente con il termine dialetto. I sei idiomi sono sostanzialmente privi di codificazione sia a livello locale sia, e ancor di più, a
livello di una norma di riferimento sovralocale. Considerato lingua “severly endangered”
dall’UNESCO (cf. Moseley 32010: 40), l’istrioto nel suo complesso conta attualmente, secondo
le stime di Cergna (2014: 317), “non più di circa 1070 individui” parlanti attivi residenti in
Istria,4 a fronte di stime di circa 20.000 parlanti a inizio 1800 (cf. Buršić Giudici 2017: 59). In
realtà, lo “stato di salute” nelle sei località in cui si parla(va)no idiomi istrioti è piuttosto di-
verso: mentre per Valle e Gallesano la trasmissione intergenerazionale pare resistere (come ISSN 1615-3014 Luca Melchior: La fraseologia nei dizionari istrioto-italiano 119 mostrano dati di una ricerca inedita effettuata dallo scrivente con Isabella Matticchio nel 2021),
a Dignano5 la lingua sembra fortemente in pericolo, andando ad assumere funzioni postverna-
colari (cf. Shandler 2004) più che comunicative. Per l’ultima località, Fasana, Cergna (2014:
317) la indica addirittura come completamente scomparsa ed effettivamente, nel corso del pro-
getto DERSII – Documentazione e rivitalizzazione dei sei idiomi istrioti, attivo dal 2013, non
sono stati individuati parlanti di fasanese. Una ricerca più recente riguarda Sissano: qui si regi-
stra un deciso calo sia di uso sia di trasmissione intergenerazionale del sissanese a favore di
istroveneto, ciacavo e croato standard (cf. Giudici 2018: 103–105), ma al contempo offre dati
meno pessimistici di precedenti ricerche per quanto riguarda il numero di parlanti. Sia dal punto di vista linguistico sia da quello sociolinguistico i sei idiomi istrioti si trovano in
una situazione assai complessa. Dal primo, una classificazione di tali idiomi come parte del
continuum italoromanzo settentrionale o come gruppo autonomo è tuttora dibattuta6 – e non
scevra di implicazioni politiche –, come si vedrà anche più avanti. 5 In questa località si registra però un certo fiorire di produzioni letterarie in istrioto. 7 L’italiano standard, scrive Giudici (2018: 106) richiamandosi a Blagoni (2012: 452) “non ha mai avuto la forza
di imporsi al di fuori degli ambiti scolastici e istituzionali nemmeno durante il periodo fascista […], tantomeno
dopo la II Guerra Mondiale con il passaggio del territorio istriano al governo jugoslavo, è relegato all’ultimo posto,
di fatto escluso dalla sfera familiare”. Come riportato da Blagoni (2012: 452, corsivo nell’originale), infatti, “Ita-
lian o Talian” sono i termini utilizzati per indicare l’istroveneto, mentre per l’italiano standard si utilizza l’espres-
sione parlar in cicara, che si ritrova in forme simili anche nell’Italia settentrionale con il significato di ‘sforzarsi
di parlare in un italiano ricercato’. L’istrioto Lo spiccato plurilinguismo
che caratterizza l’Istria rende anche il quadro sociolinguistico non meno intricato: oltre al croato
standard, nelle località in cui si parla ancora istrioto, l’italiano ha co-ufficialità; queste due lin-
gue vanno a coprire il polo alto della situazione poliglossica. Fanno infatti parte del repertorio
sociale – e spesso di quello individuale dei parlanti di istrioto – anche varietà ciacave, ben
distanti dallo standard croato, nonché dialetti istroveneti locali, che sono di regola la prima
lingua dei parlanti appartenenti alla Comunità Nazionale Italiana (CNI)7 e lingua della quoti-
dianità comunicativa “romanza” e infine un istroveneto koineizzante, sorta di “lingua franca”
per gli “italofoni” della penisola istriana. Alla CNI appartengono di regola anche i parlanti
istrioto, che non di rado hanno a lungo considerato e talora ancora “considerano l’istrioto una
delle varianti dell’istroveneto” (Filipi 2009), subordinandolo sociolinguisticamente a questo
(sul senso di appartenenza, anche identitaria, cf. anche Filipi 2005).8 Come accennato, l’uso dei
termini istrioto e istroromanzo può prestarsi – e si è purtroppo prestato – a possibili interpreta-
zioni ideologico-politiche: mentre al primo, infatti, è stata attribuita talora la funzione di postu-
lare una continuità geolinguistica tra l’istrioto e i dialetti italoromanzi, al termine istroromanzo
è stata contestata una presunta affermazione dell’autonomia di questo gruppo (cf. al riguardo
Fanciullo 2011: 116s., ripreso anche in Covino 2019: 84s.).9 A prescindere da tali questioni, g
p (
)
7 L’italiano standard, scrive Giudici (2018: 106) richiamandosi a Blagoni (2012: 452) “non ha mai avuto la forza
di imporsi al di fuori degli ambiti scolastici e istituzionali nemmeno durante il periodo fascista […], tantomeno
dopo la II Guerra Mondiale con il passaggio del territorio istriano al governo jugoslavo, è relegato all’ultimo posto,
di fatto escluso dalla sfera familiare”. Come riportato da Blagoni (2012: 452, corsivo nell’originale), infatti, “Ita-
lian o Talian” sono i termini utilizzati per indicare l’istroveneto, mentre per l’italiano standard si utilizza l’espres-
sione parlar in cicara, che si ritrova in forme simili anche nell’Italia settentrionale con il significato di ‘sforzarsi
di parlare in un italiano ricercato’. ISSN 1615-3014
5 In questa località si registra però un certo fiorire di produzioni letterarie in istrioto.
6 Una discussione al riguardo in Filipi (2002).
7 L’italiano standard, scrive Giudici (2018: 106) richiamandosi a Blagoni (2012: 452) “non ha mai avuto la forza
di imporsi al di fuori degli ambiti scolastici e istituzionali nemmeno durante il periodo fascista […], tantomeno
dopo la II Guerra Mondiale con il passaggio del territorio istriano al governo jugoslavo, è relegato all’ultimo posto,
di fatto escluso dalla sfera familiare”. Come riportato da Blagoni (2012: 452, corsivo nell’originale), infatti, “Ita-
lian o Talian” sono i termini utilizzati per indicare l’istroveneto, mentre per l’italiano standard si utilizza l’espres-
sione parlar in cicara, che si ritrova in forme simili anche nell’Italia settentrionale con il significato di ‘sforzarsi
di parlare in un italiano ricercato’.
8 Sulla situazione linguistica e sociolinguistica dell’Istria, cf., tra gli altri, Blagoni/Poropat Jeletić/Blecich (2018).
Le iniziative di promozione dell’istrioto hanno comunque contribuito a far crescere la coscienza linguistica dei
parlanti, cf. anche Giudici (2018: 107–109).
9 Il che non significa che la scelta dell’uno o dell’altro termine implichi necessariamente una componente ideolo-
gico-politica; così Giudici (2022: 13s.) nega valore politico all’uso del termine istroromanzo da parte di Pavao
Tekavčić. In questo contributo si preferisce istrioto, che pare il termine maggiormente utilizzato nella ricerca. sione parlar in cicara, che si ritrova in forme simili anche nell Italia settentrionale con il significato di sforzarsi
di parlare in un italiano ricercato’.
8 Sulla situazione linguistica e sociolinguistica dell’Istria, cf., tra gli altri, Blagoni/Poropat Jeletić/Blecich (2018).
Le iniziative di promozione dell’istrioto hanno comunque contribuito a far crescere la coscienza linguistica dei
parlanti, cf. anche Giudici (2018: 107–109). 6 Una discussione al riguardo in Filipi (2002). 10 I quattro vocabolari, così come quello di Dalla Zonca (1978) e quelli, di varietà istrovenete, di Manzini/Rocchi
(1995), di Dussich (2008, 2019) e di Orbanić/Giudici (2009), sono liberamente scaricabili in formato PDF. Al
riguardo si vedano le rassegne bibliografiche a cura di Muljačić (1969, 1981, 1988, 1998, 2009) e di Buršić Giudici
(1996). Lessicografia istriota 3 Non solo esiste una notevole mole di lavori scientifici dedicati a diversi aspetti delle parlate
istriote,10 ma vi è anche una certa produzione lessicografica, in particolare a partire dalla fine
del secolo scorso, sebbene le parole di Tekavčić (1986: 5) non abbiano perso la loro attualità:
“non abbiamo ancora nemmeno un vocabolario moderno dell’insieme dei dialetti istroro-
manzi”, bensì esistono dizionari bilingui delle singole parlate (in coppia con l’italiano).11 Così
si hanno per la parlata di Valle il dizionario di Cernecca (1986) e quello di Cergna (2015), che
del primo “rappresenta la naturale continuazione” (Cergna 2015: 9); per il rovignese esiste il
ponderoso vocabolario in due volumi a cura di Pellizzer/Pellizzer (1992), mentre alla parlata di
Gallesano è dedicato il vocabolario di Balbi/Moscarda Budić (2003). Tutti i vocabolari pre-
sentano la direzione di consultazione varietà istriota > italiano, di cui costituiscono delle sorte
di repertori lessicali dal carattere più documentario che d’uso, come esplicitamente dichiarato
nella prefazione di Pellizzer/Pellizzer (1992: 15): Tuttavia sarebbe ingiusto non riconoscere la motivazione affettiva nella quale è da ricercarsi il
primo vigoroso impulso impresso alla volontà, se non di “fermare”, ma almeno di documentare
nel tempo, un aspetto della vita, quella specificatamente “ruvigniʃa”, nella dimensione che era ed
è, congiuntamente con i suoi usi e costumi e con il suo patrimonio culturale tout court, attraverso
la sua più autentica realtà storica e sociale: quella della comunicazione, in presenza di una
diaspora sofferta e mutilante su entrambi i versanti. 12 A quelli citati va aggiunto il Vocabolario giuliano (Rosamani 1958), che contiene anche elementi afferenti i
diversi idiomi istrioti (al riguardo, cf. Tekavčić 1986; Holtus 1987). 11 Crevatin (2015: 159) scrive al proposito della lessicografia istriota: “[l]’Istria meridionale, il cui dialetto è defi-
nito in genere ‘istrioto’, è coperta in maniera ragionevole anche se non completa”. tuttavia, si è visto che, percettivamente, i parlanti tendono a riconoscere la pertinenza delle
proprie varietà al sistema italoromanzo, senza peraltro disconoscerne le peculiarità. tuttavia, si è visto che, percettivamente, i parlanti tendono a riconoscere la pertinenza delle
proprie varietà al sistema italoromanzo, senza peraltro disconoscerne le peculiarità. L’istrioto g
p (
)
7 L’italiano standard, scrive Giudici (2018: 106) richiamandosi a Blagoni (2012: 452) “non ha mai avuto la forza
di imporsi al di fuori degli ambiti scolastici e istituzionali nemmeno durante il periodo fascista […], tantomeno
dopo la II Guerra Mondiale con il passaggio del territorio istriano al governo jugoslavo, è relegato all’ultimo posto,
di fatto escluso dalla sfera familiare”. Come riportato da Blagoni (2012: 452, corsivo nell’originale), infatti, “Ita-
lian o Talian” sono i termini utilizzati per indicare l’istroveneto, mentre per l’italiano standard si utilizza l’espres-
sione parlar in cicara, che si ritrova in forme simili anche nell’Italia settentrionale con il significato di ‘sforzarsi
di parlare in un italiano ricercato’. 8 Sulla situazione linguistica e sociolinguistica dell’Istria, cf., tra gli altri, Blagoni/Poropat Jeletić/Blecich (2018). Le iniziative di promozione dell’istrioto hanno comunque contribuito a far crescere la coscienza linguistica dei
parlanti, cf. anche Giudici (2018: 107–109). 8 Sulla situazione linguistica e sociolinguistica dell’Istria, cf., tra gli altri, Blagoni/Poropat Jeletić/Blecich (2018). Le iniziative di promozione dell’istrioto hanno comunque contribuito a far crescere la coscienza linguistica dei
parlanti, cf. anche Giudici (2018: 107–109). 9 Il che non significa che la scelta dell’uno o dell’altro termine implichi necessariamente una componente ideolo-
gico-politica; così Giudici (2022: 13s.) nega valore politico all’uso del termine istroromanzo da parte di Pavao
Tekavčić. In questo contributo si preferisce istrioto, che pare il termine maggiormente utilizzato nella ricerca. ISSN 1615-3014 Linguistik online 125, 1/24 120 3.1 Le somiglianze tra i vocabolari analizzati sono numerose. In tutti e quattro, la lingua utilizzata
nei testi esterni, nella descrizione microstrutturale e nei rinvii nella mediostruttura è l’italiano,
che è anche la lingua obiettivo dei dizionari, che, come detto, sono bilingui, ma monodirezio-
nali. Tutti i dizionari hanno una serie di testi esterni, che comprende una presentazione o intro-
duzione dell’autore/degli autori, una lista delle abbreviazioni e una bibliografia delle opere con-
sultate, sostituita in Cergna (2015) da una lista delle persone intervistate. A Pellizzer/Pellizzer
(1992) e a Cergna (2015) è premessa una presentazione per opera del linguista Franco Crevatin. In Balbi/Moscarda Budić (2003), premessa alla lista delle abbreviazioni (e dunque al lemmario)
vi è una sezione con cenni storici sulla varietà in oggetto; posposte al lemmario invece sono
alcune note grammaticali. Cernecca (1996) contiene un’appendice posposta al lemmario, nella
quale sono presentati i paradigmi di verbi regolari e irregolari, alcuni testi in vallese e un breve
riassunto dell’opera in croato (Sažetak). Sia Cernecca (1986) per Valle sia Balbi/Moscarda Budić (2003) per Gallesano hanno raccolto
il materiale linguistico sulla base di fonti esclusivamente orali – informanti, di regola anziani – ,
poiché affermano che il rispettivo idioma non conosce usi scritti. Pellizzer/Pellizzer (1992) per
Rovigno e Cergna (2015) per Valle si basano invece sia su fonti orali che scritte, ma mentre i
primi fanno uno spoglio il più completo possibile della scritturalità, di regola letteraria, rovi-
gnese, il secondo si basa sostanzialmente su “dieci Quaderni manoscritti di Giovanni Obrovaz
– ‘Zaneto’, compilati dallo stesso tra gli anni Sessanta e Settanta del Novecento ed oggi accu-
ratamente custoditi nell’archivio del Centro di ricerche storiche di Rovigno” (Cergna 2015: 10,
corsivo nell’originale), concepiti a loro volta come sorta di repertorio lessicale. I quattro dizio-
nari hanno una mole – e di conseguenza un lemmario – dalle dimensioni assai diverse tra loro:
140 pagine in totale Cernecca (1986), di cui 106 dedicate al lemmario, 293 pagine (di cui 266
di lemmario, con diverse illustrazioni) Balbi/Moscarda Budić (2003), 421 pagine Cergna
(2015), di cui 400 dedicate al lemmario (con alcune illustrazioni), e ben 1140 pagine Pelliz-
zer/Pellizzer (1992), di cui 1096 di lemmario (518 nel primo, 578 nel secondo volume). Diversa
non è solo l’estensione del lemmario, ma anche, come visto, la ricchezza di testi esterni, il
numero di abbreviazioni registrate – su cui si tornerà tra poco – e l’esaustività della microstrut-
tura. 13 Su tale vocabolario si veda Tekavčić (1986; con particolare riguardo alla trattazione di elementi fraseologici in
senso lato, ibd.: 14s.). (ibd: 15, corsivo nell’originale) Ai vocabolari citati si aggiungono inoltre l’edizione, nel 1978, del Vocabolario dignanese-ita-
liano, compilato dall’erudito istriano Giovanni Andrea Dalla Zonca a metà Ottocento, e infine
il Vocabolario italiano-rovignese di Benussi (2013) che, pur contenendo un’appendice a Pelliz-
zer/Pellizzer (1992) rovignese-italiano, è altrimenti concepito nella direzione opposta. Manca-
no repertori lessicografici delle varietà di Fasana e di Sissano. Per quest’ultima è in preparazio-
ne un repertorio etimologico e fraseologico (cf. Buršić Giudici 2018, 2021; Giudici/ Buršić
Giudici 2022).12 Con l’eccezione di Benussi (2013), tutti i vocabolari sono usciti per il Centro
di Ricerche Storiche di Rovigno, emanazione dell’Unione Italiana, che rappresenta i membri
della Comunità Nazionale Italiana di Croazia e Slovenia. Per lo stesso Centro di Ricerche sono
apparsi anche dizionari dei dialetti istroveneti di Capodistria (Manzini/Rocchi 1995), di Buie
d’Istria (Dussich 2008, 2019) e di Pola (Orbanić/Giudici 2009). Sebbene questi non si occupino
di varietà istriote, sono accomunabili ai primi non solo perché ne condividono la casa editrice,
ma perché gli autori degli uni e degli altri pertengono allo stesso ambiente culturale. I diversi 10 I quattro vocabolari, così come quello di Dalla Zonca (1978) e quelli, di varietà istrovenete, di Manzini/Rocchi
(1995), di Dussich (2008, 2019) e di Orbanić/Giudici (2009), sono liberamente scaricabili in formato PDF. Al
riguardo si vedano le rassegne bibliografiche a cura di Muljačić (1969, 1981, 1988, 1998, 2009) e di Buršić Giudici
(1996). 11 Crevatin (2015: 159) scrive al proposito della lessicografia istriota: “[l]’Istria meridionale, il cui dialetto è defi-
nito in genere ‘istrioto’, è coperta in maniera ragionevole anche se non completa”. ISSN 1615-3014 Luca Melchior: La fraseologia nei dizionari istrioto-italiano 121 dizionari mostrano chiaramente il debito reciproco che hanno tra loro. Nel presente contributo
si illustreranno i risultati dell’analisi dei quattro vocabolari con direzione istrioto > italiano
(Balbi/Moscarda Budić 2003 per il gallesanese, Cergna 2015; Cernecca 1986, entrambi per il
vallese; e Pellizzer/Pellizzer 1992 per il rovignese); in fase di ricerca non sono mancati tuttavia
numerosi confronti con i restanti. Si è scelto di escludere Dalla Zonca (1978)13 in quanto esso
non solo riporta uno stato della lingua decisamente anteriore agli altri, ma anche nella sua strut-
turazione risponde a criteri ottocenteschi; Benussi (2013) è stato invece escluso perché la dire-
zione di lemmatizzazione e ricerca è opposta a quella dei precedenti, non è dunque con questi
facilmente comparabile. 3.1 Così, mentre in Cernecca (1986), nella microstruttura, oltre all’indicazione della parte del
discorso o del genere (per i nomi) si trova pressoché solamente il traducente italiano, Sia Cernecca (1986) per Valle sia Balbi/Moscarda Budić (2003) per Gallesano hanno raccolto
il materiale linguistico sulla base di fonti esclusivamente orali – informanti, di regola anziani – ,
poiché affermano che il rispettivo idioma non conosce usi scritti. Pellizzer/Pellizzer (1992) per
Rovigno e Cergna (2015) per Valle si basano invece sia su fonti orali che scritte, ma mentre i
primi fanno uno spoglio il più completo possibile della scritturalità, di regola letteraria, rovi-
gnese, il secondo si basa sostanzialmente su “dieci Quaderni manoscritti di Giovanni Obrovaz – ‘Zaneto’, compilati dallo stesso tra gli anni Sessanta e Settanta del Novecento ed oggi accu-
ratamente custoditi nell’archivio del Centro di ricerche storiche di Rovigno” (Cergna 2015: 10,
corsivo nell’originale), concepiti a loro volta come sorta di repertorio lessicale. I quattro dizio-
nari hanno una mole – e di conseguenza un lemmario – dalle dimensioni assai diverse tra loro:
140 pagine in totale Cernecca (1986), di cui 106 dedicate al lemmario, 293 pagine (di cui 266
di lemmario, con diverse illustrazioni) Balbi/Moscarda Budić (2003), 421 pagine Cergna
(2015), di cui 400 dedicate al lemmario (con alcune illustrazioni), e ben 1140 pagine Pelliz-
zer/Pellizzer (1992), di cui 1096 di lemmario (518 nel primo, 578 nel secondo volume). Diversa
non è solo l’estensione del lemmario, ma anche, come visto, la ricchezza di testi esterni, il
numero di abbreviazioni registrate – su cui si tornerà tra poco – e l’esaustività della microstrut-
tura. Così, mentre in Cernecca (1986), nella microstruttura, oltre all’indicazione della parte del
discorso o del genere (per i nomi) si trova pressoché solamente il traducente italiano, ISSN 1615-3014 122 Linguistik online 125, 1/24 accompagnato, sporadicamente, da esempi, compresi tra le virgolette «» e non tradotti, in Pel-
lizzer/Pellizzer (1992) la microstruttura prevede esempi e attestazioni, con indicazione della
fonte e traduzione, confronti con altri idiomi (istrioti e non solo), talora l’etimologia o informa-
zioni di stampo enciclopedico. Tra questi due estremi si pongono Balbi/Moscarda Budić (2003)
e Cergna (2015), che presentano un buon apparato di esempi e attestazioni (con traduzione). Il trattamento della fraseologia Punto di partenza per l’analisi è stato il più corposo vocabolario di Pellizzer/Pellizzer (1992),
per poi allargare le osservazioni agli altri tre. L’analisi è stata effettuata in due modi: innanzi-
tutto, si è osservato come vengano utilizzate, nella microstruttura, le abbreviazioni che si rife-
riscono alla fraseologia, cioè quali elementi vengano da queste introdotti e quale sistematica vi
stia alla base; dall’altra parte è stato analizzato il trattamento dei fraseologismi menzionati sotto
determinati lemmi (càn, bàrca, lògo), se e quali marcature essi presentano e se vi sono eventuali
rimandi mediostrutturali. 3.2
Riferimenti espliciti alla fraseologia Solamente Pellizzer/Pellizzer (1992: 17) fanno esplicito, seppur indiretto, riferimento alla fra-
seologia nell’introduzione al loro lavoro. Essi scrivono infatti: “Certi lemmi vengono presentati
in più varianti, appunto perché si è voluto dare un quadro completo (alle volte anche contrad-
ditorio rispetto alla normativa linguistica) della situazione odierna del dialetto, alla quale si è
voluto subordinare anche la fraseologia.” A loro volta, Balbi/Moscarda Budić (2003: 10) si limitano ad affermare di aver raccolto anche
elementi fraseologici: “Non abbiamo preteso entrare nel campo dell’etimologia, poiché non di
nostra competenza, ma ci siamo limitate alla raccolta documentata di voci, ma anche frasi tipi-
che, detti, proverbi, modi di dire, usanze che riflettono momenti e aspetti della vita quotidiana,
contadina, familiare della nostra gente.” Con l’eccezione di Cernecca (1986), nei restanti vocabolari sono registrate, nella rispettiva lista,
abbreviazioni utilizzate nella microstruttura per indicare elementi fraseologici. Queste sono
“loc.” e “loc. avv.” in Cergna (2015: 14), “locuz.”, “locuz. avv.” e “loc. inf.” (glossata come
“locuzione infantile”) in Balbi/Moscarda Budić (2003: 15). In Pellizzer/Pellizzer (1992) vi è
una maggiore copia di abbreviazioni relative agli elementi fraseologici. Si trovano infatti
“espress.” per espressione ed espressivo; “loc., locuz.” per locuzione, “locuz. avv.” e “locuz. verb.” per locuzione avverbiale e locuzione verbale, oltre che “fras.” per fraseologia, fraseolo-
gico (ibd.: 21s.). L’abbondanza di soluzioni sembra creare più confusione che portare chiarezza,
anche se in realtà l’abbreviazione “espress.” viene usata solamente in casi in cui essa non va ad
assumere, nella microstruttura, la funzione dell’indicazione sintattico-semantica, ma si trova
nel commento semantico o introduce esempio d’uso, come nei casi di danàro e danèri (cf. figure 2–3). ISSN 1615-3014
Figura 2: Voce danàro (Pellizzer/Pellizzer 1992: 298)
Figura 3: Voce danèri (Pellizzer/Pellizzer 1992: 298) Figura 2: Voce danàro (Pellizzer/Pellizzer 1992: 298)
Figura 3: Voce danèri (Pellizzer/Pellizzer 1992: 298) ISSN 1615-3014 Luca Melchior: La fraseologia nei dizionari istrioto-italiano 123 In realtà, l’analisi delle voci mostra che in Balbi/Moscarda Budić (2003), nella microstruttura,
sia presente due volte anche “loc. avv.” (mentre l’abbreviazione “loc.” normalmente significa
‘località’); in Pellizzer/Pellizzer (1992), inoltre, modi di dire e proverbi sono introdotti dalla
nota “Detti e prov. rov.”. 14 L’allineamento spostato a destra nelle riproduzioni degli articoli lessicografici sta a indicare che l’articolo
stesso, nel dizionario, continua su una seconda colonna o una seconda pagina. 4.1 È stato possibile indagare l’uso delle abbreviazioni tramite una ricerca automatica delle stesse
nei file PDF. Dell’abbreviazione “espress.” e del suo uso in Pellizzer/Pellizzer (1992) si è detto
sopra. Per quanto riguarda l’abbreviazione “fras.”, essa si trova solamente tre volte, alle voci
Càiser, ecùte e ièsi (cf. figure 4–6). Interessante che nel primo caso tale abbreviazione introduca
un fraseologismo in dialetto bisiaco, portato a confronto con l’istrioto rovignese, e non per in-
trodurre quello in quest’ultimo idioma, che viene presentato solo come esempio. Per quanto
riguarda i fraseologismi alla voce ièsi, stupisce il fatto che essi siano presentati solamente sotto
il lemma della copula e non sotto quello degli altri elementi che li compongono. Figura 4: Voce Càiser (Pellizzer/Pellizzer 1992: 166)14
Figura 5: Voce ecùte (Pellizzer/Pellizzer 1992: 345s.) Figura 5: Voce ecùte (Pellizzer/Pellizzer 1992: 345s.) 14 L’allineamento spostato a destra nelle riproduzioni degli articoli lessicografici sta a indicare che l’articolo
stesso, nel dizionario, continua su una seconda colonna o una seconda pagina. ISSN 1615-3014 124 Linguistik online 125, 1/24 Figura 6: Voce ièsi (Pellizzer/Pellizzer 1992: 449s.) Figura 6: Voce ièsi (Pellizzer/Pellizzer 1992: 449s.) Nel caso di ecùte, la classificazione come fraseologismo non pare comprensibile: si tratta infatti
di un’interiezione monolessicale, formata da un avverbio e da un pronome oggetto, la cui fun-
zione può sicuramente essere formulaica, ma che non ricade sotto una definizione assai ampia
di fraseologismo. La caratterizzazione come “fras. avv.”, interpretabile come ‘fraseologismo
avverbiale’, pare inoltre essere un ἄπαξ nella lessicografia e fraseografia: L’abbreviazione “locuz. avv.” viene utilizzata in Pellizzer/Pellizzer (1992) per diversi tipi di
espressioni, in realtà solo in parte con valore avverbiale, come nei casi di a baloûchi, a bruʃaca-
meîʃa e in canpanièla, come si vede nelle figure 7–9. Figura 7: Voce baloûchi (Pellizzer/Pellizzer 1992: 88)
Figura 8: Voce bruʃacameîʃa (Pellizzer/Pellizzer 1992: 141) Figura 7: Voce baloûchi (Pellizzer/Pellizzer 1992: 88) ISSN 1615-3014
Figura 7: Voce baloûchi (Pellizzer/Pellizzer 1992: 88)
Figura 8: Voce bruʃacameîʃa (Pellizzer/Pellizzer 1992: 141) Figura 7: Voce baloûchi (Pellizzer/Pellizzer 1992: 88) Figura 8: Voce bruʃacameîʃa (Pellizzer/Pellizzer 1992: 141) Figura 8: Voce bruʃacameîʃa (Pellizzer/Pellizzer 1992: 141) 15 Fermo restando che biʃièfe è componente unicale, come giustamente nota un recensore del presente articolo,
sebbene tale caratteristica non sia rilevante per l’analisi presentata in questo punto. Sui componenti unicali cf. tra
gli altri Veland (2005, 2006) e Konecny (2018). L’indicazione dell’unicalità per gli elementi da questa caratteriz-
zati non è presente nei dizionari in esame, come si vede anche nella figura 24. 16 Solamente la maggiore trasparenza di dabànda rispetto a cusiveîa pare forse poter giustificare la decisione per
un trattamento diverso da parte degli autori del dizionario. ISSN 1615-3014 125 Luca Melchior: La fraseologia nei dizionari istrioto-italiano Figura 9: Voce canpanièla (Pellizzer/Pellizzer 1992: 181) Figura 9: Voce canpanièla (Pellizzer/Pellizzer 1992: 181) Per quanto riguarda la lemmatizzazione, in questi tre casi il fraseologismo è riportato sotto il
termine con maggiore valore lessicale, mentre la preposizione, parte integrale della locuzione,
viene indicata tra parentesi in coda al lemma, prima dell’indicazione sintattica. In altri casi,
strutturalmente non diversi da quelli appena discussi, la strutturazione dell’articolo lessicogra-
fico è diversa. Si veda il caso di a biʃièfe (figura 10): anche qui il fraseologismo è registrato
sotto l’elemento con valore lessicale, il quale viene già indicato come “locuz.”, salvo poi spe-
cificare “Usato nella locuz. avv.: a biʃièfe”. La strutturazione dell’articolo risulta dunque assai
diversa dalle precedenti, pur essendo gli elementi descritti, per struttura morfosintattica, pres-
soché identici.15 Figura 10: Voce biʃièfe (Pellizzer/Pellizzer 1992: 122) Figura 10: Voce biʃièfe (Pellizzer/Pellizzer 1992: 122) In altri casi ancora, l’indicazione “locuz. avv.” caratterizza elementi univerbali con valore av-
verbiale. Si osservi l’esempio in figura 11: Figura 11: Voce cusuveîa (Pellizzer/Pellizzer 1992: 289) Figura 11: Voce cusuveîa (Pellizzer/Pellizzer 1992: 289) Interessante è che, per elementi simili, si trovino anche soluzioni diverse, come nel caso di
dabànda (figura 12), senza che sia comprensibile quali ragioni stiano alla base della disparità
di trattamento:16 15 Fermo restando che biʃièfe è componente unicale, come giustamente nota un recensore del presente articolo,
sebbene tale caratteristica non sia rilevante per l’analisi presentata in questo punto. Sui componenti unicali cf. tra
gli altri Veland (2005, 2006) e Konecny (2018). L’indicazione dell’unicalità per gli elementi da questa caratteriz-
zati non è presente nei dizionari in esame, come si vede anche nella figura 24. 16 Solamente la maggiore trasparenza di dabànda rispetto a cusiveîa pare forse poter giustificare la decisione per
un trattamento diverso da parte degli autori del dizionario. 17 Seppure il rovignese pare sia storicamente, tra gli idiomi istrioti, l’unico a presentare una serie quasi completa
di pronomi enclitici (cf. Giudici 2022: 195); tuttavia tale tematica non viene trattata nella grammatica di Benussi
(2015), il che fa presumere “che questo tratto sia ormai uscito dall’uso” (Giudici 2022: 196). 18 Da notare però che la forma con pronome atono femminile ʃìla è trattata ben diversamente. Anch’essa è lem-
matizzata come tale, seguita da “(ùla)”, ma la microstruttura è la seguente: “f. verb. - Forma verbale contratta
corrispondente a: dove è lei. Ùla ʃìla, dove (lei) è?; i nu la trùvo, dùve ʃìla? non la trovo, dove è (lei)” (Pelliz-
zer/Pellizzer 1992: 911). ISSN 1615-3014 ISSN 1615-3014 126 Linguistik online 125, 1/24 Figura 12: Voce dabànda (Pellizzer/Pellizzer 1992: 293)
Infine, l’indicazione “locuz. avv.” viene utilizzata anche per elementi che non hanno valore
avverbiale, come nel caso seguente (figura 13): Fi
12 V
d bà d (P lli
/P lli
1992 293) Figura 12: Voce dabànda (Pellizzer/Pellizzer 1992: 293) Infine, l’indicazione “locuz. avv.” viene utilizzata anche per elementi che non hanno valore
avverbiale, come nel caso seguente (figura 13): Figura 13: Voce bài (Pellizzer/Pellizzer 1992: 84) Figura 13: Voce bài (Pellizzer/Pellizzer 1992: 84) L’abbreviazione “locuz. verb.” è presente un’unica volta; essa introduce, in realtà, una forma
interrogativa con inversione di verbo alla terza persona singolare (da ièsi ‘essere’) e pronome
soggetto enclitico maschile, che fa parte del verbo alla forma interrogativa, presentata nel con-
testo di una domanda e lemmatizzata come tale. La scelta di caratterizzare questo elemento
come locuzione verbale risulta incomprensibile; si può ipotizzare che l’inversione interrogativa,
cristallizzata e non più in uso,17 sia ritenuta poco riconoscibile ai parlanti.18 Figura 14: Voce ʃìlo (Pellizzer/Pellizzer 1992: 911) Figura 14: Voce ʃìlo (Pellizzer/Pellizzer 1992: 911) Sia “locuz.” che “locuz. avv.” vengono utilizzate però non solo immediatamente dopo il lemma,
ma anche all’interno della descrizione microstrutturale per introdurre elementi fraseologici
composti con il lemma in questione, come nell’esempio in figura 15, al lemma cuòrda. ISSN 1615-3014 127 Luca Melchior: La fraseologia nei dizionari istrioto-italiano Figura 15: Voce cuòrda (Pellizzer/Pellizzer 1992: 277) Figura 15: Voce cuòrda (Pellizzer/Pellizzer 1992: 277) Analoghe incoerenze si trovano anche in Balbi/Moscarda Budić (2003), dove peraltro il numero
di voci introdotte da una delle indicazioni elencate nell’indice delle abbreviazioni è assai limi-
tato: appena 31, di cui 28 da “locuz. avv.” e tre da “locuz.”. Si confronti il diverso trattamento
di a cavaloto, a cao e n batùda nelle figure 16–18. Figura 16: Voce cavaloto (Balbi/Moscarda Budić 2003: 56) Figura 16: Voce cavaloto (Balbi/Moscarda Budić 2003: 56) Figura 17: Voce cao (Balbi/Moscarda Budić 2003: 56) Figura 17: Voce cao (Balbi/Moscarda Budić 2003: 56) ISSN 1615-3014 128 Linguistik online 125, 1/24 Non vi sono lemmi marcati con l’abbreviazione “locuz. inf.”, presente tuttavia nell’indice delle
abbreviazioni. In alcuni casi l’uso dell’abbreviazione “locuz.” risulta difficilmente comprensi-
bile. Si osservi per esempio il caso in figura 19: qui “locuz.” caratterizza un secondo significato
del verbo. Fraseologico non è però l’utilizzo del verbo istrioto gallesanese, bensì il suo tradu-
cente italiano; a sua volta, però, l’esempio proposto viene tradotto con un altro fraseologismo
italiano: Figura 19: Voce ʃbafà (Balbi/Moscarda Budić 2003: 217) Figura 19: Voce ʃbafà (Balbi/Moscarda Budić 2003: 217) Più coerenza nella caraterrizzazione degli elementi introdotti dalle abbreviazioni “loc.” e “loc. avv.” mostra Cergna (2015), che però lemmatizza le espressioni fraseologiche talora nell’arti-
colo di uno degli elementi che le compongono, talaltra come lemmi autonomi, talaltra sotto
l’elemento lessicale, seguito dalla preposizione (o in alcuni casi da un verbo) tra parentesi. Si
confrontino le figure 20–23: ISSN 1615-3014
Figura 20: Voce ala una el can lavòra (Cergna 2015: 22)
Figura 21: Voce babalòca (Cergna 2015: 34)
Figura 22: Voce badinè (Cergna 2015: 35)
Figura 23: Voce basilà (Cergna 2015: 40) Figura 20: Voce ala una el can lavòra (Cergna 2015: 22) Figura 20: Voce ala una el can lavòra (Cergna 2015: 22) 19 Da non confondere con l’omonimo can ‘murice’. Figura 22: Voce badinè (Cergna 2015: 35) Luca Melchior: La fraseologia nei dizionari istrioto-italiano 129 Come si è accennato più sopra, Cernecca (1986) non contiene un indice delle abbreviazioni. Una scansione del testo nel documento PDF con possibili abbreviazioni relative a fraseologismi
permette di individuare un caso di utilizzo dell’abbreviazione “locuz.”, come nella figura 24: Come si è accennato più sopra, Cernecca (1986) non contiene un indice delle abbreviazioni. Una scansione del testo nel documento PDF con possibili abbreviazioni relative a fraseologismi
permette di individuare un caso di utilizzo dell’abbreviazione “locuz.”, come nella figura 24: 22 Nei lemmi pubblicati in anteprima del futuro dizionario di sassanese vi è il lemma can (cf. Buršic Giudici/Giu-
dici 2022: 129). Nella microstruttura dell’articolo vi è un elemento che pare avere carattere proverbiale (Tùti i càni
bàja) con rispettiva traduzione. Tale elemento non è introdotto da alcuna indicazione, ma è in corsivo come tutti
gli esempi presentati in questi stralci di dizionario. 21 Il vocabolario lemmatizza anche fiòl d’oùn cein, caratterizzato come “locuz.” e spiegato come “Forma attenuata
di ‘fiòl d’oûn can’” (Pellizzer/Pellizzer 1992: 374). 20 Che peraltro non sono tutti lemmatizzati: nel vocabolario non si trova infatti il lemma bòia. 4.2
Il trattamento dei fraseologismi all’interno degli articoli Oltre all’uso delle abbreviazioni, la ricerca ha previsto anche un’analisi esemplare del tratta-
mento dei fraseologismi all’interno degli articoli lessicografici. Si è scelto di indagare le voci
riguardanti tre sostantivi (barca, can ‘cane’,19 logo ‘luogo’), tre verbi (cantà ‘cantare’, magnà
‘mangiare’, ʃeî (o equivalente ʃì) ‘andare’) e tre aggettivi (alto ‘alto’, bon ‘buono’, viècio (o
equivalente vècio) ‘vecchio’). Punto di partenza è stato anche in questo caso il vocabolario rovignese di Pellizzer/Pellizzer
(1992), per poi verificare la presenza delle unità eventualmente identificate negli altri dizionari. Come si può vedere in figura 25, al lemma can sono presenti diversi elementi fraseologici in
senso lato: Figura 25: Voce can (Pellizzer/Pellizzer 1992: 176) Figura 25: Voce can (Pellizzer/Pellizzer 1992: 176) La presentazione di tali elementi non avviene in modo sistematico e coerente: a una serie di
unità fraseologiche (di tipo diverso) priva di qualunque indicazione sulla loro natura segue una
sezioni più breve introdotta da “Modi di dire, detti e prov. rov.”. In questa, oltre a due proverbi
si trova tuttavia anche una voce polirematica (can da vàche). La stessa voce è lemmatizzata
anche come voce a sé stante (Pellizzer/Pellizzer 1992: 179), ma non è registrata alla voce vàca. Solamente can barbòn e can da firma sono, infatti, presenti anche ai lemmi corrispondenti agli ISSN 1615-3014 130 Linguistik online 125, 1/24 altri elementi che li formano:20 il primo al lemma barbòn, ma sotto forma di esempio, il secondo
alla voce firma, a sua volta in un esempio che ha però la funzione di spiegazione semantica di
questo significato del lemma. Come can da vàche, anche fiòl d’oûn can è presentato inoltre
come lemma autonomo e caratterizzato come “locuz. scherz.” (ibd: 374).21 All’interno
dell’articolo relativo si trova un rimando mediostrutturale al lemma fiòl (non però a can). Alla
voce fiòl il fraseologismo è presente solo in un commento di stampo comparatistico, senza che
se ne spieghi il significato (cf. figura 26): Figura 26: Voce fiòl (Pellizzer 1992: 373s.) Figura 26: Voce fiòl (Pellizzer 1992: 373s.) Figura 26: Voce fiòl (Pellizzer 1992: 373s.) Cernecca (1986) presenta, alla voce can, il solo fraseologismo vita de can (cf. figura 27). Questo
è introdotto da virgolette, assieme all’espressione can del s’ciopo, dove can ha però significato
di ‘grilletto’.22 Più ricco è Balbi/Moscarda Budić (2003), che al lemma can presenta voci polirematiche, non
introdotte come categoria a sé stante, mentre Xe lavor de cani viene indicato come modo di dire
e Fiol de un can! come imprecazione. Segue una sezione di proverbi, alcuni dei quali corri-
spondono a quelli contenuti anche in Pellizzer/Pellizzer (1992). ISSN 1615-3014 131 Figura 29: Voce bàrca (Pellizzer/Pellizzer 1992: 95s.) Luca Melchior: La fraseologia nei dizionari istrioto-italiano Luca Melchior: La fraseologia nei dizionari istrioto-italiano Figura 28: Voce can (Balbi/Moscarda Budić 2003: 4 Figura 28: Voce can (Balbi/Moscarda Budić 2003: 45) Per quanto riguarda gli altri elementi che formano tali espressioni, le voci polirematiche non
sono riportate ai rispettivi lemmi; lavor, bando e fiol non sono registrati nemmeno come lemmi. Lemma a sé stante forma invece fiolduncan, univerbato, e indicato come aggettivo con il signi-
ficato di ‘bricconcello’; non vi sono rinvii alla voce can (cf. Balbi/Moscarda Budić 2003: 91). Cergna (2015: 69) non presenta elementi fraseologici alla voce can. Anche alla voce bàrca, troviamo diverse forme fraseologiche, incluse collocazioni e proverbi
– questi ultimi introdotti dall’indicazione “Modi di dire e prov. rov.”. Di tutte le unità fraseolo-
giche, solo Bàrca rùta cònti fàti e bàrca nìta nu vadàgna sono menzionati anche altrove, rispet-
tivamente alla voce rùto (Pellizzer/Pellizzer 1992: 793) e alla voce vadagnà (ibd.: 1099). Stupisce dunque il rinvio mediostrutturale a paròn, sotto il quale l’elemento fraseologico non è
presente (cf. ibd.: 658). Figura 29: Voce bàrca (Pellizzer/Pellizzer 1992: 95s.) Figura 29: Voce bàrca (Pellizzer/Pellizzer 1992: 95s.) ISSN 1615-3014 Linguistik online 125, 1/24 132 Cernecca (1986: 20) e Cergna (2015: 38) non hanno elementi fraseologici alla voce barca, che
non è lemmatizzata in Balbi/Moscarda Budić (2003: 28), dove si trova solo barcon.23 Al lemma lògo Pellizzer/Pellizzer (1992) riporta il fraseologismo ni fògo ni lògo e la colloca-
zione avì lògo, non introdotti da alcuna indicazione. Entrambi sono registrati soltanto al lemma
lògo. Anche in questo caso, le due espressioni sono presenti solo qui. Figura 30: Voce lògo (Pellizzer/Pellizzer 1992: 534) Figura 30: Voce lògo (Pellizzer/Pellizzer 1992: 534) Negli altri tre dizionari il fraseologismo è attestato, al rispettivo lemma (lógo in Cernecca 1986:
60; lògo in Balbi/Moscarda Budić 2003: 124; lógo in Cergna 2015: 186). In Cernecca (1986)
tale unità è presentata come “no vé né fógo né l.”, il cui traducente è indicato come “essere
povero in canna”. Si è già visto più sopra che in Cernecca (1986) le virgolette hanno molteplici
funzioni, introducendo esempi, fraseologia, ecc.; analogamente polifunzionale è in Cergna
(2015) e Balbi/Moscarda Budić (2003) il corsivo, con cui è indicata l’unità fraseologica in
oggetto. 23 Il lemma barca è uno di quelli che andranno a formare il futuro dizionario di sissanese presentati in anteprima
in Buršic Giudici (2021: 554); alla rispettiva voce non vi sono però fraseologismi. 24 Il lemma cantà dal futuro dizionario di sissanese è presentato in anteprima in Buršic Giudici/Giudici (2022:
131); nell’articolo corrispondente non vi sono elementi fraseologici. Luca Melchior: La fraseologia nei dizionari istrioto-italiano In Balbi/Moscarda Budić (2003) essa è presente solo in un esempio, tradotto prima
letteralmente, poi nel senso figurato: Figura 31: Voce lògo (Balbi/Moscarda Budić 2003: 124) Figura 31: Voce lògo (Balbi/Moscarda Budić 2003: 124) Al lemma cantà si trovano in Pellizzer/Pellizzer (1992: 182) un fraseologismo comparativo,
sotto forma di esempio (el cànta cùme oûn deîo), e due proverbi, questi ultimi introdotti con
l’indicazione, già menzionata sopra, “Modi di dire, detti e prov. rov.”. Tutti e tre i fraseologismi
sono registrati solo al lemma cantà. ISSN 1615-3014 133 25 Nella bibliografia di Pellizzer/Pellizzer (1992: 1134) è indicata la ristampa della seconda edizione del dizionario
del Boerio, datata 1861, che non è a mia disposizione. Il brano citato in Pellizzer/Pellizzer (ibd.: 165) si trova nella
seconda edizione (Boerio 1856: 114). Luca Melchior: La fraseologia nei dizionari istrioto-italiano Figura 35: Voce ʃeî (Pellizzer/Pellizzer 1992: 880) Figura 35: Voce ʃeî (Pellizzer/Pellizzer 1992: 880) ISSN 1615-3014 Luca Melchior: La fraseologia nei dizionari istrioto-italiano 135 Di questa copia di fraseologismi, solo sei sono registrati anche ai lemmi corrispondenti di altri
elementi che li compongono – tra questi tre sono verbi sintagmatici. Alla voce uòrsa si trova il
fraseologismo in un esempio: “Si ti ʃbàndi màsa, va a l’uòrsa, se sbandi troppo, va all’orza;
uòrsa” (Pellizzer/Pellizzer 1992: 1087, corsivo nell’originale). Lo stesso sintagma è riportato
tuttavia nella forma base e in un altro esempio anche con un significato diverso: “ʃeî a l’uòrsa,
prendere una storta: i son ʃeî a l’uòrsa cul peîe e dièso el ma sa ʃgiònfa, mi sono preso una
storta al piede e adesso mi si gonfia”. Alla voce ʃboûʃa si trova “ʃeî ʃboûʃa, andare buca, andare
male, non aver successo” (ibd.: 832, corsivo nell’originale), seguito da due esempi con relativa
traduzione. Alla voce foûmo, nella sezione “Motti, detti e prov. rov.” si trova, dopo altre due
unità fraseologiche, “ʃeî in foûmo, svanire, sparire, nel nulla” (ibd.: 381), corsivo nell’originale). Per quanto riguarda i verbi sintagmatici, alla voce vànti (ibd.: 1105) vi sono alcuni esempi per
i quali si propongono da una parte il traducente letterale ‘andare avanti’, in altri casi però anche
quello di ‘continuare, proseguire’; alla voce dreîo è presente ʃeî dreîo (ibd.: 335, corsivo
nell’originale), ma con due significati (e relativi traducenti) diversi da quello proposto alla voce
ʃeî, ovverosia ‘dare ascolto, credere, seguire, prestar fede, badare’ e ‘corteggiare, far la corte’. Infine, ʃeî ʃù è presente, in un esempio, alla voce ʃù: “i ʃèmo ʃù, (o in ʃù), andiamo giù e anche
usciamo” (ibd.: 991, corsivo nell’originale). Di questa copia di fraseologismi, solo sei sono registrati anche ai lemmi corrispondenti di altri
elementi che li compongono – tra questi tre sono verbi sintagmatici. Alla voce uòrsa si trova il
fraseologismo in un esempio: “Si ti ʃbàndi màsa, va a l’uòrsa, se sbandi troppo, va all’orza;
uòrsa” (Pellizzer/Pellizzer 1992: 1087, corsivo nell’originale). Lo stesso sintagma è riportato
tuttavia nella forma base e in un altro esempio anche con un significato diverso: “ʃeî a l’uòrsa,
prendere una storta: i son ʃeî a l’uòrsa cul peîe e dièso el ma sa ʃgiònfa, mi sono preso una
storta al piede e adesso mi si gonfia”. Luca Melchior: La fraseologia nei dizionari istrioto-italiano Luca Melchior: La fraseologia nei dizionari istrioto-italiano Figura 32: Voce cantà (Pellizzer/Pellizzer 1992: 182) Figura 32: Voce cantà (Pellizzer/Pellizzer 1992: 182) In Cergna (2015: 70) alla voce cantà non vi sono fraseologismi, come nemmeno in Bal-
bi/Moscarda Budić (2003: 47). In Cernecca (1986: 28) si trova “c. da galo” (non riportato alla
voce galo, cf. Cernecca 1986: 51), presentato senza traducenti, che non è conosciuto ai parlanti
vallesi interpellati come fraseologismo e che pare dunque una combinazione libera.24 Anche al lemma magnà (Pellizzer/Pellizzer 1992: 558) si trovano alcune espressioni fraseolo-
giche. Tra queste, la collocazione magnà fòra ‘mangiare al ristorante’ e la locuzione idiomatica
magnà la fòia ‘mangiare la foglia, scoprire l’inganno’ sono presentati senza particolari indica-
zioni, il primo in un esempio, il secondo alla forma base. Seguono tre proverbi, appositamente
introdotti. Tra questi, magnà puòco e duòrmi mòndo è presente anche al lemma duòrmi (ibd.:
341) e ti iè vulìsto, màgna da quìsto al lemma vulì; gli altri non sono presenti altrove. Figura 33: Voce magnà (Pellizzer/Pellizzer 1992: 558) Figura 33: Voce magnà (Pellizzer/Pellizzer 1992: 558) Figura 33: Voce magnà (Pellizzer/Pellizzer 1992: 558) Figura 33: Voce magnà (Pellizzer/Pellizzer 1992: 558) ISSN 1615-3014 134 Linguistik online 125, 1/24 Cernecca (1986: 61), alla voce magnà riporta “‘m. la fùia’ accorgersi dell’inganno”, non regi-
strato alla voce fùia (Cernecca 1986: 49). Cergna (2015: 194) riporta il detto o magna l oso o
salta l foso, il cui carattere fraseologico è indicato non tramite un’abbreviazione o mezzi tipo-
grafici, ma con un’esplicita spiegazione nel commento semantico (cf. figura 34). Il detto è men-
zionato solo a questa voce, non a quelle degli altri elementi che lo compongono. Figura 34: Voce magnà (Cergna 2015: 194) Figura 34: Voce magnà (Cergna 2015: 194) In Balbi/Moscarda Budić (2003: 128, corsivo nell’originale), alla voce magnà troviamo “Prov. gall.: Chi magna pian, lavora pian; chi magna ʃvelto, lavora ʃvelto. Chi mangia piano, lavora
piano; chi mangia veloce, lavora veloce”. Il proverbio è registrato solo a questa voce (il lemma
ʃvelto manca nel dizionario). Alla voce ʃei in Pellizzer/Pellizzer (1992: 880) vengono presentati, in maniera indifferenziata,
numerose unità fraseologiche di tipo diverso. 26 Il lemma álto dal futuro dizionario di sissanese è presentato in anteprima in Buršic Giudici (2018: 211); nell’ar-
ticolo relativo non vi sono elementi fraseologici.
27 Il pronome cheî non è lemmatizzato, viene introdotto al lemma chi come allomorfo interrogativo dello stesso
(Pellizzer/Pellizzer 1992: 215). Benussi (2015: 75) indica che chei si riferisce a persone, chi a cose o animali. 26 Il lemma álto dal futuro dizionario di sissanese è presentato in anteprima in Buršic Giudici (2018: 211); nell’ar-
ticolo relativo non vi sono elementi fraseologici.
27 Il pronome cheî non è lemmatizzato viene introdotto al lemma chi come allomorfo interrogativo dello stesso ticolo relativo non vi sono elementi fraseologici.
27 Il pronome cheî non è lemmatizzato, viene introdotto al lemma chi come allomorfo interrogativo dello stesso
(Pellizzer/Pellizzer 1992: 215). Benussi (2015: 75) indica che chei si riferisce a persone, chi a cose o animali. Luca Melchior: La fraseologia nei dizionari istrioto-italiano Alla voce ʃboûʃa si trova “ʃeî ʃboûʃa, andare buca, andare
male, non aver successo” (ibd.: 832, corsivo nell’originale), seguito da due esempi con relativa
traduzione. Alla voce foûmo, nella sezione “Motti, detti e prov. rov.” si trova, dopo altre due
unità fraseologiche, “ʃeî in foûmo, svanire, sparire, nel nulla” (ibd.: 381), corsivo nell’originale). Per quanto riguarda i verbi sintagmatici, alla voce vànti (ibd.: 1105) vi sono alcuni esempi per
i quali si propongono da una parte il traducente letterale ‘andare avanti’, in altri casi però anche
quello di ‘continuare, proseguire’; alla voce dreîo è presente ʃeî dreîo (ibd.: 335, corsivo
nell’originale), ma con due significati (e relativi traducenti) diversi da quello proposto alla voce
ʃeî, ovverosia ‘dare ascolto, credere, seguire, prestar fede, badare’ e ‘corteggiare, far la corte’. Infine, ʃeî ʃù è presente, in un esempio, alla voce ʃù: “i ʃèmo ʃù, (o in ʃù), andiamo giù e anche
usciamo” (ibd.: 991, corsivo nell’originale). Cernecca (1986: 102) alla voce ʃeî presenta solo un esempio e parte del paradigma; in Bal-
bi/Moscarda Budić (2003: 233), nell’articolo di ʃì si trovano solo il traducente e alcuni esempi
con relativi traducenti. Cergna (2015: 346, corsivo nell’originale) riporta come secondo signi-
ficato del verbo ʃì ‘(Figura) ʃì per le longhe, prolungare una questione, un impegno’, seguito da
un’attestazione. Delle unità fraseologiche proposte alla voce àlto (Pellizzer/Pellizzer 1992: 44, cf. figura 36),
delle quali alcune sono introdotte dall’indicazione “Come loc. avv. e fig.”, solo una è presente
anche al lemma corrispondente a un altro elemento costituente, ovverosia “Àlta de cagadùr”,
per la quale vi è anche un rinvio mediostrutturale al lemma cagadùr. A questa voce viene però
presentato solamente un brano dal dizionario veneziano di Boerio (31867), che registra la stessa
unità fraseologica in veneziano, spiegandone il significato.25 ISSN 1615-3014 136 Linguistik online 125, 1/24 Figura 36: Voce àlto (Pellizzer/Pellizzer 1992: 44) 27 Il pronome cheî non è lemmatizzato, viene introdotto al lemma chi come allomorfo interrogativo del
(Pellizzer/Pellizzer 1992: 215). Benussi (2015: 75) indica che chei si riferisce a persone, chi a cose o ani Figura 36: Voce àlto (Pellizzer/Pellizzer 1992: 44) Gli altri dizionari lemmatizzano l’aggettivo nella forma alto (Cernecca 1986; Balbi/Moscarda
Budić 2003: 20; Cergna 2015: 24), alle rispettive voci non sono però riportati elementi fraseo-
logici.26 Gli altri dizionari lemmatizzano l’aggettivo nella forma alto (Cernecca 1986; Balbi/Moscarda
Budić 2003: 20; Cergna 2015: 24), alle rispettive voci non sono però riportati elementi fraseo-
logici.26 Nell’ampio articolo dedicato al lemma bon (Pellizzer/Pellizzer 1992: 126s.) vi sono diversi
elementi fraseologici. Indicati come tali (“nelle locuz. avv. e agg ibd.: 127) sono quelli in cui
l’aggettivo viene in realtà nominalizzato alla forma femminile e quattro “modi di dire e pro-
verbi”, non però le collocazioni tignìse bon, da bon grado e stà bon. Tra tutti gli elementi fra-
seologici, solo il proverbio Biègna stà in bòna cui treîsti ca cui bòni nu mànca mài si trova
registrato anche altrove, alla voce treîsto (ibd.: 1062).27 È inoltre classificato come “loc. avv.”
bon bon, in realtà segnale discorsivo, per cui si propongono i traducenti “sì, sì, d’accordo”. ISSN 1615-3014 137 Luca Melchior: La fraseologia nei dizionari istrioto-italiano Luca Melchior: La fraseologia nei dizionari istrioto-italiano Figura 37: Voce bon (Pellizzer/Pellizzer 1992: 126s.) Cernecca (1986: 23) riporta il fraseologismo comparativo “b. como l pan” e le due collocazioni
“ʃì cun le bone”, per il quale propone il traducente ‘persuadere con le buone maniere’, e ‘tégnise
n bonÄ, per cui propone il traducente ‘vantarsi’. Tutte e tre le unità fraseologiche sono registrate ISSN 1615-3014 138 Linguistik online 125, 1/24 solo al lemma bon. Cergna (2015: 51, corsivo nell’originale) ha il fraseologismo comparativo
“Bon como il pan” e la collocazione “tégnise n bona”. Entrambi sono presenti solo al lemma
bon e non sono classificati come elementi fraseologici. Balbi/Moscarda Budić (2003: 34,
corsivo nell’originale) caratterizza il lemma bon come “agg. e locuz. avv.” e presenta, come
esempi, i fraseologismi “Al xe bon como ’l pan” e “I xe n bona”, con i relativi traducenti (per il
secondo “Hanno dei buoni rapporti”). Anche in questo dizionario si registra, in un esempio,
bon bon, non ulteriormente caratterizzato, per cui si propone il traducente “Va bene”. Infine, per viècio, che conosce un’unica lemmatizzazione come aggettivo e sostantivo (i due
elementi non vengono differenziati nemmeno nell’articolo), in Pellizzer/Pellizzer (1992: 1119)
si trovano quattro proverbi (più altri due in cui l’elemento è sostantivo), introdotti da “Detti e
prov. rov.”. Di tali proverbi, tre su quattro sono presenti anche sotto lemmi corrispondenti ad
altri elementi che li compongono: galeîna viècia fa bon brù (ibd.: 138, corsivo nell’originale)
si trova anche al lemma brù, can viècio nu bàia a la loûna (ibd.: 84, corsivo nell’originale) ai
lemmi baià e loûna (ibd.: 536), e i moûri vièci fà pànsa al lemma pànsa (ibd.: 649s., corsivo
nell’originale; i proverbi con viècio sostantivo sono registrati solo a questa voce). In
Balbi/Moscarda Budić (2003: 276) non vi sono unità fraseologiche alla voce vecio. Cernecca
(1986: 116) ha solo “pan v.” e “l me vècio” ‘mio padre’, che non sono elementi fraseologici;
gli stessi sono riportati da Cergna (2015: 410, corsivo nell’originale), che ha anche la
collocazione “vècio cudinaro, vecchissimo”. L’articolo del lemma cudinaro (Cergna 2015: 99)
consiste solo di un rinvio mediostrutturale a vècio. Figura 38: Voce viècio (Pellizzer/Pellizzer 1992: 1119) Figura 38: Voce viècio (Pellizzer/Pellizzer 1992: 1119) 5
Conclusioni 5 L’analisi dei quattro dizionari ha rivelato diverse incoerenze, sia nell’utilizzo delle abbrevia-
zioni per indicare elementi fraseologici, sia nel trattamento degli stessi. In alcuni casi le abbre-
viazioni – poco differenziate per le diverse categorie di unità fraseologiche – introducono ele- ISSN 1615-3014 Luca Melchior: La fraseologia nei dizionari istrioto-italiano 139 menti che non sono fraseologici, spesso invece mancano completamente; all’interno dell’arti-
colo lessicografico vengono utilizzate in modo e posizione diversi. La maggior parte delle unità
fraseologiche è presentata solo sotto un lemma; in pochi casi anche ai lemmi corrispondenti ad
altri elementi che la formano. Anche in tal caso però si notano numerose incongruenze: talora
sotto un lemma non si trovano tutte le possibili unità fraseologiche registrate invece all’altro o
non sono presenti tutti i traducenti possibili. Altre volte i traducenti proposti per la stessa unità
fraseologica, attestata sotto diversi lemmi, differiscono tra loro. Infine, alcune unità fraseologi-
che vanno a costituire lemmi a sé stanti (eventualmente in forma grafica diversa), senza che sia
comprensibile il criterio che sta alla base della scelta di quali siano autonomamente lemmatiz-
zati e quali no. Pressoché assenti sono i rinvii mediostrutturali; se presenti, talora paiono fuor-
vianti. Dei quattro dizionari, Pellizzer/Pellizzer (1992) presenta una fraseologia relativamente
ricca, trattata in maniera differenziata, anche se in questo caso spesso non coerente. Balbi/Mo-
scarda Budić (2003: 276) e Cergna (2015) hanno una discreta presenza di fraseologismi, date
le dimensioni più ridotte di questi dizionari, però sicuramente inferiore a quella del precedente. Pur costituendo indubbiamente fonti documentarie preziose per le varietà di istrioto in esse
trattate, i dizionari mostrano un certo carattere di “amatorialità” lessicografica, che conduce
appunto alle incoerenze strutturali qui evidenziate. Auspicabile è dunque che gli interessanti
materiali qui e altrove raccolti (cf. p. e. Moscarda 1998; Trgovčić 1998; Moscarda Mirković
2001, 2002, 2013; Cergna 2013; Tamaro 2021), integrati con ulteriori materiali da raccogliere
sul campo, vengano ulteriormente sistematizzati in future opere lessicografiche e fraseogra-
fiche. Bibliografia Ascoli, Graziadio Isaia (1873): “Saggi ladini”. Archivio Glottologico Italiano 1: 1–556. Ascoli, Graziadio Isaia (1873): “Saggi ladini”. Archivio Glottologico Italiano 1: 1–556 Banfi, Emanuele (2003): “Evoluzione delle frontiere delle lingue romanze: Romania del Sud-
Est”. In: Ernst, Gerhard et al. (eds.): Romanische Sprachgeschichte./Histoire linguistique de
la Romania. Vol. 1. Berlin/New York, de Gruyter: 622–631. (= Handbücher zur Sprach-
und Kommunikationswissenschaft 23.1). Benussi, Libero (2015): Grammatica del dialetto di Rovigno d’Istria. Rovigno: Comunità degli
italiani “Pino Budicin” – Rovigno. Blagoni, Robert (2012): “L’italiano in Croazia e in Istria tra opportunità giurilinguistiche, at-
teggiamenti sociolinguistici e squilibri linguistico-politici”. In: Telmon, Tullio/Raimondi,
Gianmario/Revelli, Luisa (eds.): Coesistenze linguistiche nell’Italia pre- e postunitaria. Atti
del XLV Congresso internazionale della Società di Linguistica Italiana (Aosta/Bard/Torino,
26–28 settembre 2011). Roma, Bulzoni: 449–458. (= Pubblicazioni della Società di Lingui-
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Productive Gaming
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To cite this version: Ulrich Brandstätter, Christa Sommerer. Productive Gaming. 15th International Conference on Enter-
tainment Computing (ICEC), Sep 2016, Wien, Austria. pp.260-265, 10.1007/978-3-319-46100-7_27. hal-01640274 Distributed under a Creative Commons Attribution 4.0 International License 1 “[. . . ] a game’s value proposition is in how it might make its player think and feel and
fun is the ultimate emotional state that they expect to experience as a consequence of
playing.” [2] Ulrich Brandstätter and Christa Sommerer Interface Culture, University of Art and Design Linz, Austria
{Ulrich.Brandstaetter, Christa.Sommerer}@ufg.at Abstract. Video games can be appropriated for productive purposes. Commercial games and game engines are often used for video productions,
and game development companies provide development kits and modding
environments to gaming communities and independent developers. With
gamification, game principles are deployed in non-game contexts for
benefits beyond pure entertainment. Most approaches are more focused
on using games and their design elements rather than the process of
playing. We propose a video game category wherein productivity is
achieved by playing video games, and present a forthcoming productive
game as an example. Keywords: Serious games; media production; video games; play HAL Id: hal-01640274
https://inria.hal.science/hal-01640274v1
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abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Productive gaming Ulrich Brandstätter and Christa Sommerer 1
Introduction We propose a new video game category for video games that yield productive
results by being played: productive gaming transforms playing activities into
creative processes, facilitating creations with validity beyond game virtualities. In the language of McLuhan and Nevitt, who conceived the concept for electronic
technology in 1972, we allow gamers to become Prosumers [1], i.e., consumers
turned into producers. The proposed video game category increases the value propositions1 of video
games. Implicitly, “all games express and embody human values” [3]. However,
with the exception of serious games [4], the explicit gain of playing video games
beyond pure recreation is disputable: as Malaby points out, play dissociates
from everyday life by being consequence free and pleasurable and, especially,
dissociates from work by staying within a magic circle [5]. Caillois divides play
and productivity, and implies that game play must not become productive or be
conducted in pursuit of profit, otherwise it becomes corrupted [6]. Consequently,
the conjunction of play and productivity makes the introduction of specific frame-
work conditions necessary. These are described as a set of attributes that apply
to video games that enable productive gaming. However, we refrain from giving
an ultimate definition to describe productive gaming. The reasoning behind this
approach is given by Wittgenstein, who argues that “elements of games, such Ulrich Brandstätter and Christa Sommerer 2 as play, rules, and competition, all fail to adequately define what games are” [7],
and by Sutton-Smith, who considers the description and definition of play and
games in non-paradoxical terms almost impracticable [8]. Games facilitating productive gaming are serious games with additional charac-
teristics: – productivity is a subject matter of the game, not to be enforced as game
goals, but enabled as goals of the meta game.l – Consistently, productivity is not reflected in victory conditions, creational
objectives are set and determined before or during play sessions by the players
themselves, who retain unrestrained artistic freedom.l Creation processes are aligned towards (and do not break) the game flow. – Gameplay results are perceivable without the game and, ideally, even without
knowledge of the game. These nominal characteristics describe a non-exclusive video game category with
emphasis on the game-play. Consistently, productive gaming requires games that
provide mechanisms to start and realize creative processes, but also constructive-
minded players. In this paper, we describe related work with a concise disambiguation in
section 2. 1
Introduction In section 3, we provide a more detailed introduction to productive
gaming. We then present Forever loops (section 4), an ongoing project that
facilitates productive gaming for the creation of audio-visual compositions. In
section 5, we discuss our findings and conclusions and present suggestions for
future work. 2
Related work We differentiate from similar concepts using the previously introduced productive
gaming characteristics. A related concept can be found in the industrial use of
construction games, e.g. the utilization of Lego [9] for architectural prototypes,
or using Minecraft [10] (in creative mode) as a 3D modeling environment. In
both cases, productivity is a subject matter. The main distinction originates
from the objectives. The industrial use of construction games typically involves
predefined ambitions given by constituents, whereas in productive gaming the
players themselves determine creational objectives before or during play. Another
approach can be found in human-based computation games, or games with a
purpose, where “people playing computer games could, without consciously doing
so, simultaneously solve large-scale problems” [11]. Here, human game interaction
is used to acquire labeling data for specific meta game goals, such as the analysis
of gene sequences in Play to Cure: Genes in Space [12]. By being played, results
with validity beyond the game virtuality are achieved. However, the creational
goals usually are fixed by the developers and cannot be influenced by the players. A different, yet very popular concept is the utilization of game engines and
modifications for media productions, e.g. Machinima communities using suitable
computer games for video productions. Here, results typically are perceivable 3 Productive gaming without the game and yield audio-visual makings. Nevertheless, productivity
is achieved by using games rather than playing games. Finally, we dissociate
productive gaming from gamification (i.e., the use of game design elements in
non-game contexts [13]) because the latter usually aims at goal-oriented play:
“Finally, academic as well as industry critiques of gamified applications have
repeatedly emphasized that these focus almost exclusively on design elements for
rule-bound, goal-oriented play (i.e., ludus), with little space for open, exploratory,
free-form play (i.e., paidia)” [14] 3
Productive gaming A game example facilitating productive gaming can be found in Sim Tunes [15]. It resembles a drawing game in which players populate a canvas with up to four
virtual bugs of different color. These AI-controlled bugs traverse the displayed
virtual space and trigger music playback. Specific pixel colors represent different
musical notes that are played by the instrument according to what is assigned to
the bug. In terms of our proposed characteristics, Sim Tunes does not employ
victory conditions, i.e., players set their own objectives and produce audio-visual
creations by playing the game. In general, productive gaming is closer to free form (paidia) than to more
structured (ludus) playing and takes advantage of the games also being simulations:
“[. . . ] Espen Aarseth has argued that [. . . ] simulation is what drives most”serious
games" [. . . ]. In simulations, and I quote, “knowledge and experience is created by
the player’s actions and strategies”. Aarseth calls for recognition of simulation as
“a major new hermeneutic discourse mode, coinciding with the rise of computer
technology, and with roots in games and playing.” [16] The proximity to simulations makes a clear distinction from productivity software
necessary. Barr describes the main differences as follows: “First, the motivations
for playing video games differ from productivity application use. Specifically,
players play games for their own sake, while they generally use productivity
applications to achieve some other task. Second, video game interfaces are not
neutral, presenting carefully designed narratives and complex graphics to the
player. Third, video games frequently dictate goals to players, while productivity
applications generally facilitate user goals. Finally, video game designs purpose-
fully involve conflict and constraints on the player, while productivity applications
are designed to minimize them.” [17] Juul and Norton consider game obstacles
and challenges as the distinguishing features of games as opposed to usability
for productivity software [18]. For our purposes, the capability of seducing users
into a lusory attitude [19] is the distinctive feature, i.e., play requires a proper
mindset and engagement, which is established upon and complements game
rules [20]. Concerning both the level of engagement and the level of game
structure, productive gaming involves substantial oscillation effects on the paidia-
ludus continuum [21] and play structure flexibility. During play sessions, users
experiment and play with the simulations without pre-defined goals, and then
apply their discoveries and new techniques to their performances. 3
Productive gaming Ulrich Brandstätter and Christa Sommerer 4 In this context, the notion of serious storytelling must be contemplated, as well
as its relation between serious games and productive games. Serious storytelling
refers to “storytelling outside the entertainment context, where the narration
as artefact is impressive in quality and relates to a matter of importance and
seriousness.” [22] A narrative, i.e. the “[. . . ] chain of events related by cause
and effect occurring in time and space and involving some agency” [23], is
important for both serious storytelling and productive gaming. Serious storytelling
constitutes a broader concept with several application scenarios, including serious
games, but also eLearning, qualitative journalism, virtual training environments,
or forensics. Productive gaming on the other hand builds upon serious games
and at the same time concerns only a subset. In this context, the notion of serious storytelling must be contemplated, as well
as its relation between serious games and productive games. Serious storytelling
refers to “storytelling outside the entertainment context, where the narration
as artefact is impressive in quality and relates to a matter of importance and
seriousness.” [22] A narrative, i.e. the “[. . . ] chain of events related by cause
and effect occurring in time and space and involving some agency” [23], is
important for both serious storytelling and productive gaming. Serious storytelling
constitutes a broader concept with several application scenarios, including serious
games, but also eLearning, qualitative journalism, virtual training environments,
or forensics. Productive gaming on the other hand builds upon serious games
and at the same time concerns only a subset. 5
Conclusion To some extent, video games are already used for productive purposes, e.g.,
supporting the modification of existing games, asset export and import capability,
or video capture support, which are popular game features that facilitate usage
scenarios beyond pure entertainment. Specific video game genres, such as active
games, educational games, and art games, attempt to offer additional benefits. The proposed new video game category specifically describes video games that
facilitate productivity by being played. Productive games as described in this
paper are biased towards free-form play and can be considered as a specialization
of serious games. Another approach towards productive gaming is the segregation
of victory conditions and productivity, i.e., results correlate with and are affected
by game progression, as they can actively be influenced by the player. Creative
freedom is more limited, whereas the underlying game genre can be chosen more
arbitrarily. 4
Forever loops Forever loops by Marlene and Ulrich Brandstätter is a productive gaming project
that produces audio-visual compositions. The underlying simulation involves an
interface that primarily comprises graphical gears, as shown in Figure 1. Fig. 1. Forever loops screen-shot. A gear train comprising multiple interconnected gears
is playfully arranged and programmed by a user. The canvas in the upper left corner
gives a visual representation of the audio-visual composition. The window to the right
is required for media selection. Fig. 1. Forever loops screen-shot. A gear train comprising multiple interconnected gears
is playfully arranged and programmed by a user. The canvas in the upper left corner
gives a visual representation of the audio-visual composition. The window to the right
is required for media selection. 5 Productive gaming Relevant mechanisms are based on real-world gears and their machinery, which
is well known and understood by most people. Users begin with a fundamental
understanding of the system mechanics, which quickly evolves into impressive
skills regarding (musical) composition and performance. Concerning productivity,
the virtual gears can be interfaced with audio-visual material, including sounds,
images, and videos. Playback of the media goes hand in hand with playfully
arranging gears and gear trains with their respective programming. Game play results include musical compositions, video remixes, slide shows, VJ
acts, and even performances. In contrast to other productivity games, including
the aforementioned Sim Tunes, players can import and are encouraged to use
personal media material. On top of this, satisfactory results can be exported as a
video file at user request, which can than be viewed without the game and even
without knowledge of the game. Users are encouraged to experiment with the underlying physics of the gear
simulation, and to use their findings to produce new compositions at their own
pace. A central aspect of our approach is the rejection of the notions of winning
or losing. From a design perspective, it is common for video games to blur the
borders between interface and game play [18]. Here the gear wheels and their
mechanics become the central interface, and are tightly interwoven with the
game-play. 3. Mary Flanagan and Helen Niessenbaum. Values at Play in Digital Games. The
MIT Press, Cambridge, Massachusetts, London, England, 2014. 1. M. McLuhan and B. Nevitt. Take Today: The Executive As Dropout. Harcourt,
Brace, Jovanovich, Incorporated, 1972. ISBN 9780151878307. 1. M. McLuhan and B. Nevitt. Take Today: The Executive As Dropout. Harcourt,
Brace, Jovanovich, Incorporated, 1972. ISBN 9780151878307.
2. Laura Ermi and Frans Mäyrä. Fundamental components of the gameplay experience:
Analysing immersion. In In DIGRA. DIGRA. 2005.
3. Mary Flanagan and Helen Niessenbaum. Values at Play in Digital Games. The
MIT Press, Cambridge, Massachusetts, London, England, 2014. 2. Laura Ermi and Frans Mäyrä. Fundamental components of the gameplay experience:
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Changes of Spatial Characteristics: Socio-Cultural Sustainability in Historical Neighborhood in Beijing, China
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sustainability sustainability sustainability sustainability
Keywords: socio-cultural sustainability; typological approach; urban morphology; neighborhood;
spatial characteristics; urban transformation Academic Editor: Manuel
Duarte Pinheiro Received: 23 April 2021
Accepted: 26 May 2021
Published: 31 May 2021 Article
Changes of Spatial Characteristics: Socio-Cultural
Sustainability in Historical Neighborhood in Beijing, China Huiming Liu
and Bin Li * Laboratory for Future City, School of Urban Planning and Design, Shenzhen Graduate School, Peking University,
Shenzhen 518055, China; liuhuiming@pkusz.edu.cn
* Correspondence: libin@pkusz.edu.cn; Tel.: +86-186-2870-6470 Laboratory for Future City, School of Urban Planning and Design, Shenzhen Graduate School, Peking University,
Shenzhen 518055, China; liuhuiming@pkusz.edu.cn
* Correspondence: libin@pkusz.edu.cn; Tel.: +86-186-2870-6470 Abstract: This paper uses a typological approach as a tool to establish an analytical framework from
a physical perspective to understand ‘place‘ and to identify key spatial characteristics that could
adapt to local needs to deliver socio-cultural sustainability. Six representative housing types with
their spaces and uses that were introduced in a historic neighborhood in Beijing, China are selected as
case studies. Their morphological characteristics at the building, open space and neighborhood scales
are examined, and typological transformations among the cases in terms of the degree of spatial
continuity are identified. The paper proposes an analytical framework consisting of fifteen indicators
to assess socio-cultural sustainability at the different morphological scales (building, open space and
block/neighborhood) of the residents of the six cases. The score of changes from its original design is
brought into calculations of continuities of spatial characteristics, which present the transitions and
transformations of morphological characteristics in relation to adaptation of local needs and uses. The analysis results show that the spatial characteristics were changed when political-socioeconomic
ideologies changed, and local needs and uses were transformed to follow these mutations, and
finally, the methods of use in different morphological scales mostly differed from historical norms. Although the continuities of spatial characteristics were significantly changed, they are positively
and continually accommodating the transformations and transitions of local needs and uses. On the
other hand, the invariant spatial characteristics are important, which last despite transformation of
the city development and changing of political-social-economic ideologies, and could be maintained
for future development to enhance sociocultural sustainability. Citation: Liu, H.; Li, B. Changes of
Spatial Characteristics: Socio-Cultural
Sustainability in Historical
Neighborhood in Beijing, China.
Sustainability 2021, 13, 6212.
https://doi.org/10.3390/su13116212 1. Introduction In the past few decades, the international architecture has been accused of incom-
patibility with the local cultures [1], and is commonly believed to be responsible for
placelessness, particularly in China [2–5]. Many Chinese cities are suffering from a typolog-
ical crisis and a loss of adaptation to local needs. Especially in Beijing, due to dramatically
increased population, traditional courtyard housing types were largely transformed into
court-no-yard houses, and turned from one family owned to multi-family occupied [6]. Meanwhile, since the Modernist movement, mass production of standardized houses was
free planned into historical neighborhoods to fulfill emerging housing demands. As a
result, they devised a built environment, and transformed the spatial network organically
to balance the changing needs and transitions of the physical setting [7,8]. This leads to
two questions which need to be asked: Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). (1)
Is current built environment (both historic and early modern) responsive to socio-
cultural needs in use? (2)
What spatial characteristics should be continued, which could adapt local socio-
cultural needs to guide future development? https://www.mdpi.com/journal/sustainability Sustainability 2021, 13, 6212. https://doi.org/10.3390/su13116212 2 of 28 Sustainability 2021, 13, 6212 In order to achieve local socio-cultural sustainability in historical neighborhood devel-
opment, the theory of place helps to understand what people’s needs should be considered. Butina-Watson and Bentley [9] (p. 6) define place as a ‘set of meanings associated with any
particular cultural landscape which any particular person or group of people draws on
in the construction of their own personal or social identities.’ Castello [10] (p. 62) goes
further, reinforcing the idea of time and suggesting the importance of heritage by defining
place as ‘a collection of images that can transmit, at a glance, a suggestive reminder of
the historical urban forms the city has acquired all along its evolution’ [11]. Although the
concept of place is rooted in history, it is continuously changing and is neither static nor
uniform [9,12]. 1. Introduction Morphologists explain these changes of urban form as typological studies
that is cumulative and continuous [13], and consider that continuities of urban form and
typological transformation could enhance socio-cultural sustainability and benefit people’s
satisfaction with living [14–16]. Nurse [17] (p. 33) supports this argument, and states that
socio-culture is more than just the manifestations of culture, it is the ‘whole social order’,
the ‘whole way of life’ and a dynamic process driving sustainable development with the
forms of engagement [18]. Therefore, typological approach can be seen as a great tool for
defining spatial characteristics and distinctiveness of old and new physical forms based on
spatial forms, urban culture and social practice through transformation of the built form
that delivers socio-cultural sustainability [19–22]. Recent typological studies in China have not brought much socio-cultural sustain-
ability to light. Gu et al. [23] studied the transformation process of traditional houses
in Guangzhou, but focused on the building level. Feng [24] studied the continuities of
spatial changes on shikumen (linong) houses with their relationships to layout, plot and
street. Chen and Thwaites [14] employed Nanjing as a case in which urban forms were
comprehensively studied and described its transformation in relation to a design process
for identifying key factors to improve local policy making. Whitehand et al. [7] compared
the transformation process of terrace house types and lilong houses between England
and Shanghai, and aims to identify whether the latter had been influenced by the former
around the late nineteenth and early twentieth centuries. However, although they have contributed to either large-scale city developments or
regeneration of specific urban heritage building typology, none of studies look at what
spatial characteristics could adapt to local needs and continue to be applied to future devel-
opment to enhance socio-cultural sustainability, especially in the context of Beijing, China. In addition, it has always been challenging to identify types through a transformation pro-
cess, firstly due to the degree of judgement in relation to determining the types of changes
to be continued, which is largely subject to the individual researcher’s viewpoints [7]. Secondly, it is unclear which types of spatial characteristics and the level of specificity in
the investigation that should be considered. Thirdly, there are limitations in empirical
studies of spatial types at articulated scales, although many studies focus on individual
scales with limited levels of specificity (such as [25]). 1. Introduction Based on discussions above, this paper develops an analytical framework from a
physical perspective to define changes of spatial characteristics, which could adapt to
the changing of needs to deliver socio-cultural sustainability in historical neighborhoods
(vernacular neighborhoods) in Beijing. Six housing types with their spaces and uses were
investigated. The research findings contribute to a conceptual and analytical framework for
assessing local needs from a physical perspective in order to sustain socio-cultural sustain-
ability; the discontinuity of adaptation of local needs requires more attention to design in
order to improve the physical setting to improve socio-cultural sustainability. In addition,
the developed analytical framework improves efficiency of analysis in identifications of
which spatial characteristics adapt to local needs through physical transformations. This paper proposes three strategies for analysis: 2. Research Strategies This paper proposes three strategies for analysis: Sustainability 2021, 13, 6212 3 of 28 1. Based on the understanding of place, define key spatial characteristics by employing
a typological approach 1. Based on the understanding of place, define key spatial characteristics by employing
a typological approach 2. To select cases through identified morphological periods in the neighborhood for
analysis 2. To select cases through identified morphological periods in the neighborhood for
analysis 3. To apply the analytical framework to cases that identify continuity of spatial charac-
teristics that could adapt local needs in order to achieve socio-cultural sustainability
in historical neighborhoods in Beijing In the first step, an analytical framework is developed for delivering socio-cultural
sustainability in historical development in Beijing by employing a typological approach
to define continuity of spatial characteristics. It reviews literature on place [9,10,12,26–28]
and seeks to understand links to typological transformation [22,29–32] from a physical
perspective to look at how built forms could adapt to local uses and needs. In the second step, case selections should be representative of their specific historical
and geographic characteristics in each morphological period. The identification of residen-
tial building types based on different morphological periods is defined by the analysis of
the historical transformation period of Beijing from imperial times (before 1911) through
the period of Soviet influence (1949–1978) and post Reform period (1978–1990) to the
contemporary period (after 1990) [33]. Starting with the building types with their spaces
and uses that correspond to imperial traditions and continuing contemporary lifestyles,
the case study neighborhood has thus accommodated a greater variety of building types
than other areas in Beijing (details refer to analysis in later sections). The third step implements the analytical framework developed in the first step, to
cases identified in the second step. It first studies the neighborhood/ block layout to
understand the context, then it analyzes the transformation of cases from its original design
to contemporary configurations by employing a typological approach, which identifies the
adaptation of changes of local cultural values, social patterns and uses experiences and
perceptions following a chronological order. It then compares the degree of changes in the
spatial transformation. Based on each spatial characteristic in three morphological layers,
this research considers continue, partly continue, and change as evaluation measurements,
and a formula is developed in order to calculate the precise value to manage the continuities
of these spatial characteristics between cases. 2. Research Strategies Finally, the changes between typological
studies as to which spatial characteristics could adapt to local needs and sustain socio-
cultural sustainability are presented. 3. Development of Analytical Framework Key Theories to Understand Place
Key Authors
Sense of belonging
[2,37]
Potpourri of memories, interpretations and feelings in relation to the
physical setting
[38]
Character, uniqueness, distinctiveness and sense of place are evolving
[9,26,27]
Metal registration of physical space ‘mind map’
[39]
Set of meanings associated with cultural landscape and
morphological characteristics
[9]
Act of embedding meanings in a place, rootedness, memories and
feelings within layers of the physical setting
[2,9,27,39,40]
Physical environment and the meanings feelings associates with it
[2]
Physical representation of place
[10,15]
Unique places with strong images
[15]
Result of a continuous evolutionary process over time, that includes
socio-economic and political relationships and dynamics
[12,28,40–44]
,
p
g
ation to the physical setting
[38]
iqueness, distinctiveness and sense of
place are evolving
[9,26,27]
ration of physical space ‘mind map’
[39]
associated with cultural landscape and
orphological characteristics
[9]
ding meanings in a place, rootedness,
d feelings within layers of the physical
setting
[2,9,27,39,40]
onment and the meanings feelings asso-
ciates with it
[2]
sical representation of place
[10,15]
ue places with strong images
[15]
inuous evolutionary process over time,
cio-economic and political relationships
and dynamics
[12,28,40–44]
al setting needs to adapt to a sense of place rootedness of feelings and Key Theories to Understand Place
g
iveness and sense of The physical setting needs to adapt to a sense of place, rootedness of feelings and
memories and perceptions through urban transformation [2,9,10,15,37,38], while in order to
carry out these experiences of use, physical form needs to be developed that corresponds
to spatial characteristics through different morphological scales (block, open spaces and
buildings) [9,30]. Thereby, the place can be simply seen as the result of the interactions
between people and the physical setting. The changes to the physical setting need to adapt
to people’s needs, and to sustain people’s needs, the regeneration of physical environment
then is as an opportunity to enhance social and economic satisfaction [45,46]. These needs
are sense of place, sense of belonging, set of meanings and memories and feelings (Figure 1). erceptions through urban transformation [2,9,10,15,37,38], while in order
se experiences of use, physical form needs to be developed that corre-
al characteristics through different morphological scales (block, open
dings) [9,30]. Thereby, the place can be simply seen as the result of the
ween people and the physical setting. 3. Development of Analytical Framework In order to achieve the first research goal, developing an analytical framework, it is
first important to understand people’s needs in place (Table 1), and then to clarify whether
these needs are adapted to by physical form through time—this is the key to achieving
socio-cultural sustainability. Christian Norberg-schulz [34] suggests that the connection
between people and places involves a sort of guardian-spirit that accompanies them from
birth to death, a genius loci, that determines their characters, which is identity for a place. Therefore, the relationship between an individual and a place is not just between oneself
and one’s surroundings, but it has to do with a much deeper process to identify where
people create a friendly connection with a particular environment [2,9,15]. The process
of shaping place is then formed by interactions between people and the physical setting;
human actions influence physical settings, and, in return, physical settings stimulate human
actions [35,36]. Sustainability 2021, 13, 6212
Table 1. Un 4 of 28 Table 1. Understanding of needs in place. emories, interpretations and feelings in
ti
t
th
h
i
l
tti Table 1. Understanding of needs in place. 3. Development of Analytical Framework The changes to the physical setting
people’s needs, and to sustain people’s needs, the regeneration of physi-
t then is as an opportunity to enhance social and economic satisfaction
eds are sense of place, sense of belonging, set of meanings and memories
gure 1). nging of people’s needs in a local place
Figure 1. The changing of people’s needs in a local place. nging of people’s needs in a local place
Figure 1. The changing of people’s needs in a local place. nging of people’s needs in a local place
Figure 1. The changing of people’s needs in a local place. whether a place is considered as socio-culturally sustainable depends on
ort or sustainability of the local needs: sets of meanings, sense of belong-
ce, and memories from and feelings about a physical setting. The core of
However, whether a place is considered as socio-culturally sustainable depends on the
level of support or sustainability of the local needs: sets of meanings, sense of belonging,
sense of place, and memories from and feelings about a physical setting. The core of
socio-cultural sustainability [47] then involves two aspects: futurity and equity. The most
often-quoted definition is “(sustainable development) is development that meets the needs
of the present without compromising the ability of future generations to meet their own
needs”[48] (p. 28). However, this definition lacks consideration of the distinction between
equity within a generation, and equity between generations [49]. The needs of local people
should be emphasized for the least advantaged in society and a fair treatment of future
generations should be ensured [50]. Lim [51] states that culture can be considered a complex
matrix of collective experiences and knowledge that formulates a meaning of life, so the
values that people hold, the rules and norms they obey and the material objects they use
are keys to shaping the built environment into a sustainable place [52,53]. Zetter and Butina
Watson [54] go further to say that how people shape their own places and spaces, how Sustainability 2021, 13, 6212 5 of 28 they adapt local technologies and traditions, and how they deploy innate capacities to
adapt cultural precepts to the modern idiom are crucial for sustainability implementations. 3. Development of Analytical Framework Therefore, the sets of meanings, sense of place, sense of belonging and memories and
feelings that people attach in shaping a place becomes the essence of the local needs, while
continually fulfilling these needs for contemporary and future development is the key of
the theory of socio-cultural sustainability [17,55]. On the other hand, when we go to study in detail how the transformations of physical
settings could adapt local needs to achieve socio-cultural sustainability, the difficulties
include how to precisely define which spatial characteristics could continually contribute
to uses in public open spaces, and what spatial characteristics of urban forms should be
involved, and which can fulfill the local socio-cultural needs. Therefore, the typological
approach is a tool because it goes beyond the analysis of physical forms of urban trans-
formation and studies how physical transformation adapts to the changing of needs to
understand human actions [22,56]. Moudon [57] states that urban forms can be understood
by investigating layout and configuration of forms and spaces in order to clarify the pro-
cesses of local change—how forms and spaces are locally built and why. Zetter and Butina
Watson [54] argue that physical forms are the products of social consensus and productions
of urban forms are the process by which socio-spatial construction take place. The typological approach aims to provide an understanding of the built environment
by examining the historical process of its formation and its hierarchical structure, in order
to identify the adaptation to changing needs. It interprets the history of the civilization
and its needs for transformation through architecture [58] by analyzing building materials,
the structure of buildings and the arrangement of rooms [32]. Then, it uses buildings as
elements in order to understand forms and their memories and values, and the meanings
beyond. On the other hand, Goodey [59] claims that physical forms should create a sense
of belonging and a sense of place that adapts to people’s feelings as though individuals
belong to the people, to the buildings and to the life there (Figure 2). 6 of 31 Figure 2 The interrelationship between spatial characteristics and changing of needs
Figure 2. The interrelationship between spatial characteristics and changing of needs. re 2 The interrelationship between spatial characteristics and changing of needs
Figure 2. The interrelationship between spatial characteristics and changing of needs. p
p
g
g
ng to Kropf [30] (p. 3. Development of Analytical Framework The diachronic type is relevant to the typological study, where the types evolve
and adapt to the transformation of socio-cultural needs through different historical periods,
with the essential spatial characteristics which remain becoming a foundation type [19]. while others are more robust and can survive over consecutive time periods in the same
place [32]. The diachronic type is relevant to the typological study, where the types evolve
and adapt to the transformation of socio-cultural needs through different historical periods,
with the essential spatial characteristics which remain becoming a foundation type [19]. Based on the discussion above, the understanding of an interactional relationship between
people and physical setting is clear—in order to design a socio-culturally sustainable place,
one must fully understand how people’s changing needs associate with continuities of spatial
characteristics that deliver socio-cultural sustainability to the local place (Figure 3). In other words,
in order to identify what spatial characteristics can be continued in the transformation process, it
must comprehensively understand what spatial characteristics continually associate with local
uses. Then, the key components of the analytical framework are identified and associated with
each morphological scale (building, open space and neighborhood block), and will be used for
analyzing cases, identified throughout the morphological period in this research. 7 of 31 Figure 3 Achievement of socio cultural sustainability with continuities of spatial characteris
Figure 3. Achievement of socio-cultural sustainability with continuities of spatial characteristics. Figure 3 Achie ement of socio cultural sustainability with continuities of spatial characteris
Figure 3. Achievement of socio-cultural sustainability with continuities of spatial characteristics. y
p
o understand people’s changing needs, and the adaptation of these needs
characteristic through physical transformation, the study of physical form
ee morphological scales: building structures, open spaces, and neighbor-
out. The spatial characteristics related to each morphological scale are ex-
heories (Tables 2 and 3). Researching the neighborhood/block layout in-
ntification of the relationship of the buildings to the block, size and shape
rhood, land coverage, density and spatial flow and street configurations. open space concentrates on building/plot arrangements along public open
ng height and width, active front coverage and spatial access patterns and
ween building-building and building-open space. The building structure
y involves the building layout, functions, number of floors, spatial sequence
to private property and efficiency of access. 3. Development of Analytical Framework 112), ‘forms found at different levels are identified as
are conceived as cultural entities rooted in, and specific to the local process
velopment’ [60]. In Caniggia and Maffei’s [29] work, they divided the types
gories: synchronic and diachronic. In synchronic types, production decreases
and remains strictly confined to the period in which it was introduced, while
c type undergoes a series of progressive transformations throughout an im-
period [32]. Some of types can be observed in different places within a par-
According to Kropf [30] (p. 112), ‘forms found at different levels are identified as types,
which are conceived as cultural entities rooted in, and specific to the local process of cultural
development’ [60]. In Caniggia and Maffei’s [29] work, they divided the types into two
categories: synchronic and diachronic. In synchronic types, production decreases through
time and remains strictly confined to the period in which it was introduced, while the
diachronic type undergoes a series of progressive transformations throughout an important
time period [32]. Some of types can be observed in different places within a particular time, p
p
g
g
ing to Kropf [30] (p. 112), ‘forms found at different levels are identified as
h are conceived as cultural entities rooted in, and specific to the local process
evelopment’ [60]. In Caniggia and Maffei’s [29] work, they divided the types
egories: synchronic and diachronic. In synchronic types, production decreases
e and remains strictly confined to the period in which it was introduced, while
nic type undergoes a series of progressive transformations throughout an im-
period [32]. Some of types can be observed in different places within a par-
According to Kropf [30] (p. 112), ‘forms found at different levels are identified as types,
which are conceived as cultural entities rooted in, and specific to the local process of cultural
development’ [60]. In Caniggia and Maffei’s [29] work, they divided the types into two
categories: synchronic and diachronic. In synchronic types, production decreases through
time and remains strictly confined to the period in which it was introduced, while the
diachronic type undergoes a series of progressive transformations throughout an important
time period [32]. Some of types can be observed in different places within a particular time, Sustainability 2021, 13, 6212 6 of 28 while others are more robust and can survive over consecutive time periods in the same
place [32]. 3. Development of Analytical Framework Figure 4 presents these spatial
In order to understand people’s changing needs, and the adaptation of these needs
to each spatial characteristic through physical transformation, the study of physical form
focuses on three morphological scales: building structures, open spaces, and neighbor-
hood/block layout. The spatial characteristics related to each morphological scale are
extracted from theories (Tables 2 and 3). Researching the neighborhood/block layout
involves the identification of the relationship of the buildings to the block, size and shape
of the neighborhood, land coverage, density and spatial flow and street configurations. The analysis of open space concentrates on building/plot arrangements along public open
spaces, building height and width, active front coverage and spatial access patterns and
hierarchy between building-building and building-open space. The building structure
analysis mainly involves the building layout, functions, number of floors, spatial sequence
from entrance to private property and efficiency of access. Figure 4 presents these spatial
characteristics and their relationship with each morphological scale that constitutes the
framework of adaptation of changing of use in place. s and their relationship with each morphological scale that constitutes the
adaptation of changing of use in place. l characteristics of typological study. aracteristics
Key Authors
g layout
e.g., [23,25,31,61–63]
g façade
e.g., [23,31,62,63]
r of floors
e.g., [61]
Table 2. Spatial characteristics of typological study. Spatial Characteristics
Key Authors
Building layout
e.g., [23,25,31,61–63]
Building façade
e.g., [23,31,62,63]
Number of floors
e.g., [61]
Building materials
e.g., [58,62] and their relationship with each morphologica
adaptation of changing of use in place. Table 2. Spatial characteristics of typological study. 7 of 28 Sustainability 2021, 13, 6212 Table 2. Cont. Spatial Characteristics
Key Authors
Spatial hierarchy
e.g., [24,31,64]
Access efficiency
e.g., [35,58]
Open space structures
e.g., [65,66]
Plot and block layout, size and shapes
e.g., [25,30,62,65,67,68]
Density and land coverage
e.g., [64,65]
Spatial relationship between building, plot, open space and block
e.g., [24,31,62,69]
Buildings with its relations to open spaces
e.g., [31,35,64,69]
Site configurations
e.g., [67]
Table 3. The components of Analytical framework. Layout
Building/Plot Arrangement
Site/Block Arrangement
Public/private zones are strictly
separated, partly separated or not
separated? Buildings’ spatial relationships
(Back-to-back/Side-to-side);
Any set back, front garden, back garden? Diversity and continuity of buildings
Identical or irregular plots in shape and size? Open plan or gated? Any defined site entrance or
boundary? 4. Identification of Cases in Transformation of the Neighborhood
4. Identification of Cases in Transformation of the Neighborhoo In the second research step, six cases of building typologies are identified in a trans-
formation study of the neighborhood. Qian Yuan’en Si neighborhood (Figure 5) is one of
the historical living areas where mainly vernacular but also a variety of other building
typologies were free planned after the 1980s [6,69]. During the urban transformation, the
main driver of changes in socio-spatial patterns was the shift in formation of the economy
from a politically-controlled to a market-driven mechanism [70–72]. Although horizon-
tally the original fabric can still be recognized in the current urban tissue, vertically, the
building facades and high-rise plots show dramatic changes (Figure 6). The neighborhood
contributes to the overall urban analysis because its built form combined a variety of
building types in a vernacular context. Table 4 illustrates four morphological periods in the
neighborhood that present transformation of political administration, economic formation,
as well as socio-cultural transitions. g
In the second research step, six cases of building typologies are identified in a trans-
formation study of the neighborhood. Qian Yuan’en Si neighborhood (Figure 5) is one of
the historical living areas where mainly vernacular but also a variety of other building
typologies were free planned after the 1980s [6,69]. During the urban transformation, the
main driver of changes in socio-spatial patterns was the shift in formation of the economy
from a politically-controlled to a market-driven mechanism [70–72]. Although horizon-
tally the original fabric can still be recognized in the current urban tissue, vertically, the
building facades and high-rise plots show dramatic changes (Figure 6). The neighborhood
contributes to the overall urban analysis because its built form combined a variety of
building types in a vernacular context. Table 4 illustrates four morphological periods in
the neighborhood that present transformation of political administration, economic for-
mation as well as socio-cultural transitions (a)
(b)
Figure 5. The location of Qian Yuan’en Si neighborhood. The location of the neighborhood in the city (a), the area of the
neighborhood (b). Figure 5. The location of Qian Yuan’en Si neighborhood. The location of the neighborhood in the city (a), the area of the
neighborhood (b). (b) (b) (a) (a) (b) Figure 5. The location of Qian Yuan’en Si neighborhood. The location of the neighborhood in the city (a), the area of the
neighborhood (b). Figure 5. The location of Qian Yuan’en Si neighborhood. 3. Development of Analytical Framework Spatial Hierarchy
Functions
Building height/street width
Size and shape
Functions are defined/partly defined/
not defined
W = H/2; W = H/3; W = 2H; W = H
Size defined by footprint in terms of
accessibility
Numbers of floors
Active frontage
Land coverage
Original designed/ professional retrofit/
self-regenerated
Active front coverage (%)? Land construction coverage (%)
(20%—low/50%—medium/over
70%—high)
Numbers of residents
Spatial Sequence
Spatial hierarchy
Density and Spatial Flow
From the public entrances to private
property;
Spatial relationship from public to private
Location of the case in density areas;
Accessibility efficiency and privacy;
Access efficiency to open space
Spatial Movement
Street configurations
Total number of steps from private
property to public open spaces;
Spatial transition of access;
Steps from buildings to private property
Total number of steps from private property
to out of neighborhood
Types of roads/streets/paths in site;
Are they mix of pedestrian and
vehicles or separate? Table 2. Cont. 8 of 28
vehi- Sustainability 2021, 13, 6212
Spatial transition of acce
Steps from buildings to p Figure 4. The analytical framework of changing of needs. R REVIEW
9
Figure 4. The analytical framework of changing of needs. Figure 4. The analytical framework of changing of needs. Figure 4. The analytical framework of changing of needs. 4. Identification of Cases in Transformation of the Neighborhood
4. Identification of Cases in Transformation of the Neighborhood 4. Identification of Cases in Transformation of the Neighborhood
4. Identification of Cases in Transformation of the Neighborhoo The location of the neighborhood in the city (a), the area of the
neighborhood (b). 9 of 28
he Sustainability 2021, 13, 6212
Figure 5. The loca Figure 6. The section of Qian Yuan’en Si neighborhood. Figure 6. The section of Qian Yuan’en Si neighborhood. Figure 6. The section of Qian Yuan’en Si neighborhood. Figure 6. The section of Qian Yuan’en Si neighborhood. Figure 6 The section of Qian Yuan’en Si neighborhood
Figure 6. The section of Qian Yuan’en Si neighborhood. Figure 6 The section of Qian Yuan’en Si neighborhood
Figure 6. The section of Qian Yuan’en Si neighborhood. g
g
g yp
Morphological Periods and Transforming of Buildings
Period 1
Imperial Period—before 1949
Morphological Periods and Transforming of Buildings
Period 1
Imperial Period—before 1949
Traditional housing types, open space structure and block layout, which
address imperial power and social hierarchy in spatial structure. Period 2
Soviet Influence Period—1949–1978
Soviet socialist idea is spread into China, the housing types changed to
early modern style, to emphasize socialism and collectivism in lifestyle. Period 3
Post-reform and Contemporary—1978-current period
Modern building types with more private property layout design were
imported into China, and the lifestyles were transformed into market lead The neighborhood came into existence during the Ming dynasty (1368–1644). It was
mainly developed during the Qing dynasty (1636–1912) in terms of its morphological
tissues. The neighborhood was developed for government institutions and government
owned workshops. Thus, the road network within the neighborhood was characterized
by one-way roads (cul-de-sacs) designed to have one entrance accessible to the hutong
(internal road within the historic block) from the main streets, but all hutongs ended
inside in order to maintain a high level of privacy [73,74]. The significant change of the
neighborhoods started during the Communist period, when there was a movement to
remove the ‘Four Olds’ and the population dramatically increased, and therefore, the
function of the neighborhoods completely changed from institutional and residential use
to mostly residential use by adapting to the changes in the political and social environment
in order to respond to political needs [8,73,75]. Thus, the housing types were changed from
private to shared houses, and hutong networks were transformed from private design to
open access. 4. Identification of Cases in Transformation of the Neighborhood
4. Identification of Cases in Transformation of the Neighborhoo Before the Post-Reform (1970s), due to population growth, the work-unit
based social structure and resources allocation had a serious effect on property allocation,
so a movement to reform the political, economic and social structure was formed [75]. However, before the City Planning Law of People’s Republic of China (1989) was enacted,
due to the dramatically increased population, the residential houses grew organically with
occupation of public space and leveled up horizontally in order to meet the enormous
demand for living spaces [8,75]. Meanwhile, during the transformation process, economic
influences started to play an important role because daily commercial needs increased
due to population density growth, the changing needs in public space based on uses of
automobile, and the lifestyle transit because of cultural effects from globalization, which all
meant the area started to engage with urban growth in a more complex way [71] (Figure 7). 10 of 28
31 Sustainability 2021, 13, 6212
nability 2021, 13, x FOR PEER R Figure 7. Land use transformation of the neighborhoods. Figure 7. Land use transformation of the neighborhoods. d
f the neighborhoods. Figure 7. Land use transformation of the neighborhoods. Through morphological layers for identifying continuities of spatial characteristics
deliver socio-cultural sustainability, Table 5 presents the analysis of a contemporary
hborhood/block structure, which provides current uses in the neighborhood as
ground and context. In the following sub-sections, each case is identified based on
hological period, and is introduced and analyzed at building scale and open space
in order to find out changes of spatial characteristics in relation to uses. Through morphological layers for identifying continuities of spatial characteristics that
deliver socio-cultural sustainability, Table 5 presents the analysis of a contemporary neigh-
borhood/block structure, which provides current uses in the neighborhood as background
and context. In the following sub-sections, each case is identified based on morphological
period, and is introduced and analyzed at building scale and open space scale in order to
find out changes of spatial characteristics in relation to uses. Table 5 Neighborhood layout ana
4.1. Imperial Period: Case 1 and Case 2 Table 5. Neighborhood layout analysis. Neighborhood Layout Analysis
Site/Block Arrangement
Open side (organically and internally grown)
Externally no strict defined boundaries
Size and shape
Max. 7 turns (approx. 180 m) from primary streets to private prop-
erty. Regular and historical continued layout shape with walkable
The entire neighborhood was developed based on imperial context; traditional court-
yard houses and open space networks are commonly discovered in the neighborhoods. However, since 1949, due to changing socio-political ideology, dramatically increased
populations and other factors, the traditional courtyard houses were transformed into
chaotic-no-yard houses, and one family-owned houses become multi-family occupied
residential plots [6,69]. Therefore, its original spatial design is largely changed to fulfill the
transitions of access, uses of facilities and ways of living (Figures 8 and 9). Case 1 and 2
are all originally part of traditional courtyard houses, but during urban transformation
and socio-political transition, the original inner private yard is built with more rooms to
fulfill increased demands for housing, and existing rooms start opening up access directly
to the public street (Tables 6 and 7). The differentiations between these cases are: case 1
has occupied public space and built an extension garden as transitional space; and case 2
has converted from residential to commercial uses with transparent doors that visually
increase spatial connections between indoor and outdoor. These types of cases represent
the largest number of residential houses within the neighborhood. 11 of 28
en space
en space Sustainability 2021, 13, 6212 Table 5. Neighborhood layout analysis. Neighborhood Layout Analysis
scale in order to find out changes of spatial characteristics in relation to uses. Table 5. Neighborhood layout analysis. Neighborhood Layout Analysis
Site/Block Arrangement
Open side (organically and internally grown)
Externally no strict defined boundaries
Size and shape
Max. 7 turns (approx. 180 m) from primary streets to private
property. Regular and historical continued layout shape with
walkable size
(1)→(2)→(3)→(4)→(5)→(6)→home
Land coverage
Land construction coverage is approx. over 80%
Residents approx. 900
Site/Block Arrangement
Open side (organically and internally grown)
Externally no strict defined boundaries
Table 5. Neighborhood layout analysis. Neighborhood Layout Analysis
Site/Block Arrangement
Open side (organically and internally grown)
Externally no strict defined boundaries
Size and shape
Max. 7 turns (approx. 180 m) from primary streets to private
property. Regular and historical continued layout shape with
walkable size
(1)→(2)→(3)→(4)→(5)→(6)→home
Land coverage
Land construction coverage is approx. over 80%
Residents approx. 900
Size and shape
Max. Table 5 Neighborhood layout ana
4.1. Imperial Period: Case 1 and Case 2 7 turns (approx. 180 m) from primary streets to private
property. Regular and historical continued layout shape with
walkable size
(1)→(2)→(3)→(4)→(5)→(6)→home
Land coverage
Land construction coverage is approx. over 80%
Residents approx. 900
Sustainability 2021, 13, x FOR PEER REVIEW
12 of 31
Density and spatial flow
Relatively, the higher density is located in the west which is
connected to the primary street for efficient transportation and
facilities, instead of the east that links to historical
neighborhoods, where large facilities (such as shopping malls)
and public transport cannot be built. The cul-de-sacs provide a good level of privacy and easy access
to public areas in terms of block size. Street configurations
The primary street directly leads the secondary street that
forms efficient access. The cul-de-sacs link to the case VI plot
that creates third-levels of spatial class. Mixed pedestrian and vehicle parking and access, which
creates risks and safety issues for pedestrians
Sustainability 2021, 13, x FOR PEER REVIEW
12 of 31
Density and spatial flow
Relatively, the higher density is located in the west which is
connected to the primary street for efficient transportation and
facilities, instead of the east that links to historical
neighborhoods, where large facilities (such as shopping malls)
and public transport cannot be built. The cul-de-sacs provide a good level of privacy and easy access
to public areas in terms of block size. Street configurations
The primary street directly leads the secondary street that
forms efficient access. The cul-de-sacs link to the case VI plot
that creates third-levels of spatial class. Mixed pedestrian and vehicle parking and access, which
creates risks and safety issues for pedestrians
Density and spatial flow
Relatively, the higher density is located in the west which is
connected to the primary street for efficient transportation and
facilities, instead of the east that links to historical
neighborhoods, where large facilities (such as shopping malls)
and public transport cannot be built. The cul-de-sacs provide a good level of privacy and easy access
to public areas in terms of block size. Sustainability 2021, 13, x FOR PEER REVIEW
12 of 31
Density and spatial flow
Relatively, the higher density is located in the west which is
connected to the primary street for efficient transportation and
facilities, instead of the east that links to historical
neighborhoods, where large facilities (such as shopping malls)
and public transport cannot be built. Table 5 Neighborhood layout ana
4.1. Imperial Period: Case 1 and Case 2 The cul-de-sacs provide a good level of privacy and easy access
to public areas in terms of block size. Street configurations
The primary street directly leads the secondary street that
forms efficient access. The cul-de-sacs link to the case VI plot
that creates third-levels of spatial class. Mixed pedestrian and vehicle parking and access, which
creates risks and safety issues for pedestrians
Street configurations
The primary street directly leads the secondary street that forms
efficient access. The cul-de-sacs link to the case VI plot that
creates third-levels of spatial class. Mixed pedestrian and vehicle parking and access, which creates
risks and safety issues for pedestrians Table 5. Neighborhood layout analysis. Table 5. Neighborhood layout analysi
Neighborhood Layout Analysis Table 5. Neighborhood layout analysis. Table 5. Neighborhood layout analysi
Neighborhood Layout Analysis Neighborhood Layout Analysis
Neighborhood Layout Analy pen side (organically and internally grown)
Open side (organically and internally grown)
Externally no strict defined boundaries
pen side (organically and internally grown)
ternally no strict defined boundaries y 2021, 13, x FOR PEER REVIEW
2021, 13, x FOR PEER REVIEW
2021, 13, x FOR PEER REVIEW nd coverage
nd construction coverage is approx over 80%
nd construction coverage is approx. over 80%
sidents approx 900
Land construction coverage is approx. over 80%
Residents approx. 900
nsity and spatial flow sidents approx. 900
ensity and spatial flow
nsity and spatial flow
Density and spatial flow
latively, the higher dens e ative y, t e
ig e de sity is ocated i
t e west w ic
is
nnected to the primary street for efficient transportation an
cilities, instead of the east that links to historical
ighborhoods, where large facilities (such as shopping mall
nd public transport cannot be built. y,
g
y
nected to the primary street for efficient transportation and
lities, instead of the east that links to historical
ghborhoods, where large facilities (such as shopping malls
d public transport cannot be built. Relatively, the higher density is located in the west which is
connected to the primary street for efficient transportation and
facilities, instead of the east that links to historical
neighborhoods, where large facilities (such as shopping malls)
and public transport cannot be built. nnected to the primary street for efficient transportation an
ilities, instead of the east that links to historical
ghborhoods, where large facilities (such as shopping malls
d public transport cannot be built. Table 5 Neighborhood layout ana
4.1. Imperial Period: Case 1 and Case 2 Mixed pedestrian and vehicle parking and access, which creates
risks and safety issues for pedestrians 12 of 28
visually
t 12 of 28
visually
t Sustainability 2021, 13, 6212 (a)
(b)
Figure 8. The transformation of courtyard house. The left subfigure shows the original design of the courtyard house (a),
and the right subfigure presents how multi-families occupied the courtyard houses currently (b). Figure 8. The transformation of courtyard house. The left subfigure shows the original design of the courtyard house (a),
and the right subfigure presents how multi-families occupied the courtyard houses currently (b). ity 2021, 13, x FOR PEER REVIEW (a)
ty 2021, 13, x FOR PEER REVIEW (b) (b) (a)
EVIEW Figure 8. The transformation of courtyard house. The left subfigure shows the original design of the courtyard house (a),
and the right subfigure presents how multi-families occupied the courtyard houses currently (b). Figure 8. The transformation of courtyard house. The left subfigure shows the original design of the courtyard house (a),
and the right subfigure presents how multi-families occupied the courtyard houses currently (b). igure 9. The transformation of courtyard houses in relation to block. Original spatial design of the block (a), spat
ationship of original courtyard houses and hutong (b), private space in original courtyard house design (c), huto
d courtyard houses in 1980s (d), spatial changes of courtyard houses and hutongs (e), spaces in courtyard houses
multi-families occupied (f). Figure 9. The transformation of courtyard houses in relation to block. Original spatial design of the block (a), spatial
relationship of original courtyard houses and hutong (b), private space in original courtyard house design (c), hutongs
and courtyard houses in 1980s (d), spatial changes of courtyard houses and hutongs (e), spaces in courtyard houses after
multi-families occupied (f). igure 9. The transformation of courtyard houses in relation to block. Original spatial design of the block (a), spati
ationship of original courtyard houses and hutong (b), private space in original courtyard house design (c), huto
courtyard houses in 1980s (d), spatial changes of courtyard houses and hutongs (e), spaces in courtyard houses
multi-families occupied (f). Figure 9. The transformation of courtyard houses in relation to block. Table 5 Neighborhood layout ana
4.1. Imperial Period: Case 1 and Case 2 e cul-de-sacs provide a good level of privacy and easy acce p
p
he cul-de-sacs provide a good level of privacy and easy acce
public areas in terms of block size
p
p
e cul-de-sacs provide a good level of privacy and easy acces
public areas in terms of block size
p
p
The cul-de-sacs provide a good level of privacy and easy access
to public areas in terms of block size. e cul de sacs provide a good level of privacy and easy acce
public areas in terms of block size. p
Street configurations
The primary street directly leads the secondary street that
forms efficient access. The cul-de-sacs link to the case VI plot
that creates third-levels of spatial class. Mixed pedestrian and vehicle parking and access, which
creates risks and safety issues for pedestrians
p
Street configurations
The primary street directly leads the secondary street that
forms efficient access. The cul-de-sacs link to the case VI plot
that creates third-levels of spatial class. Mixed pedestrian and vehicle parking and access, which
creates risks and safety issues for pedestrians
Street configurations
The primary street directly leads the secondary street that
forms efficient access. The cul-de-sacs link to the case VI plot
that creates third-levels of spatial class. Mixed pedestrian and vehicle parking and access, which
creates risks and safety issues for pedestrians
Street configurations
The primary street directly leads the secondary street that forms
efficient access. The cul-de-sacs link to the case VI plot that
creates third-levels of spatial class. Mixed pedestrian and vehicle parking and access, which creates
risks and safety issues for pedestrians he primary street directly leads the secondary street that
rms efficient access. The cul-de-sacs link to the case VI plot
at creates third-levels of spatial class. e primary street directly leads the secondary street that
ms efficient access. The cul-de-sacs link to the case VI plot
t creates third-levels of spatial class. e primary street directly leads the secondary street that
ms efficient access. The cul-de-sacs link to the case VI plot
at creates third-levels of spatial class. xed pedestrian and vehicle parking and access, which
The primary street directly leads the secondary street that forms
efficient access. The cul-de-sacs link to the case VI plot that
creates third-levels of spatial class. Table 5 Neighborhood layout ana
4.1. Imperial Period: Case 1 and Case 2 Original spatial design of the block (a), spatial
relationship of original courtyard houses and hutong (b), private space in original courtyard house design (c), hutongs
and courtyard houses in 1980s (d), spatial changes of courtyard houses and hutongs (e), spaces in courtyard houses after
multi-families occupied (f). 13 of 28
ngs
fter
er Sustainability 2021, 13, 6212
relationsh
and courty
and courtyard h Table 6. Case 1 and Case 2—building structure analysis. g
y
Building Structu
Building Structure A Table 6. Case 1 and Case 2
building structure analysis. Table 5 Neighborhood layout ana
4.1. Imperial Period: Case 1 and Case 2 Soviet Influence Period: Case 3, Case 4, and Case 5
In this particular time (1949–1979), soviet socialism and collectivism have seriously
influenced not only urban construction and development, but also socio-political ideologies
through people’s life [75]. During this period, physical urban settings are the major change in
China; a large number of traditional buildings were demolished and replaced by soviet style
raised buildings, and traditional urban tissue is transited to suit new sociopolitical ideologies,
modern facilities (train station, automobile, and motorway) and increased population [75]. The building typologies in this period are mainly presented as two or three storage soviet
style buildings, with shared kitchen and bathrooms in each floor. However, in contemporary Table 7. Case 1 and Case 2—open space analysis. Open Space Analysis
Open Space Analysis
Open Space Analysis
Ca e 1
Open Space Analysis
C
1
Open Space Analysis
Open Space Analysi Open Space Analysis
1
Case 1
Case 1
Case 1 Number of Case o Bui
Buildin
height/
Bu
he
Building height/street
width
r
w
wid 1:1.2 Active front S
hi
Spat
hierah
Spatial movement nt
fluence Period: Case 3 Case 4 and Case 5
uence Period: Case 3, Case 4, and Case 5
oviet Influence Period: Case 3, Case 4, and Case 5
4.2. Soviet Influence Period: Case 3, Case 4, and Case 5 fluence Period: Case 3, Case 4, and Case 5
2. Soviet Influence Period: Case 3, Case 4, and Case 5
nce Period: Case 3, Case 4, and Case 5
nfluence Period: Case 3, Case 4, and Case 5
Soviet Influence Period: Case 3, Case 4, and Case 5
4.2. Soviet Influence Period: Case 3, Case 4, and Case 5
fluence Period: Case 3, Case 4, and Case 5
Soviet Influence Period: Case 3, Case 4, and Case 5
uence Period: Case 3, Case 4, and Case 5
oviet Influence
eriod Case 3, Case , and Case 5
fl
In this particular time (1949–1979), soviet socialism and collectivism have seriously
influenced not only urban construction and development, but also socio-political ideologies
through people’s life [75]. During this period, physical urban settings are the major change in
China; a large number of traditional buildings were demolished and replaced by soviet style
raised buildings, and traditional urban tissue is transited to suit new sociopolitical ideologies,
modern facilities (train station, automobile, and motorway) and increased population [75]. Table 5 Neighborhood layout ana
4.1. Imperial Period: Case 1 and Case 2 Building Structure Analysis
Number of Case
Case 1
Case 2
Building Structure Analysis
Number
of Case
Case 1
Case 2
Building Structure Analysis
Number
of Case
Case 1
Case 2
Layout
OR PEER REVIEW
14 of 3
Layout
Functions
Number
of floors
Spatial
sequence
Access
efficiency
to open
space
EER REVIEW
14 of 31
Layout
Functions
Number
of floors
Spatial
sequence
Access
efficiency
to open
space
Functions
OR PEER REVIEW
14 of 3
Layout
Functions
Number
of floors
Spatial
sequence
Access
efficiency
to open
space
ER REVIEW
14 of 31
Layout
Functions
Number
of floors
Spatial
sequence
Access
efficiency
to open
space
Number of floors
OR PEER REVIEW
14 of 3
Layout
Functions
Number
of floors
Spatial
sequence
Access
efficiency
to open
space
EER REVIEW
14 of 31
Layout
Functions
Number
of floors
Spatial
sequence
Access
efficiency
to open
space
Spatial sequence
OR PEER REVIEW
14 of 3
Layout
Functions
Number
of floors
Spatial
sequence
Access
efficiency
to open
space
EER REVIEW
14 of 31
Layout
Functions
Number
of floors
Spatial
sequence
Access
efficiency
to open
space
Access efficiency
to open space
OR PEER REVIEW
14 of 3
Layout
Functions
Number
of floors
Spatial
sequence
Access
efficiency
to open
space
EER REVIEW
14 of 31
Layout
Functions
Number
of floors
Spatial
sequence
Access
efficiency
to open
space Building Structure Analysis
Case 1
Case 1 Number of Case
of Case
of Case 14 of
14 of 31
14 of
14 of 31
14 o
14 of 31
14 of
14 of 31
14 o
14 of 31 F
ti
ti
Functions
Functions
nctions
u
io Number
Number
Number of floors
Number
of floors
Number
of floors
of floors
of floors Spatial
Spatial
Spatial sequence
Spatial
sequence
Spatial
sequence
sequence
sequence Access
Access
Access
efficiency
Access
efficiency
Access
efficiency
to o e
Access
fficiency
to o e
Access efficiency
to open space
efficiency
to open
efficiency
to open 14 of 28 Sustainability 2021, 13, 6212
Number
of Case Table 7. Case 1 and Case 2—open space analysis. Open Space Analysis
Number of Case
Case 1
Case 2
Table 7. Case 1 and Case 2—open space analysis. Open Space Analysis
Number
of Case
Case 1
Case 2
Building
/plot
arrange
ment
Building
height/st
reet
width
1 : 1.2
1 : 1
Active
front
Spatial
hierarch
y
Spatial
moveme
nt
4.2. Soviet Influence Period: Case 3, Case 4, and Case 5
Table 7. Table 5 Neighborhood layout ana
4.1. Imperial Period: Case 1 and Case 2 Case 1 and Case 2—open space analysis. Open Space Analysis
Number
of Case
Case 1
Case 2
Building
/plot
arrange
ment
Building
height/st
reet
width
1 : 1.2
1 : 1
Active
front
Spatial
hierarch
y
Spatial
moveme
nt
4.2. Soviet Influence Period: Case 3, Case 4, and Case 5
Building/plot
arrangement
Open Space Analysis
Number
of Case
Case 1
Case 2
Building
/plot
arrange
ment
Building
height/st
reet
width
1 : 1.2
1 : 1
Active
front
Spatial
hierarch
y
Spatial
moveme
nt
4.2. Soviet Influence Period: Case 3, Case 4, and Case 5
Open Space Analysis
Number
of Case
Case 1
Case 2
Building
/plot
arrange
ment
Building
height/st
reet
width
1 : 1.2
1 : 1
Active
front
Spatial
hierarch
y
Spatial
moveme
nt
4.2. Soviet Influence Period: Case 3, Case 4, and Case 5
Table 7. Case 1 and Case 2—open space analysis. Open Space Analysis
Number
of Case
Case 1
Case 2
Building
/plot
arrange
ment
Building
height/st
reet
width
1 : 1.2
1 : 1
Active
front
Spatial
hierarch
y
Spatial
moveme
nt
4.2. Soviet Influence Period: Case 3, Case 4, and Case 5
Table 7. Case 1 and Case 2—open space analysis. Open Space Analysis
Number
of Case
Case 1
Case 2
Building
/plot
arrange
ment
Building
height/st
reet
width
1 : 1.2
1 : 1
Active
front
Spatial
hierarch
y
Spatial
moveme
nt
4.2. Soviet Influence Period: Case 3, Case 4, and Case 5
Building height/street
width
1:1.2
1:1
Active front
Number
of Case
Case 1
Case 2
Building
/plot
arrange
ment
Building
height/st
reet
width
1 : 1.2
1 : 1
Active
front
Spatial
hierarch
y
Spatial
moveme
nt
4.2. Soviet Influence Period: Case 3, Case 4, and Case 5
of Case
Building
/plot
arrange
ment
Building
height/st
reet
width
1 : 1.2
1 : 1
Active
front
Spatial
hierarch
y
Spatial
moveme
nt
4.2. Soviet Influence Period: Case 3, Case 4, and Case 5
Spatial hierarchy
Building
/plot
arrange
ment
Building
height/st
reet
width
1 : 1.2
1 : 1
Active
front
Spatial
hierarch
y
Spatial
moveme
nt
4.2. Soviet Influence Period: Case 3, Case 4, and Case 5
Building
/plot
arrange
ment
Building
height/st
reet
width
1 : 1.2
1 : 1
Active
front
Spatial
hierarch
y
Spatial
moveme
nt
4.2. Soviet Influence Period: Case 3, Case 4, and Case 5
Spatial movement
4.2. Table 5 Neighborhood layout ana
4.1. Imperial Period: Case 1 and Case 2 Building Building Structure Analysis
p
p
p
p
g
y
p
g
and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. Building Building Structure Analysis Table 8. Case 3, Case 4 and Case 5–Building structure analysis. Building Building Structure Analysis
Building Building Structure Analysis
Building Building Structure Analysis
Building Building Structure Analysis
C
3
C
4
C
5
Building Building Structure Analysis
3
C
4
C
5
Building Building Structure Analysis
3
C
4
C
5
ase 3
Case 4
Case 5
ase 3
Case 4
Case 5
se 3
Case 4
Case 5 Table 8. Case 3, Case 4 and Case 5–Building structure analysis. Building Building Structure Analysis
Building Building Structure Analysis
Building Building Structure Analysis
Building Building Structure Analysis
3
C
4
C
5
Building Building Structure Analysis
3
C
4
C
5
Building Building Structure Analysis
3
C
4
C
5
ase 3
Case 4
Case 5
ase 3
Case 4
Case 5
e 3
Case 4
Case 5 Table 8. Case 3, Case 4 and Case 5–Building structure analysis. Building Building Structure Analysis
Building Building Structure Analysis
Building Building Structure Analysis
Building Building Structure Analysis
C
C
4
Building Building Structure Analysis
3
C
4
Building Building Structure Analysis
3
C
4
ase 3
Case 4
ase 3
Case 4
se 3
Case 4 Table 8. Case 3, Case 4 and Case 5–Building structure analysis. Table 5 Neighborhood layout ana
4.1. Imperial Period: Case 1 and Case 2 The building typologies in this period are mainly presented as two or three storage soviet
style buildings, with shared kitchen and bathrooms in each floor. However, in contemporary
use (Tables 8 and 9), many of these buildings are regenerated; the representative examples are: Sustainability 2021, 13, 6212 15 of 28
ny of the
y of thes
of these
ny of the
al buildi
y of thes
l buildin
of these
building
al buildin
opulatio
l buildin
opulation
building
ulations 15 of 28
ny of the
y of thes
of these
ny of the
al buildi
y of thes
l buildin
of these
building
al buildin
opulatio
l buildin
opulation
building
ulations case 3—the original building has extended its height and length with changed façade to adapt
to increased populations and transitions of needs to suit new lifestyles and technologies such
as automobile use; case 4—develops its own semi-private space for improving activity needs
and parking; and case 5—keeps its original design. and transitions of needs to suit new lifestyles and technologies such as automobile us
case 4—develops its own semi-private space for improving activity needs and parkin
and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. and transitions of needs to suit new lifestyles and technologies such as automobile us
case 4—develops its own semi-private space for improving activity needs and parkin
and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. and transitions of needs to suit new lifestyles and technologies such as automobile use;
case 4—develops its own semi-private space for improving activity needs and parking;
and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. case 4—develops its own semi-private space for improving activity needs and parkin
and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. case 4—develops its own semi-private space for improving activity needs and parking
and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. case 4—develops its own semi-private space for improving activity needs and parking;
and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. p
p
p
p
g
y
p
and case 5—keeps its original design. Table 8. Table 5 Neighborhood layout ana
4.1. Imperial Period: Case 1 and Case 2 Case 3, Case 4 and Case 5–Building structure analysis. Building Building Structure Analysis
p
p
p
p
g
y
p
and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. Building Building Structure Analysis
p
p
p
p
g
y
p
g
and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. Building Building Structure Analysis case 3—the original building has extended its height and length with changed façade to adapt
to increased populations and transitions of needs to suit new lifestyles and technologies such
as automobile use; case 4—develops its own semi-private space for improving activity needs
and parking; and case 5—keeps its original design. and transitions of needs to suit new lifestyles and technologies such as automobile us
case 4—develops its own semi-private space for improving activity needs and parkin
and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. and transitions of needs to suit new lifestyles and technologies such as automobile us
case 4—develops its own semi-private space for improving activity needs and parkin
and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. and transitions of needs to suit new lifestyles and technologies such as automobile use;
case 4—develops its own semi-private space for improving activity needs and parking;
and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. case 4—develops its own semi-private space for improving activity needs and parkin
and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. case 4—develops its own semi-private space for improving activity needs and parking
and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. case 4—develops its own semi-private space for improving activity needs and parking;
and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. p
p
p
p
g
y
p
and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. Building Building Structure Analysis
p
p
p
p
g
y
p
and case 5—keeps its original design. Table 8. Case 3, Case 4 and Case 5–Building structure analysis. Table 5 Neighborhood layout ana
4.1. Imperial Period: Case 1 and Case 2 Case 3, Case 4 and Case 5—open space analysis. Table 9. Case 3, Case 4 and Case 5—open space analysis
Table 9. Case 3, Case 4 and Case 5—open space analysis. Open Space Analysis
Open Space Analysis
Open Space Analysis
Open Space Analysis
Open Space Analysis
Open Space Analysis Table 9. Case 3, Case 4 and Case 5—open space analysis. Open Space Analysis
Number of
Case
Case 3
Case 4
Case 5
Building/plot
arrangement
Building
height/street
width
1 : 0.1
1 : 1
1 : 0.3
Table 9. Case 3, Case 4 and Case 5—open space analysis. Open Space Analysis
Number of
Case
Case 3
Case 4
Case 5
Building/plot
arrangement
Building
height/street
width
1 : 0.1
1 : 1
1 : 0.3
Table 9. Case 3, Case 4 and Case 5—open space analysis. Open Space Analysis
Number of
Case
Case 3
Case 4
Case 5
Building/plot
arrangement
Building
height/street
width
1 : 0.1
1 : 1
1 : 0.3
Table 9. Case 3, Case 4 and Case 5—open space analysis. Table 5 Neighborhood layout ana
4.1. Imperial Period: Case 1 and Case 2 Open Space Analysis
Number of
Case
Case 3
Case 4
Case 5
Open Space Analysis
Number of
Case
Case 3
Case 4
Case 5
Building/plot
arrangement
Building
height/street
width
1 : 0.1
1 : 1
1 : 0.3
Open Space Analysis
Number of
Case
Case 3
Case 4
Case 5
Building/plot
arrangement
Building
height/street
width
1 : 0.1
1 : 1
1 : 0.3
Open Space Analysis
Number of
Case
Case 3
Case 4
Case 5
Building/plot
arrangement
Building
height/street
width
1 : 0.1
1 : 1
1 : 0.3
Building/plot
arrangement
Open Space Analysis
Number of
Case
Case 3
Case 4
Case 5
Building/plot
arrangement
Building
height/street
width
1 : 0.1
1 : 1
1 : 0.3
Open Space Analysis
Number of
Case
Case 3
Case 4
Case 5
Building/plot
arrangement
Building
height/street
width
1 : 0.1
1 : 1
1 : 0.3
Open Space Analysis
Number of
Case
Case 3
Case 4
Case 5
Building/plot
arrangement
Building
height/street
width
1 : 0.1
1 : 1
1 : 0.3
Open Space Analysis
Number of
Case
Case 3
Case 4
Case 5
Building/plot
arrangement
Building
height/street
width
1 : 0.1
1 : 1
1 : 0.3
Open Space Analysis
Number of
Case
Case 3
Case 4
Case 5
Building/plot
arrangement
Building
height/street
width
1 : 0.1
1 : 1
1 : 0.3
Open Space Analysis
Number of
Case
Case 3
Case 4
Case 5
Building/plot
arrangement
Building
height/street
width
1 : 0.1
1 : 1
1 : 0.3
Building
height/street
width
1:0.1
1:1
1:0.3
Active front
Sustainability 2021, 13, x FOR PEER REVIEW
18 of 31
Active front
Spatial
hierarchy
Spatial
movement
4.3. Post-reform and Contemporary Period: Case 6
Sustainability 2021, 13, x FOR PEER REVIEW
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Active front
Spatial
hierarchy
Spatial
movement
4.3. Post-reform and Contemporary Period: Case 6
Since 1978 the country has transformed from a planned economy to an open market
Sustainability 2021, 13, x FOR PEER REVIEW
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Active front
Spatial
hierarchy
Spatial
movement
4.3. Post-reform and Contemporary Period: Case 6
Spatial
hierarchy
Sustainability 2021, 13, x FOR PEER REVIEW
18 of 31
Active front
Spatial
hierarchy
Spatial
movement
4.3. Post-reform and Contemporary Period: Case 6
Si
1978
h
h
f
d f
l
d
k
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18 of 31
Active front
Spatial
hierarchy
Spatial
movement
4.3. Table 5 Neighborhood layout ana
4.1. Imperial Period: Case 1 and Case 2 Cont. Table 8. Cont. Building Building Structure Analysis
Number of Case
Case 3
Case 4
Case 5
Access efficiency
to open space
Spatial
squence
Access
efficiency to
open space
Spatial
squence
Access
efficiency to
open space
Spatial
squence
Access
efficiency to
open space
squence
Access
efficiency to
open space
Table 9 Case 3 Case 4 and Case 5—open space analysis
squence
Access
efficiency to
open space
Table 9 Case 3 Case 4 and Case 5—open space analysis
squence
Access
efficiency to
open space
Table 9 Case 3 Case 4 and Case 5—open space analysis
Access
efficiency to
open space
Table 9. Case 3, Case 4 and Case 5—open space analysis. Access
efficiency to
open space
Table 9. Case 3, Case 4 and Case 5—open space analysis. Access
efficiency to
open space
Table 9. Case 3, Case 4 and Case 5—open space analysis. Access
efficiency to
open space
Table 9. Case 3, Case 4 and Case 5—open space analysis. Access
efficiency to
open space
Table 9. Case 3, Case 4 and Case 5—open space analysis. Access
efficiency to
open space
Table 9. Case 3, Case 4 and Case 5—open space analysis. Building Building Structure Analysis Access efficiency
to open space
Access
efficiency to
open space
Access
efficiency to
open space
Access
efficiency to
open space
Access
fficiency to
pen space
Access
efficiency to
open space
Access
fficiency to
open space
ficiency to
pen space
fficiency to
open space
efficiency to
open space
efficiency to
open space
efficiency to
open space
fficiency to
open space Table 9. Case 3, Case 4 and Case 5—open space analysis. Table 9. Case 3, Case 4 and Case 5—open space analysis. Table 9. Case 3, Case 4 and Case 5—open space analysis. Table 9. Case 3, Case 4 and Case 5—open space analysis. Table 9. Case 3, Case 4 and Case 5—open space analysis. Table 9. Case 3, Case 4 and Case 5—open space analysis. Table 9. Case 3, Case 4 and Case 5—open space analysis. Open Space Analysis
Open Space Analysis
Open Space Analysis
Open Space Analysis
Open Space Analysis
Open Space Analysis Table 9. Case 3, Case 4 and Case 5—open space analy
Table 9. Case 3, Case 4 and Case 5—open space analys
Table 9. Case 3, Case 4 and Case 5—open space analysis
Table 9. Case 3, Case 4 and Case 5—open space analysis. Table 9. Table 5 Neighborhood layout ana
4.1. Imperial Period: Case 1 and Case 2 Building Building Structure Analysis
Number of Case
Case 3
Case 4
Case 5
Building Building Structure Analysis
Number of
Case
Case 3
Case 4
Case 5
Layout
Functions
Building Building Structure Analysis
Number of
Case
Case 3
Case 4
Case 5
Layout
Functions
Building Building Structure Analysis
Number of
Case
Case 3
Case 4
Case 5
Layout
Functions
Layout
Number of
Case
Case 3
Case 4
Case 5
Layout
Functions
Number of
Case
Case 3
Case 4
Case 5
Layout
Functions
Number of
Case
Case 3
Case 4
Case 5
Layout
Functions
Functions
Case
Case 3
Case 4
Case 5
Layout
Functions
Case
Case 3
Case 4
Case 5
Layout
Functions
Case
Case 3
Case 4
Case 5
Layout
Functions
Number of floors
Sustainability 2021, 13, x FOR PEER REVIEW
17 of 3
Number of
floors
Spatial
squence
Sustainability 2021, 13, x FOR PEER REVIEW
17 of 31
Number of
floors
Spatial
squence
Sustainability 2021, 13, x FOR PEER REVIEW
17 of 31
Number of
floors
Spatial
squence
Spatial squence
Sustainability 2021, 13, x FOR PEER REVIEW
17 of 3
Number of
floors
Spatial
squence
Sustainability 2021, 13, x FOR PEER REVIEW
17 of 31
Number of
floors
Spatial
squence
Sustainability 2021, 13, x FOR PEER REVIEW
17 of 31
Number of
floors
Spatial
squence
A Number of Case
Case
Case
Case L
t
L
t
L
t
Layout
Layout
Layout
Layout
Layout
Layout
Layout Functions
Functions
Functions
Functions
Functions
Functions
Functions
Functions
Functions
Functions
Number of floors
Sustainability 2021, 13, x FOR PEER REVIEW
17 of
Number of
floors
Spatial
squence
Sustainability 2021, 13, x FOR PEER REVIEW
17 of 3
Number of
floors
Spatial
squence
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Number of
floors
Spatial
squence
Spatial squence
Sustainability 2021, 13, x FOR PEER REVIEW
17 of
Number of
floors
Spatial
squence
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17 of 3
Number of
floors
Spatial
squence
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Number of
floors
Spatial
squence F
ti
F
ti
F
ti
Functions
Functions
Functions
Functions
S
t i
bilit 2
S
t i
bilit 2
t i
bilit 202
Sustainability
Sustainability 2
ustainability 20 Number of floors
Number of
floors
Number of
floors
Number of
floors
floors
floors
floors Spatial
Spatial
Spatial
Spatial squence
Spatial
squence
Spatial
squence
Spatial
squence 16 of 28 Sustainability 2021, 13, 6212 Table 8. Table 5 Neighborhood layout ana
4.1. Imperial Period: Case 1 and Case 2 Post-reform and Contemporary Period: Case 6
Si
1978 th
t
h
t
f
d f
l
d
t
k t
Sustainability 2021, 13, x FOR PEER REVIEW
18 of 31
Active front
Spatial
hierarchy
Spatial
movement
4.3. Post-reform and Contemporary Period: Case 6
Since 1978, the country has transformed from a planned economy to an open market,
Spatial
movement 1 : 1
1 : 1
1 : 1
1 : 1
1 : 1
1 : 1
1:1 1 : 0 3
1 : 0 3
1 : 0 3
1 : 0 3
1 : 0 3
1 : 0 3
1:0.3
18 of 31
18 of 31
18 of 31
18 of 31
18 of 3
18 of 31 1
1 1
1 1
1 : 1
1 1
1 1
1:1 et
1 : 0.1
1 : 1
t
1 : 0.1
1 : 1
1 : 0.1
1 : 1
1 : 0.1
1 : 1
1
1 : 0.1
1 : 1
1 : 0.1
1 : 1
1
nt Active front
Active front
Active
Active fron
Active fron
Ac i e
o Spatial
hierarchy
Spatial
movement
Spatial
hierarchy
Spatial
movement
Spatial
hierarchy
Spatial
movement
Spatial
hierarchy
Spatial
hierarchy
Spatial
movement
Spatial
hierarchy
Spatial
movement
p
hierarchy
Spatial
movement
Spatial
movement 17 of 28 17 of 28 Sustainability 2021, 13, 6212 4.3. Post-Reform and Contemporary Period: Case 6 4.3. Post-Reform and Contemporary Period: Case 6 Since 1978, the country has transformed from a planned economy to an open market,
and socio-political and economic ideology is changing under the influence of globalization. Functional apartments with a good level of privacy have been introduced since this time,
and the living style has been transformed from shared facilities (e.g., bathroom and kitchen)
to privately owned [75] (Figure 10). Case 6 is an example to present the design of the
building in this period (Table 10). The building is international style with four storages, and
due to lack of professional design with appropriate spatial relation to the surroundings,
the spaces between nearby houses are extremely narrow. In contemporary use, residents
occupied part of public areas and built extensional rooms as storage for bicycle parking at
the beginning, and pure storage later. REVIEW
18 of 31 (a)
(b)
(c)
Figure 10. Modern apartment and its layout. Top left image shows the apartment layout (a); top right image presents the
b ildi
d
i
(b)
d d
i
ill
t
t
th fl
l
f th b ildi
( )
Figure 10. Modern apartment and its layout. Top left image shows the apartment layout (a); top right image presents the
ustainability 2021, 13, x FOR PEER REVIEW
20 o
Sustainability 2021, 13, x FOR PEER REVIEW
20 (a)
(b)
(c)
Figure 10. Modern apartment and its layout. Top left image shows the apartment layout (a); top right image presents the
building design (b); and down image illustrates the floor plan of the building (c). Figure 10. Modern apartment and its layout. Top left image shows the apartment layout (a); top right image presents the
building design (b); and down image illustrates the floor plan of the building (c). ustainability 2021, 13, x FOR PEER REVIEW
20 of
ustainability 2021, 13, x FOR PEER REVIEW
20 o (b) (a)
(b)
(c)
Figure 10. Modern apartment and its layout. Top left image shows the apartment layout (a); top right image presents the
building design (b); and down image illustrates the floor plan of the building (c). Figure 10. Modern apartment and its layout. 4.3. Post-Reform and Contemporary Period: Case 6 Building Structure Analysis
Open Space Analysis
Case 6
Case 6
Layout
Layout
Building/plo
t
arrangement
Functions
Building
height/street
width
1 : 0.2
Number of
floors
Active front
Spatial
sequence
Spatial
hierarchy
Building/plot
arrangement
Layout
Building/plo
t
arrangement
Functions
Building
height/street
width
1 : 0.2
Number of
floors
Active front
Spatial
sequence
Spatial
hierarchy
Layout
Building/plo
t
arrangement
Functions
Building
height/street
width
1 : 0.2
Number of
floors
Active front
Spatial
sequence
Spatial
hierarchy
Functions
Layout
Building/plo
t
arrangement
Functions
Building
height/street
width
1 : 0.2
Number of
floors
Active front
Spatial
sequence
Spatial
hierarchy
Building height/street
width
1:0.2
Number of floors
Layout
Building/plo
t
arrangement
Functions
Building
height/street
width
1 : 0.2
Number of
floors
Active front
Spatial
sequence
Spatial
hierarchy
Active front
Layout
Building/plo
t
arrangement
Functions
Building
height/street
width
1 : 0.2
Number of
floors
Active front
Spatial
sequence
Spatial
hierarchy
Spatial sequence
Layout
Building/plo
t
arrangement
Functions
Building
height/street
width
1 : 0.2
Number of
floors
Active front
Spatial
sequence
Spatial
hierarchy
Spatial hierarchy
Sustainability 2021, 13, x FOR PEER REVIEW
21 of 31
Access
efficiency to
open space
Spatial
movement
In total, six cases were identified based on morphological transformation analysis to
cover all types of buildings and influences on the open spaces in the neighborhood (Figure
11). Access efficiency to
open space
Sustainability 2021, 13, x FOR PEER REVIEW
21 of 31
Access
efficiency to
open space
Spatial
movement
In total, six cases were identified based on morphological transformation analysis to
ll
f b
ld
d
fl
h
h
hb
h
d
Spatial movement
In total, six cases were identified based on morphological transformation analysis to cover
all types of buildings and influences on the open spaces in the neighborhood (Figure 11). Table 10. Cont. 4.3. Post-Reform and Contemporary Period: Case 6 Top left image shows the apartment layout (a); top right image presents the
b ildi
d
i
(b)
d d
i
ill
t
t
th fl
l
f th b ildi
( )
ustainability 2021, 13, x FOR PEER REVIEW
20 o
Sustainability 2021, 13, x FOR PEER REVIEW
20 (b) (c) Figure 10. Modern apartment and its layout. Top left image shows the apartment layout (a); top right image presents the
building design (b); and down image illustrates the floor plan of the building (c). Figure 10. Modern apartment and its layout. Top left image shows the apartment layout (a); top right image presents the
building design (b); and down image illustrates the floor plan of the building (c). Table 10. Case 6—building structure analysis and open space analysis. Table 10. Case 6—building structure analysis and open space analysis. Table 10. Case 6—building structure analysis and open space analysis Table 10. Case 6—building structure analysis and open space analysis. Building Structure Analysis
Open Space Analysis
Case 6
Case 6
g
y
p
p
y
Building Structure Analysis
Open Space Analysis
Case 6
Case 6
g
y
p
p
y
Building Structure Analysis
Open Space Analysis
Case 6
Case 6 Building Structure Analysis
Open Space Analysis
Case 6
Case 6
Building Structure Analysis
Open Space Analysis
Case 6
Case 6
Building Structure Analysis
Open Space Analysis
Case 6
Case 6 18 of 28 Sustainability 2021, 13, 6212 Sustainability 2021, 13, 6212
18 of 28
Table 10. Cont. 4.3. Post-Reform and Contemporary Period: Case 6 Building Structure Analysis
Open Space Analysis
Case 6
Case 6
Layout
Layout
Building/plo
t
arrangement
Functions
Building
height/street
width
1 : 0.2
Number of
floors
Active front
Spatial
sequence
Spatial
hierarchy
Building/plot
arrangement
Layout
Building/plo
t
arrangement
Functions
Building
height/street
width
1 : 0.2
Number of
floors
Active front
Spatial
sequence
Spatial
hierarchy
Layout
Building/plo
t
arrangement
Functions
Building
height/street
width
1 : 0.2
Number of
floors
Active front
Spatial
sequence
Spatial
hierarchy
Functions
Layout
Building/plo
t
arrangement
Functions
Building
height/street
width
1 : 0.2
Number of
floors
Active front
Spatial
sequence
Spatial
hierarchy
Building height/street
width
1:0.2
Number of floors
Layout
Building/plo
t
arrangement
Functions
Building
height/street
width
1 : 0.2
Number of
floors
Active front
Spatial
sequence
Spatial
hierarchy
Active front
Layout
Building/plo
t
arrangement
Functions
Building
height/street
width
1 : 0.2
Number of
floors
Active front
Spatial
sequence
Spatial
hierarchy
Spatial sequence
Layout
Building/plo
t
arrangement
Functions
Building
height/street
width
1 : 0.2
Number of
floors
Active front
Spatial
sequence
Spatial
hierarchy
Spatial hierarchy
Sustainability 2021, 13, x FOR PEER REVIEW
21 of 31
Access
efficiency to
open space
Spatial
movement
In total, six cases were identified based on morphological transformation analysis to
cover all types of buildings and influences on the open spaces in the neighborhood (Figure
11)
Access efficiency to
open space
Sustainability 2021, 13, x FOR PEER REVIEW
21 of 31
Access
efficiency to
open space
Spatial
movement
Spatial movement
In total, six cases were identified based on morphological transformation analysis to cover
all types of buildings and influences on the open spaces in the neighborhood (Figure 11). Table 10. Cont. 5. Analysis of Changes of Spatial Characteristics Between Cases
5. Analysis of Changes of Spatial Characteristics between Cases In this section, a third research strategy is implemented, in which the analytical
framework is continually employed to analyze the continuities of spatial characteristics
between cases. Based on the analysis of all six cases, the study identified a degree of con-
tinuity of spatial characteristics in building structure and open space level in chronologi-
cal order, which illustrates which spatial characteristics are continued, partly continued
or changed from the previous morphological period to the next one. It is then clear to see,
based on the transformation of socio-political and economic ideology, how different types
of forms have changed local needs, and how local needs have changed these spatial char-
acteristics. During the process, many spatial characteristics are discontinued due to tran-
sitions of needs, but it also shows available spatial characteristics that can be continued to
sustain socio-cultural sustainability in future development
In this section, a third research strategy is implemented, in which the analytical frame-
work is continually employed to analyze the continuities of spatial characteristics between
cases. Based on the analysis of all six cases, the study identified a degree of continuity of
spatial characteristics in building structure and open space level in chronological order,
which illustrates which spatial characteristics are continued, partly continued or changed
from the previous morphological period to the next one. It is then clear to see, based on
the transformation of socio-political and economic ideology, how different types of forms
have changed local needs, and how local needs have changed these spatial characteristics. During the process, many spatial characteristics are discontinued due to transitions of
needs, but it also shows available spatial characteristics that can be continued to sustain
socio-cultural sustainability in future development. sustain socio cultural sustainability in future development. As explained above, in the third research step a method of calculation is developed
as a measurement to evaluate the degree of continuity and changes of spatial characteris-
tics: continue, partly continue and change. The formula is developed in a two-step pro-
cess: calculation of originality of spatial characteristics, and calculation of continuity of
these spatial characteristics. As shown in Figure 12, each case in each spatial characteristic
first needs to define the originality of its typology compared to its original design, whether
it maintains original, or partly original or changed to another type (in Tables 11 and 12),
to define the spatial characteristics in contemporary use. 4.3. Post-Reform and Contemporary Period: Case 6 Building Structure Analysis
Open Space Analysis
Case 6
Case 6
Layout
Layout
Building/plo
t
arrangement
Building/plot
arrangement
Layout
Building/plo
t
arrangement
Layout
Building/plo
t
arrangement
Layout
Building/plo
t
arrangement
Layout
Building/plo
t
arrangement
Layout
Building/plo
t
arrangement
Layout
Building/plo
t
arrangement Functions
unctions
nctions
Functions
Functions
Functions
Functions
Functions Building
height/street
Building
height/street
Building
height/street
Building
height/street
width
Building height/street
width
height/street
width
height/street
width
width ti
i
Functions
unctions EER REVIEW A ti
f
Active fron
Active front
Active fro
A i e
o Number of
Number of floors
Number of
floors
Number of
floors
floors Spatial
sequence
Spatial
hierarchy
Spatial
sequence
Spatial
hierarchy
Spatial
sequence
Spatial
hierarchy
Spatial
sequence
Spatial
hierarchy
Spatial
sequence
Spatial
hierarchy
Spatial
sequence
Spatial
hierarchy
Spatial sequence
Spatial
sequence
Spatial
hierarchy
Spatial hierarchy
Access
efficiency to
open space
Spatial
movement
In total, six cases were identified based on morphological transformation analysis to
Access efficiency to
open space
Sustainability 2021, 13, x FOR PEER REVIEW
21 of 31
Access
efficiency to
open space
Spatial
movement
Spatial movement
In total, six cases were identified based on morphological transformation analysis to cover Spatial
Spatial
Spatial
hierarchy
Spatial hierarchy Spatial
Spatial
Spatial sequence
Spatial
sequence q
efficiency to
open space
Access efficiency to
open space
Access
efficiency to
open space Access efficiency to
open space
Access
efficiency to
open space Spatial
movement
Spatial movement In total, six cases were identified based on morphological transformation analysis to
cover all types of buildings and influences on the open spaces in the neighborhood (Figure
11)
In total, six cases were identified based on morphological transformation analysis to cover
all types of buildings and influences on the open spaces in the neighborhood (Figure 11). 19 of 28
alysis to
(Fi Sustainability 2021, 13, 6212 Figure 11. Locations of cases and their categories. Figure 11. Locations of cases and their categories. 11. Locations of cases and their categories. Figure 11. Locations of cases and their categories. 5. Analysis of Changes of Spatial Characteristics Between Cases
5. Analysis of Changes of Spatial Characteristics between Cases Open Space Analysis
Morphological Periods
Period 1
Period 2
Period 3
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
Building/plot
arrangement
8
8
5
9
9
9
Building height/street
width
6
9
6
9
9
6
Active frontage
6
9
6
9
9
6
Spatial hierarchy
9
3
9
9
9
9
Spatial movement
6
3
9
9
9
9 a e i i
( )
Table 11. Calculation results of originality between cases in building analysis by using formula. tinuity score (O) is calculated using the following formula:
=
=
m
1
ro
1
j
j
m
k
O
(1)
represents the originality of physical characteristics, k is a constant number
represents the total number of sub-items in each spatial characteristic, o rep-
originalities of the sub-item, and r represents the score of originalities of the
hen r = 3, it means the case maintains its original design, when r = 2, it means
tly changed its design, and when r = 1, it means the case has completely
g
y
g
y
y
g
Building
Analysis
Morphological Periods
Period 1
Period 2
Period 3
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
Layout
9
3
9
9
9
9
Functions
6
3
9
9
9
9
Number of floor
6
6
3
9
9
9
Spatial sequences
9
3
9
9
9
9
Access efficiency
6
3
9
9
9
9 riginal design. J represents the number of sub-item in sequence in each spa
istics
Table 12. Calculation results of originality between cases in open space analysis. original design. J represents the number of sub item in sequence in each spa
istics. ‘O’ scores indicate original design, while lower ‘O’ scores represent change. m possible score is thus 9 [= 3 × 1/6 × (3 + 3+3 + 3+3 + 3)] if all spatial charac-
maintained in its original formation; while the minimum possible score is 3 [=
if all spatial characteristics are changed. Therefore, If 7 ≤ C < 9, the originality
cal characteristic is maintained as original; if 5 ≤ C < 7, the originality of the
racteristic is defined as partly original; if 3 ≤ C < 5, the originality of the phys-
ristic is indicated as changed. 5. Analysis of Changes of Spatial Characteristics Between Cases
5. Analysis of Changes of Spatial Characteristics between Cases Then, formula 1 is used to calcu-
late the score of originality (Represented by O) and justify the category of spatial charac-
teristics based on the originality of each case (Table 11 and Table 12). Secondly, when the
spatial characteristics in contemporary uses are defined, the originality score of calcula-
tion from formula 1 is used to clarify its categories: the clear cycle in Table 13 and Table
14 represents the spatial characteristics of each case maintained in the original, grey cycle
symbols are partly original, and black cycle means the physical characteristics have
changed. Each originality score is used in formula 2 to calculate the continuity of these
spatial characteristics between cases. The analysis results (Table 13 and Table 14) are then
As explained above, in the third research step a method of calculation is developed as
a measurement to evaluate the degree of continuity and changes of spatial characteristics:
continue, partly continue and change. The formula is developed in a two-step process:
calculation of originality of spatial characteristics, and calculation of continuity of these
spatial characteristics. As shown in Figure 12, each case in each spatial characteristic first
needs to define the originality of its typology compared to its original design, whether it
maintains original, or partly original or changed to another type (in Tables 11 and 12), to
define the spatial characteristics in contemporary use. Then, formula 1 is used to calculate
the score of originality (Represented by O) and justify the category of spatial characteristics
based on the originality of each case (Tables 11 and 12). Secondly, when the spatial
characteristics in contemporary uses are defined, the originality score of calculation from
formula 1 is used to clarify its categories: the clear cycle in Tables 13 and 14 represents the
spatial characteristics of each case maintained in the original, grey cycle symbols are partly
original, and black cycle means the physical characteristics have changed. Each originality
score is used in formula 2 to calculate the continuity of these spatial characteristics between
cases. The analysis results (Tables 13 and 14) are then clear—each physical characteristic
clearly presents the form of transformation at different morphological layers through
different periods. 20 of 28 Sustainability 2021, 13, 6212
clear—e Figure 12. The interrelationship between originalities of physical characteristics (O) and continuity
Figure 12. The interrelationship between originalities of physical characteristics (O) and continuity of spatial characteristics (C). Figure 12. 5. Analysis of Changes of Spatial Characteristics Between Cases
5. Analysis of Changes of Spatial Characteristics between Cases The interrelationship between originalities of physical characteristics (O) and continuity
Figure 12. The interrelationship between originalities of physical characteristics (O) and continuity of spatial characteristics (C). of spatial characteristics (C). The continuity score (O) is calculated using the following formula:
=
=
m
1
ro
1
j
j
m
k
O
(1)
The O represents the originality of physical characteristics, k is a constant number
equal to 3, m represents the total number of sub-items in each spatial characteristic, o rep-
resents the originalities of the sub-item, and r represents the score of originalities of the
sub-item. When r = 3, it means the case maintains its original design, when r = 2, it means
the case partly changed its design, and when r = 1, it means the case has completely
changed its original design. J represents the number of sub-item in sequence in each spa-
tial characteristics. Higher ‘O’ scores indicate original design, while lower ‘O’ scores represent change. The maximum possible score is thus 9 [= 3 × 1/6 × (3 + 3+3 + 3+3 + 3)] if all spatial charac-
teristics are maintained in its original formation; while the minimum possible score is 3 [=
3 × 1/1 × (1)] if all spatial characteristics are changed. Therefore, If 7 ≤ C < 9, the originality
of the physical characteristic is maintained as original; if 5 ≤ C < 7, the originality of the
physical characteristic is defined as partly original; if 3 ≤ C < 5, the originality of the phys-
ical characteristic is indicated as changed. By using the score of originality, Formula 2 calculates the score of continuity of spa-
tial characteristics (C)
When the analysis results between cases present continuity, use the formula:
Table 11. Calculation results of originality between cases in building analysis by using formula. Building
Analysis
Morphological Periods
Period 1
Period 2
Period 3
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
Layout
9
3
9
9
9
9
Functions
6
3
9
9
9
9
Number of floor
6
6
3
9
9
9
Spatial sequences
9
3
9
9
9
9
Access efficiency
6
3
9
9
9
9
Table 12. Calculation results of originality between cases in open space analysis. 5. Analysis of Changes of Spatial Characteristics Between Cases
5. Analysis of Changes of Spatial Characteristics between Cases g the score of originality, Formula 2 calculates the score of continuity of spa-
istics (C)
he analysis results between cases present continuity, use the formula:
Table 12. Calculation results of originality between cases in open space analysis. Open Space Analysis
Morphological Periods
Period 1
Period 2
Period 3
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
Building/plot
arrangement
8
8
5
9
9
9
Building height/street
width
6
9
6
9
9
6
Active frontage
6
9
6
9
9
6
Spatial hierarchy
9
3
9
9
9
9
Spatial movement
6
3
9
9
9
9 21 of 28
25 of 31 Sustainability 2021, 13, 6212
Sustainability 2021, 13, x FO Table 13. Building structure analysis results. Table 13. Building structure analysis results. Table 13. Building structure analysis results. Table 13. Building structure analysis results Table 13. Building structure analysis results. Table 13. Building structure analysis results structure analysis results. ng structure analysis results. Table 13. Build
Table 13. Bu 22 of 28
26 of 3 Sustainability 2021, 13, 6212
Sustainability 2021, 13, x FO Table 14. Open space analysis results. Table 14. Open space analysis result Table 14. Open space analysis results. Table 14. Open space analysis results ce analysis results. pace analysis results. p Sustainability 2021, 13, 6212 23 of 28 The continuity score (O) is calculated using the following formula: O = k × 1
m ×
m
∑
j=1
orj
(1) (1) The O represents the originality of physical characteristics, k is a constant number equal
to 3, m represents the total number of sub-items in each spatial characteristic, o represents the
originalities of the sub-item, and r represents the score of originalities of the sub-item. When
r = 3, it means the case maintains its original design, when r = 2, it means the case partly
changed its design, and when r = 1, it means the case has completely changed its original
design. J represents the number of sub-item in sequence in each spatial characteristics. g
p
q
p
Higher ‘O’ scores indicate original design, while lower ‘O’ scores represent change. 5. Analysis of Changes of Spatial Characteristics Between Cases
5. Analysis of Changes of Spatial Characteristics between Cases The maximum possible score is thus 9 [=3 × 1/6 × (3 + 3 + 3 + 3 + 3 + 3)] if all spatial
characteristics are maintained in its original formation; while the minimum possible score
is 3 [=3 × 1/1 × (1)] if all spatial characteristics are changed. Therefore, If 7 ≤C < 9,
the originality of the physical characteristic is maintained as original; if 5 ≤C < 7, the
originality of the physical characteristic is defined as partly original; if 3 ≤C < 5, the
originality of the physical characteristic is indicated as changed. g
y
p y
g
By using the score of originality, Formula 2 calculates the score of continuity of spatial
characteristics (C). When the analysis results between cases present continuity, use the formula: C =
Nc +
−
O
(2) (2) When the analysis results between cases present partial continuity, use the formula: When the analysis results between cases present partial continuity, use the formula: C =
Npc +
−
O
(3) (3) When the analysis results between cases present discontinuity, use the formula: When the analysis results between cases present discontinuity, use the formula: C =
Nd +
−
O
(4) (4) C represents the continuity of spatial characteristics, Nc represents continue and equals 25,
Npc presents partly continue and equals 15, Nd means discontinue and equals 5 and −represents
interrelations between two compared cases. p
Higher ‘C’ scores represent continuity of physical characteristic, while lower “C”
scores indicate discontinuity of physical characteristic. The maximum possible score is
therefore 31 [= 25 + |9−3|] if all spatial characteristics are continued between cases; while
the minimum possible score is 5 [5 + |9−9|] if all spatial characteristics are discontinued. Thus, the categories are divided—if 5 ≤C ≤11, the physical characteristic maintains
continuity; if 11 < C ≤21, the physical characteristic has partly continued; if 21 < C ≤31,
the physical characteristic discontinued. The transformations among six cases were found to be at different degrees at the
levels of building structure analysis and open space analysis (Figure 13). With regard to
building structure level, five spatial characteristics were found to strictly continue through
morphological transformation between case 4–case 5. The relations between the rest of
cases were partly continued. 6. Key Findings
Based on th
6. Key Findings Based on the analysis above, the key findings are very clear. Through the transfor
mation of the urban development and changes of political-socio-economic ideologies
both historically and due to current globalization influences, the layout, design and func-
tions of buildings are transformed in each period to follow the changes and influences of
these ideologies and fulfill permanent or contemporary needs. Due to transformations of
political-economic-socio-cultural ideologies, the spatial structure of the neighborhood is
dramatically changed through time. The political ideology is key throughout the design
of the buildings and planning of the city in imperial times to emphasize socio-politica
priority, in which the higher social class was located in the center of the block and along
the main streets, while lower social classes gradually decentralized to the edge of the block
and mostly occupied secondary streets and small lanes. The same design principle of so-
cio-political hierarchy applied in the spatial design of houses to represent the social class
of each family, and thus, the spatial characteristics of the buildings were originally de-
signed to address socio-political ideologies, and their spatial structure involved areas in
the front and back (where servants and female members of the families lived) that gradu
ally centralized to the center (where senior or male members of the family lived). I
i d t
liti
l id
l
till
l
d th k
l th
h
t th d
l
Based on the analysis above, the key findings are very clear. Through the transfor-
mation of the urban development and changes of political-socio-economic ideologies,
both historically and due to current globalization influences, the layout, design and func-
tions of buildings are transformed in each period to follow the changes and influences of
these ideologies and fulfill permanent or contemporary needs. Due to transformations of
political-economic-socio-cultural ideologies, the spatial structure of the neighborhood is
dramatically changed through time. The political ideology is key throughout the design
of the buildings and planning of the city in imperial times to emphasize socio-political
priority, in which the higher social class was located in the center of the block and along
the main streets, while lower social classes gradually decentralized to the edge of the block
and mostly occupied secondary streets and small lanes. 5. Analysis of Changes of Spatial Characteristics Between Cases
5. Analysis of Changes of Spatial Characteristics between Cases While at open space level, there was discontinuity found
between case 2-case 3, and case 2-case 5, in which spatial characteristics were muted
completely. In addition, spatial characteristics were continually maintained between case
4–case 5. In the understanding of continuity of spatial characteristics, this not only means
the spatial characteristics physically and directly continued to another (such as spatial
characteristics found in the same morphological period), but also presents in different types
of forms with shared common design principles. For example, the spatial relationship Sustainability 2021, 13, 6212 24 of 28
y means 24 of 28
y means of public→semi-private→private was found in imperial case 1, but it was also found at
cases 4, 5 and 6. Although their typologies were different, at a spatial relationship scale the
physical characteristic was continued. types of forms with shared common design principles. For example, the spatial relation-
ship of public→semi-private→private was found in imperial case 1, but it was also found
at cases 4, 5 and 6. Although their typologies were different, at a spatial relationship scale
the physical characteristic was continued (a)
(b)
Figure 13. Overall analysis results in continuity of spatial characteristics between cases. (a) shows the spatial changes
between periods at building scale; and (b) presents the spatial changes between periods at open space scale. 6 K
Fi di
Figure 13. Overall analysis results in continuity of spatial characteristics between cases. (a) shows the spatial changes
between periods at building scale; and (b) presents the spatial changes between periods at open space scale. (b) (a) (a) (b) Figure 13. Overall analysis results in continuity of spatial characteristics between cases. (a) shows the spatial changes
between periods at building scale; and (b) presents the spatial changes between periods at open space scale. Figure 13. Overall analysis results in continuity of spatial characteristics between cases. (a) shows the spatial changes
between periods at building scale; and (b) presents the spatial changes between periods at open space scale. 6. Key Findings
Based on th
6. Key Findings The same design principle of
socio-political hierarchy applied in the spatial design of houses to represent the social
class of each family, and thus, the spatial characteristics of the buildings were originally
designed to address socio-political ideologies, and their spatial structure involved areas in
the front and back (where servants and female members of the families lived) that gradually
centralized to the center (where senior or male members of the family lived). In period two, political ideology still played the key role throughout the development
of the city, although the design ideas were different from imperial times. The socialist
ideas and collectivism were largely applied to regeneration of historical neighborhoods
which transformed the traditional private houses into shared properties to address the
core socio-political ideology—social equity—and the spatial layout of houses was then
organically regenerated by each family who occupied the property, without regulatory
control. As a consequence, the regeneration of traditional courtyard houses in the area
In period two, political ideology still played the key role throughout the development
of the city, although the design ideas were different from imperial times. The socialist ideas
and collectivism were largely applied to regeneration of historical neighborhoods, which
transformed the traditional private houses into shared properties to address the core socio-
political ideology—social equity—and the spatial layout of houses was then organically
regenerated by each family who occupied the property, without regulatory control. As
a consequence, the regeneration of traditional courtyard houses in the area converted
from one family owned homes to multi-family occupied residences (case 1 and 2), and part
of originally residential buildings are now being turned from pure residential to mixed
commercial functions (case 2), in order to satisfy increased commercial activities and needs. In the meantime, in terms of its spatial structure, it first transformed internal shared space
and then expanded horizontally to external public open spaces, and grew up vertically to
respond continually increased population (case 1 and 2), which also caused the internal
road network of the block to be transformed from open access to blind alley. On the other
hand, soviet style modern buildings for laborers started to be built freely by work units Sustainability 2021, 13, 6212 25 of 28 25 of 28 in the neighborhood without planning and spatial control, but also to emphasize socialist
ideas and the collectivist lifestyle (cases 3, 4 and 5). 6. Key Findings
Based on th
6. Key Findings Due to the lack of building regulations
to control the construction in historical neighborhoods, the transformation of the buildings
and even the entire neighborhood was seen as a result of negotiations between neighbors. Since post-reform, the economic ideologies of the global market have started to replace
the socio-political ideologies that led the development of the neighborhood. The impact of
mass global culture brought enormous socio-cultural conflict, changing urban development,
and resulting in higher density international style modern buildings (case 6). Large scale
individual modern architecture began in Chinese cities. Due to lack of building and
planning regulation control and design, these high raised modern buildings created huge
visual diversity in narrow lanes, which further limited the external spaces, and largely
changed the way of living and pattern of use, both internal and external, of buildings. In contemporary use of all cases, the interesting part is after the transformation of
political-economic-socio-cultural ideologies and forms of building typologies, the spatial
sequence remains the same as vernacular design, which follows the public-semi-public-
private pattern. Even if the houses have been changed from private to shared ownership,
the residents will have built up a transitional semi-private space themselves slowly through
time, which remains the preferred spatial sequence (case 1). Therefore, the spatial sequence, plot arrangement and spatial movement of most
cases continually adapt to local needs, and maintain memories and meaning associated
with local place. On the other hand, the other aspects only remain partly adapted to the
local preferences in use, and whether they can be continued depends on transformation
of political-socio-economic ideologies, and this explains the most important mutation
between period 1 and 2. 7. Conclusions This study has developed an analytical framework to investigate changes of spatial
characteristics in adaptation to the changing of local needs for improving socio-cultural
sustainability in historical neighborhoods in the context of Beijing, China. The key contribu-
tions of the research are twofold: firstly, due to lack of studies on adaptation of local needs
from a physical perspective for sustaining socio-cultural sustainability in the context of
China, particularly Beijing, this paper identifies continuities and discontinuities of spatial
characteristics, which are as follows: (1)
Continuities of spatial characteristics—continually supporting transformation of local
needs through time and mutations of political-economic-socio-cultural ideologies. The analysis results could be used to support regulations and policies that fill the
gaps of legislation in spatial design for sustaining socio-cultural sustainability. (2)
Discontinuities of spatial characteristics—discover the gaps to adapt local socio-
cultural needs in spatial design. On the other hand, some of them may also see
new lifestyles identified in case studies, such as automobile use, and demands for in
parking facilities. Secondly, the proposed method for analyzing physical forms of cases improves clarity
and consistency in defining and analyzing typological cases at an articulated scale, pair-
wise comparison and categorization of the three degrees of morphological changes. This
analytical method is applicable to other cities and countries that improve efficiency to
quickly define continuities and discontinuities of spatial characteristics for maintaining or
improving, which improve socio-cultural sustainability of a neighborhood. The limitations of the study might involve three aspects. Firstly, one may ask to what
degree each spatial characteristic analytical framework contributes to local needs in terms
of socio-cultural sustainability, and whether there are other factors affecting these needs
that influence socio-cultural sustainability. Secondly, the analytical framework was only
tested in a historical neighborhood (covers vernacular, soviet style and international style
raised buildings, but high raised towers are not available in this area) in Beijing. Although
the results include clear identifications of continuity, partial continuity or discontinuity Sustainability 2021, 13, 6212 26 of 28 26 of 28 of spatial characteristics in adaptation of local needs, it still needs more evaluations of
variant types of neighborhoods in different context. Finally, the decision made for a case in
regard to continuity and mutation in its spatial transformation was made based on score
calculation. However, if the calculation range was set to a larger number, then partial
continuity might reduce and continuity would increase. 7. Conclusions y
g
y
In order to reduce limitations of the study, further research with a bigger sample
size with more house types in different contexts could be explored to provide a fuller
picture of how changes of spatial characteristics could improve socio-cultural sustainability
by evaluating the adaptation to local needs. The degree of each spatial characteristic’s
contribution to the impact of spatial changes in relation to adaptation of local needs would
also be interesting to study. Larger score range of calculation of continuity and discontinuity
of spatial characteristics should be tested with a larger sample to justify the stability and
applicability of the analytical framework. Author Contributions: Conceptualization, H.L. and B.L.; methodology, H.L.; software, H.L.; val-
idation, H.L. and B.L.; formal analysis, H.L.; investigation, H.L.; resources, H.L.; data curation,
H.L.; writing—original draft preparation, H.L. and B.L.; writing—review and editing, H.L. and
B.L.; visualization, H.L.; supervision, B.L.; project administration, B.L.; funding acquisition, B.L. All
authors have read and agreed to the published version of the manuscript. Funding: This research is supported by National Natural Science Foundation of China: 41871154. Institutional Review Board Statement: Not applicable. 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: The data presented in this study are available on request from the
corresponding author. The data are not publicly available due to privacy concerns. Acknowledgments: Great appreciation to Lu Yi, who offers great assist in development of formulas
in the study. Acknowledgments: Great appreciation to Lu Yi, who offers great assist in development of formulas
in the study. Conflicts of Interest: The authors declare no conflict of interest. nflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Krier, R.; Rowe, C. Urban Space; Academy editions London: London, UK, 1979. 1. Krier, R.; Rowe, C. Urban Space; Academy editions London: London, UK, 1979. 2. Relph, E. Place and Placelessness; Pion: London, UK, 1976. p
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69.
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https://openalex.org/W4387742935
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https://www.nature.com/articles/s41598-023-44880-3.pdf
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English
| null |
Contributions of viral oncogenes of HPV-18 and hypoxia to oxidative stress and genetic damage in human keratinocytes
|
Scientific reports
| 2,023
|
cc-by
| 10,154
|
orts | (2023) 13:17734
| https://doi.org/10.1038/s41598-023-44880-3
Contributions of viral oncog
of HPV‑18 and hypoxia to ox
stress and genetic damage
in human keratinocytes
Jimena Hochmann 1,2*, Magdalena Millán 1, Paola Hernández 3, Laura La
Natali D’ Aiuto 1,5, Alejandro Silva 6, Juan Llaguno 6, Julia Alonso 6, Ariel Fe
Vanesa Pereira‑Prado 2, José Sotelo‑Silveira 1,7, Ronell Bologna‑Molina 2 &
Infection with high-risk human papillomaviruses like HPV-16 and HPV-18 is high
the development of cervical and other cancers. Malignant transformation requir
E5, E6 and E7, which promote cell proliferation and increase DNA damage. Oxid
hypoxia are also key factors in cervical malignant transformation. Increased leve
of oxygen (ROS) and nitrogen (RNS) are found in the hypoxic tumor microenviro
genetic instability and invasiveness. In this work, we studied the combined effe
and hypoxia in increasing oxidative stress and promoting DNA damage and nuc
alterations. HaCaT cells containing HPV-18 viral oncogenes (HaCaT E5/E6/E7-18
levels in normoxia and higher levels of RNS in hypoxia compared to HaCaT pare
higher genetic damage in hypoxia as measured by γH2AX and comet assays. In
E7-18 increased its nuclear dry mass and both cell types displayed marked heter
mass distribution and increased nuclear foci. Our results show contributions of b
and hypoxia to oxidative stress, DNA damage and altered nuclear architecture,
altered microenvironment combines with oncogenic transformation to promote
Abbreviations
(HR-HPVs)
High-Risk Human Papillomaviruses
(EGFR)
Epidermal Growth Factor
(OS)
Oxidative stress
(ROS)
Reactive Oxygen Species
(·NO)
Nitric oxide
(RNS)
Reactive Nitrogen Species
(eNOS)
Endothelial Nitric Oxide Synthase
(HIF-1/2)
Hypoxia Inducible Factor 1/2
(SCC)
Squamous cell carcinomas
(E6AP)
E6-associatedprotein
(pRb)
Protein retinoblastoma
(NOSs)
Nitric oxide synthases
(8-oxodG)
8-Oxo-7,8-dihydro-20 -deoxyguanosine
OPEN
1Departamento de Genómica, Instituto de Investigaciones Biológicas Clement
Uruguay. 2Departamento de Diagnóstico en Patología y Medicina Bucal, Facultad de
de la República, General Las Heras 1925, Montevideo, Uruguay. 3Departamento d
Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay. 4Grupo de Biofisic
de Ciencias Biológicas, Centro Universitario Regional Litoral Norte ‑Sede Salto, Uni
(CENUR LN, UdelaR), Montevideo, Uruguay. 5Departamento de Biología Odontológica,
Universidad de la República, General Las Heras 1925, Montevideo, Uruguay. 6Institu
Ingeniería, Universidad de la República, Montevideo, Uruguay. 7Sección Biología Celu
Universidad de la República, Montevideo, Uruguay. *email: jhochmann@fcien.e
gmail.com www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Contributions of viral oncogenes
of HPV‑18 and hypoxia to oxidative
stress and genetic damage
in human keratinocytes
OPEN Jimena Hochmann 1,2*, Magdalena Millán 1, Paola Hernández 3, Laura Lafon‑Hughes 3,4,
Natali D’ Aiuto 1,5, Alejandro Silva 6, Juan Llaguno 6, Julia Alonso 6, Ariel Fernández 6,
Vanesa Pereira‑Prado 2, José Sotelo‑Silveira 1,7, Ronell Bologna‑Molina 2 & Miguel Arocena 1,5* Infection with high-risk human papillomaviruses like HPV-16 and HPV-18 is highly associated with
the development of cervical and other cancers. Malignant transformation requires viral oncoproteins
E5, E6 and E7, which promote cell proliferation and increase DNA damage. Oxidative stress and
hypoxia are also key factors in cervical malignant transformation. Increased levels of reactive species
of oxygen (ROS) and nitrogen (RNS) are found in the hypoxic tumor microenvironment, promoting
genetic instability and invasiveness. In this work, we studied the combined effect of E5, E6 and E7
and hypoxia in increasing oxidative stress and promoting DNA damage and nuclear architecture
alterations. HaCaT cells containing HPV-18 viral oncogenes (HaCaT E5/E6/E7-18) showed higher ROS
levels in normoxia and higher levels of RNS in hypoxia compared to HaCaT parental cells, as well as
higher genetic damage in hypoxia as measured by γH2AX and comet assays. In hypoxia, HaCaT E5/E6/
E7-18 increased its nuclear dry mass and both cell types displayed marked heterogeneity in nuclear dry
mass distribution and increased nuclear foci. Our results show contributions of both viral oncogenes
and hypoxia to oxidative stress, DNA damage and altered nuclear architecture, exemplifying how an
altered microenvironment combines with oncogenic transformation to promote tumor progression. Abbreviations
(HR-HPVs)
High-Risk Human Papillomaviruses
(EGFR)
Epidermal Growth Factor
(OS)
Oxidative stress
(ROS)
Reactive Oxygen Species
(·NO)
Nitric oxide
(RNS)
Reactive Nitrogen Species
(eNOS)
Endothelial Nitric Oxide Synthase
(HIF-1/2)
Hypoxia Inducible Factor 1/2
(SCC)
Squamous cell carcinomas
(E6AP)
E6-associatedprotein
(pRb)
Protein retinoblastoma
(NOSs)
Nitric oxide synthases
(8-oxodG)
8-Oxo-7,8-dihydro-20 -deoxyguanosine 1Departamento de Genómica, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo,
Uruguay. 2Departamento de Diagnóstico en Patología y Medicina Bucal, Facultad de Odontología, Universidad
de la República, General Las Heras 1925, Montevideo, Uruguay. 3Departamento de Genética, Instituto de
Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay. 4Grupo de Biofisicoquímica, Departamento
de Ciencias Biológicas, Centro Universitario Regional Litoral Norte ‑Sede Salto, Universidad de la República
(CENUR LN, UdelaR), Montevideo, Uruguay. 5Departamento de Biología Odontológica, Facultad de Odontología,
Universidad de la República, General Las Heras 1925, Montevideo, Uruguay. 6Instituto de Física, Facultad de
Ingeniería, Universidad de la República, Montevideo, Uruguay. 7Sección Biología Celular, Facultad de Ciencias,
Universidad de la República, Montevideo, Uruguay. Contributions of viral oncogenes
of HPV‑18 and hypoxia to oxidative
stress and genetic damage
in human keratinocytes
OPEN *email: jhochmann@fcien.edu.uy; m.arocena.sutz@
gmail.com | https://doi.org/10.1038/s41598-023-44880-3 Scientific Reports | (2023) 13:17734 www.nature.com/scientificreports/ (HaCaT)
Spontaneously immortalized human keratinocyte
(BCRJ)
Banco de células do Rio de Janeiro
(DMEM)
Dulbecco’s Modified Eagle’s Medium
(FBS)
Fetal bovine serum
(Bcl-xL)
B-cell lymphoma extra-large
(IL-8)
Interleukin-8
(BCRJ)
Banco de Células do Rio de Janeiro
(DMEM)
Dulbecco’s Modified Eagle’s Medium
(FBS)
Fetal bovine serum
(Bcl-xL)
B-cell lymphoma extra-large
(IL-8)
Interleukin-8 Human papilloma viruses (HPVs) are non-enveloped small DNA viruses that infect keratinocytes of the skin
and mucosa at different anatomic locations. The main risk factor for the development of cervical cancer and its
precursor lesions worldwide is the persistent infection with a high-oncogenic risk HPV type (HR-HPV)1. HPV-
16 and HPV-18 are associated with about 70% of all cervical cancers. HPV16 is the most frequent one, and it is
associated with squamous cell carcinomas (SCC), while HPV18 is the second most frequent and often detected
in adenocarcinoma2. The high-risk types of HPV encode three viral oncoproteins, E5, E6 and E7 that bind to and
modulate numerous cellular proteins. These oncoproteins serve as the major initiators of cell transformation,
interacting with cellular proteins controlling key aspects of cell proliferation. For instance, E5 interacts with
members of the epidermal growth factor receptor family (EGFRs), causing an enhancement of ligand-dependent
activation of the EGFR, and EGF-dependent proliferation of cultured human keratinocytes3,4. As for E6 from
high risk HPVs, its main target is the tumor suppressor p53. E6 forms a ternary complex with E6-associated
protein (E6AP) and p53 and induce the latter degradation by the ubiquitin-mediated proteolysis pathway5,6. E7
from high risk HPV binds and induces the degradation of the tumor suppressor protein retinoblastoma (pRb),
an essential regulator cell cycle, which disrupts the interaction of pRb with E2F transcription factor, releasing
E2F and promoting the transcription of genes involved in S phase induction7. p
g
p
g
p
Multiple steps are involved in the progression from HPV infection to cellular transformation to cervical
cancer. Among the main factors that promote malignant transformation are chronic inflammation and oxidative
stress (OS). Chronic inflammation contributes to approximately 25% of human cancers8, and induces reactive
oxygen species (ROS) and nitrogen species (RNS) in inflammatory and epithelial cells that can damage DNA
leading to mutagenic lesions9. An increase in ROS production can potentially promote cervical carcinogenesis10,11. Nitric oxide (·NO) is another contributor to oxidative stress12. Contributions of viral oncogenes
of HPV‑18 and hypoxia to oxidative
stress and genetic damage
in human keratinocytes
OPEN Previous studies have shown that women with
cervical cytological changes caused by HPV infections exhibited increased cervical nitric oxide release13. Several
studies have shown a link between hypoxia and increased oxidative stress14,15 and, on the other hand, oxidative
stress has been linked to increased tumor genetic instability and invasiveness16,17. For instance, ROS and RNS
can damage DNA to form mutagenic lesions, such as 8-oxo-7,8-dihydro-20 -deoxyguanosine (8-oxodG) and
8-nitroguanine18,19. On the other hand, DNA damage is a key event in HPV-induced carcinogenesis, which can
lead to the development of genomic instability. Recently, it was observed that HR-HPV oncoproteins activate
members of the DNA damage repair pathways, and this is critical for viral replication20. However, it has been
shown that a persistent expression of E6/E7 from high risk HPVs in normal cells could result in DNA damage
and chromosomal aberrations, and various mechanisms have been proposed to explain these observations, such
as replication stress and centrosome amplifications20,21. p
pi
In this work, we studied the relationship between hypoxia, oxidative stress, and DNA damage in HaCaT E5/
E6/E7 HPV-18 cells, which are human immortalized keratinocytes transduced with the three viral oncogenes
(E5, E6 and E7) of Human Papilloma Virus type 18 (HPV-18), previously developed by our group and which
constitute a cellular model of an intermediate stage in viral oncogenesis22. When cultured in an in vitro model
of the hypoxic, tumoral microenvironment, which we have previously characterized23,24, HaCaT E5/E6/E7-18
cells markedly increase ROS and RNS production, and they simultaneously display increased DNA damage and
altered nuclear architecture. Our results show that both hypoxia and oncogenic transformation contribute to
increase oxidative stress and DNA damage, cooperating therefore in the path towards malignant transformation
in HPV-induced carcinogenesis. ROS and RNS detection in normoxia and hypoxia yp
We evaluated the levels of intracellular ROS in HaCaT parental and HaCaT E5/E6/E7-18 live cells in normoxia by
adding the ROS Detection Reagent Deep Red (640/675 nm excitation/emission) to cells and through visualization
by confocal microscopy. We observed that cells containing viral oncogenes of HPV-18 (E5/E6/E7-18) exhibited
significantly higher intracellular ROS compared to HaCaT parental cells (Fig. 1A,B). Furthermore, in agreement
with our previous results, we detected higher expression levels of Superoxide Dismutase 2 (SOD2) in HaCaT
E5/E6/E7-18 compared to HaCaT parental cells in both normoxia and hypoxia. In addition, it is interesting to
point out that hypoxia seems not to alter SOD2 expression (Fig. 1C). p
yp
p
g
On the other hand, we also measured ·NO release by Griess method, and we observed that the production
of ·NO in both HaCaT parental and E5/E6/E7-18 cells cultured under treatment with coverslips for 24 h was
increased compared to cells cultured in normoxia. In addition, ·NO content was significantly higher in HaCaT
E5/E6/E7-18 cultured in hypoxia compared to HaCaT parental cells (Fig. 1D).We also observed higher expression
of Endothelial Nitric Oxide Synthase (eNOS) in HaCaT E5/E6/E7-18 cells cultures in normoxia and hypoxia
compared to HaCaT parental cells, although it was not statistically significant (data not shown). https://doi.org/10.1038/s41598-023-44880-3 Scientific Reports | (2023) 13:17734 | www.nature.com/scientificreports/ Figure 1. Detection of ROS and ·NO in HaCaT parental and HaCaT E5/E6/E7-18 cells. (A) Representative
images of ROS Detection Reagent Deep Red signal. Scale bar: 20 μm. (B) Quantification of ROS intensity
(mean ± SD) in normoxia for HaCaT parental and HaCaT E5/E6/E7-18 cells. Student`s Test was conducted to
compare two means. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. (C) Relative expression levels of SOD2 expressed as
mean spot pixel density is shown for HaCaT parental and HaCaT E5/E6/E7-18 cells as assessed by the Human
Cell Stress Array Kit as suggested by the manufacturer (R&D Systems, MN, USA, ARY018). Averages ± standard
deviations (SDs) of two independent experiments are shown. A two-way analysis of variance (ANOVA) test
was conducted to assess statistical significance among groups along with Dunnett post-hoc test for intra-group
comparison. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. (D) Nitrate/nitrite fold increase produced in HaCaT
parental and HaCaT E5/E6/E7-18 cells in normoxia and coverslip induced hypoxia. Three independent
experiments were conducted in triplicate. Mean ± SD is shown for each cell line. ROS and RNS detection in normoxia and hypoxia A one-way analysis of variance
(ANOVA) test was conducted to assess statistical significance among groups along with Tukey’s HSD test for
intra-group comparison. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) p < 0.0001. Figure 1. Detection of ROS and ·NO in HaCaT parental and HaCaT E5/E6/E7-18 cells. (A) Representative
images of ROS Detection Reagent Deep Red signal. Scale bar: 20 μm. (B) Quantification of ROS intensity
(mean ± SD) in normoxia for HaCaT parental and HaCaT E5/E6/E7-18 cells. Student`s Test was conducted to
compare two means. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. (C) Relative expression levels of SOD2 expressed as
mean spot pixel density is shown for HaCaT parental and HaCaT E5/E6/E7-18 cells as assessed by the Human
Cell Stress Array Kit as suggested by the manufacturer (R&D Systems, MN, USA, ARY018). Averages ± standard
deviations (SDs) of two independent experiments are shown. A two-way analysis of variance (ANOVA) test
was conducted to assess statistical significance among groups along with Dunnett post-hoc test for intra-group
comparison. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. (D) Nitrate/nitrite fold increase produced in HaCaT
parental and HaCaT E5/E6/E7-18 cells in normoxia and coverslip induced hypoxia. Three independent
experiments were conducted in triplicate. Mean ± SD is shown for each cell line. A one-way analysis of variance
(ANOVA) test was conducted to assess statistical significance among groups along with Tukey’s HSD test for
intra-group comparison. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) p < 0.0001. DNA damage in normoxia and hypoxia g
yp
Next, we assessed the contributions of both hypoxia and HPV-18 viral oncogenes in promoting DNA damage. First, we evaluated γH2AX levels in HaCaT parental and E5/E6/E7-18 cells both in normoxia and hypoxia. As
shown in Fig. 2, both HaCaT parental and HaCaT E5/E6/E7-18 cells had significantly higher γH2AX fluores-
cence intensity in hypoxia compared to normoxia. However, no significant differences in γH2AX intensity were
observed between HaCaT parental and HaCaT E5/E6/E7-18 cells either in normoxia or hypoxia, which suggests
that hypoxia leads to greater γH2AX accumulation than viral oncogene expression. To further evaluate the level
of endogenous DNA damage produced, we employed the alkaline comet assay (Fig. 3A). We observed a signifi-
cant increase in the damage index in HaCaT E5/E6/E7-18 cells in hypoxia compared to normoxia, whereas the
increase in the damage index in HaCaT parental cells in hypoxia compared to normoxia was not statistically
significant (Fig. 3B). This suggests that hypoxia and viral oncogenes make a greater contribution to DNA strand
breaks as measured by the comet assay than hypoxia alone. Nuclear dry mass density, dry mass and volume in normoxia and hypoxia
We then sought to analyze global changes in nuclear structure caused by hypoxia and viral oncogenes. To this end,
we used the quantitative phase microscope Nanolive 3D Cell Explorer-Fluo, which allows to measure refractive Nuclear dry mass density, dry mass and volume in normoxia and hypoxia y
y
y
yp
We then sought to analyze global changes in nuclear structure caused by hypoxia and viral oncogenes. To this end
we used the quantitative phase microscope Nanolive 3D Cell Explorer-Fluo, which allows to measure refractive https://doi.org/10.1038/s41598-023-44880-3 Scientific Reports | (2023) 13:17734 | www.nature.com/scientificreports/ Figure 2. Detection of γH2AX signal by confocal microscopy. (A) Representative images of γH2AX signal in
parental HaCaT and HaCaT E5/E6/E7-18 cells in normoxia and under treatment with coverslips for 24 h. Scale
bar: 20 μm. (B) Quantification of γH2AX intensity (mean ± SD) in normoxia and coverslip induced hypoxia for
HaCaT parental and HaCaT E5/E6/E7-18 cells. Three independent experiments were conducted. Mean ± SD is
shown for each cell line. For all experiments, a one-way analysis of variance (ANOVA) test was conducted to
assess statistical significance among groups along with Tukey’s HSD test for intra-group comparison (*) P < 0.05,
(**) P < 0.01, (***) P < 0.001, (****) p < 0.0001. Figure 2. Detection of γH2AX signal by confocal microscopy. (A) Representative images of γH2AX signal in
parental HaCaT and HaCaT E5/E6/E7-18 cells in normoxia and under treatment with coverslips for 24 h. Scale
bar: 20 μm. (B) Quantification of γH2AX intensity (mean ± SD) in normoxia and coverslip induced hypoxia for
HaCaT parental and HaCaT E5/E6/E7-18 cells. Three independent experiments were conducted. Mean ± SD is
shown for each cell line. For all experiments, a one-way analysis of variance (ANOVA) test was conducted to
assess statistical significance among groups along with Tukey’s HSD test for intra-group comparison (*) P < 0.05,
(**) P < 0.01, (***) P < 0.001, (****) p < 0.0001. index distributions inside cells, from which dry mass density distributions can be obtained, and which pro-
duces z-stacks from which cellular and subcellular volumes can be estimated. In hypoxia, for both cell lines, we
observed marked changes in nuclei visualized by quantitative phase microscopy, with the appearance of brighter
dots throughout the nuclear volume, suggesting that areas with increased dry mass density form inside nuclei
in cells exposed to hypoxia (Fig. 4A and supplementary videos 1 and 2). We then obtained nuclear volume and
nuclear dry mass density values, observing a small decrease in nuclear volume in hypoxia (Fig. 4B, not statistically
significant), accompanied by a significant increase in nuclear mass density in hypoxia (Fig. Nuclear dry mass density, dry mass and volume in normoxia and hypoxia 4C), suggesting that
nuclei in hypoxia do not decrease their dry mass content even if they decrease in volume. Interestingly, nuclear
dry mass content actually increases significantly in hypoxia for HaCaT E5/E6/E7-18 cells (Fig. 4D). However, the
most marked change we observed was in nuclear dry mass density variance, which greatly increased in hypoxia
for both HaCaT parental and HaCaT E5/E6/E7-18 cells (Fig. 4E), indicating a more heterogeneous dry mass
distribution in nuclei in hypoxia, and suggesting that hypoxia results in marked changes in nuclear architecture. index distributions inside cells, from which dry mass density distributions can be obtained, and which pro-
duces z-stacks from which cellular and subcellular volumes can be estimated. In hypoxia, for both cell lines, we
observed marked changes in nuclei visualized by quantitative phase microscopy, with the appearance of brighter
dots throughout the nuclear volume, suggesting that areas with increased dry mass density form inside nuclei
in cells exposed to hypoxia (Fig. 4A and supplementary videos 1 and 2). We then obtained nuclear volume and
nuclear dry mass density values, observing a small decrease in nuclear volume in hypoxia (Fig. 4B, not statistically
significant), accompanied by a significant increase in nuclear mass density in hypoxia (Fig. 4C), suggesting that
nuclei in hypoxia do not decrease their dry mass content even if they decrease in volume. Interestingly, nuclear
dry mass content actually increases significantly in hypoxia for HaCaT E5/E6/E7-18 cells (Fig. 4D). However, the
most marked change we observed was in nuclear dry mass density variance, which greatly increased in hypoxia
for both HaCaT parental and HaCaT E5/E6/E7-18 cells (Fig. 4E), indicating a more heterogeneous dry mass
distribution in nuclei in hypoxia, and suggesting that hypoxia results in marked changes in nuclear architecture. Nuclear foci quantitative image analysis q
g
y
In hypoxia, the number of nuclear foci detected with Hoechst 33342 staining was significantly higher than in
normoxia for both HaCaT parental and E5/E6/E7-18 cells, indicating an altered chromatin distribution after
exposure to a hypoxic microenvironment, and confirming our previous results with quantitative phase imaging
(Fig. 5). Discussion Furthermore, hypoxia increased
DNA strand breaks in both HaCaT parental and HaCaT E5/E6/E7-18 cells, while the extent of DNA damage was
greatest in HaCaT E5/E6/E7-18 cells exposed to hypoxia, as measured by the comet assay, showing that HPV-
18 oncogenes and hypoxia in combination contribute towards increased genotoxicity in addition to increased
oxidative stress. compared to HaCaT parental cells. In addition, HaCaT E5/E6/E7-18 cells increased ·NO production in hypoxia
and had significantly higher ·NO levels in this condition compared to HaCaT parental cells. Therefore, both
HPV-18 oncogenes and hypoxia contribute to an increase in oxidative stress. Furthermore, hypoxia increased
DNA strand breaks in both HaCaT parental and HaCaT E5/E6/E7-18 cells, while the extent of DNA damage was
greatest in HaCaT E5/E6/E7-18 cells exposed to hypoxia, as measured by the comet assay, showing that HPV-
18 oncogenes and hypoxia in combination contribute towards increased genotoxicity in addition to increased
oxidative stress. HPV oncogenes have been shown to cause increased oxidative stress levels, DNA damage and resistance
to oxidative stress induced cell death: the E6 small isoform E6* increases oxidative stress and induces DNA
damage25, whereas E7 modulates the expression of catalase, B-cell lymphoma extra-large (Bcl-xL), interleukin-8
(IL-8), Fas and Bad, resulting in resistance to oxidative stress-induced cell death in human keratinocyte cells26. More recently, it was reported that HPV-16 E6 and E7 induce oxidative stress and DNA damage in head and
neck cancer cells27. We have also previously observed by flow cytometry that E5 in cooperation with E6 and E7
of HPV-18 promotes intracellular ROS production22, which we have now confirmed by confocal microscopy
as well. In the present study, we have also observed increased expression in HaCaT E5/E6/E7-18 cells of SOD2. This enzyme catalyzes the dismutation of the superoxide (O−2) radical into ordinary molecular oxygen (O2)
and hydrogen peroxide, it has key roles in controlling the redox status of tumor cells and is upregulated in several
HPV-associated tumors, including penile and cervical carcinomas28,29, which is consistent with our results. We
have also observed an increase, albeit not significant, in eNOS expression in HaCaT E5/E6/E7-18 cells, which
is consistent with the increase in ·NO production by HaCaT E5/E6/E7-18 cells in hypoxia compared to HaCaT
parental cells in the same condition. Discussion In this work, we have studied the contributions of hypoxia and HPV-18 viral oncogenes to promoting oxidative
stress and DNA damage, and therefore advancing the process of malignant transformation. HaCaT cells trans-
duced with the E5, E6 and E7 HPV-18 oncogenes exhibited significantly higher intracellular ROS production https://doi.org/10.1038/s41598-023-44880-3 Scientific Reports | (2023) 13:17734 | www.nature.com/scientificreports/ Figure 3. Comet assay in HaCaT parental and HaCaT E5/E6/E7-18 cell lines. (A) Representative comet images
of HaCaT parental and HaCaT E5/E6/E7-18 cell lines stained with DAPI, in normoxia and in hypoxia visualized
by epifluorescence microscopy following alkaline electrophoresis. The length of the comet tail is an indicator
of the level of DNA damage (red arrows). (B) DNA damage index of HaCaT parental and HaCaT E5/E6/E7-18
cells measured in normoxia and coverslip-induced hypoxia. Two independent experiments were conducted
in triplicate. Mean ± SD is shown for each cell line and condition. For all experiments, a one-way analysis of
variance (ANOVA) test was conducted to assess statistical significance among groups along with Tukey’s HSD
test for intra-group comparison. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. Figure 3. Comet assay in HaCaT parental and HaCaT E5/E6/E7-18 cell lines. (A) Representative comet images
of HaCaT parental and HaCaT E5/E6/E7-18 cell lines stained with DAPI, in normoxia and in hypoxia visualized
by epifluorescence microscopy following alkaline electrophoresis. The length of the comet tail is an indicator
of the level of DNA damage (red arrows). (B) DNA damage index of HaCaT parental and HaCaT E5/E6/E7-18
cells measured in normoxia and coverslip-induced hypoxia. Two independent experiments were conducted
in triplicate. Mean ± SD is shown for each cell line and condition. For all experiments, a one-way analysis of
variance (ANOVA) test was conducted to assess statistical significance among groups along with Tukey’s HSD
test for intra-group comparison. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. compared to HaCaT parental cells. In addition, HaCaT E5/E6/E7-18 cells increased ·NO production in hypoxia
and had significantly higher ·NO levels in this condition compared to HaCaT parental cells. Therefore, both
HPV-18 oncogenes and hypoxia contribute to an increase in oxidative stress. Discussion Increased levels of ·NO have been observed in women with cervical cytologi-
cal changes likely related to HPV13, and this free radical can contribute to the induction of genotoxic lesions and
to aspects of tumor progression such as cell growth, invasion and angiogenesis30, while also being an important
component of the microenvironment in tumors such as cervical cancer, where it modifies immune responses
and tumor behavior31,32. Therefore, the increase in ·NO produced by the combination of a hypoxic microen-
vironment and HPV oncogenes that we observed could be an important factor mediating the contribution of
·NO towards malignant transformation in HPV-related cancers, such as cervical cancers. More generally, our
results suggest that the combination of microenvironmental hypoxia and HPV oncogenes can lead to increased
oxidative stress during initial stages of tumor progression, likely contributing to advancing this process in HPV-
associated cancers33.f HPV oncogenes induce both oxidative stress and DNA damage20,21 in different tumor types frequently asso-
ciated with HPV infection, such as head and neck cancer34. Also, expression of HPV oncogenes E6 and E7 in https://doi.org/10.1038/s41598-023-44880-3 Scientific Reports | (2023) 13:17734 | www.nature.com/scientificreports/ ntificreports/
Figure 4. Quantitative phase imaging of HaCaT parental and HaCaT E5/E6/E7-18 cell lines. (A) Representative
quantitative phase images of HaCaT parental and HaCaT E5/E6/E7-18 cell lines in normoxia and coverslip-
induced hypoxia. Scale bar: 10 μm. (B) Nuclear volume in HaCaT parental and HaCaT E5/E6/E7-18 cell lines
in normoxia and coverslip-induced hypoxia. (C) Nuclear dry mass density (g/dL) in HaCaT parental and
HaCaT E5/E6/E7-18 cell lines in normoxia and coverslip-induced hypoxia. (D) Nuclear dry mass (picograms)
in HaCaT parental and HaCaT E5/E6/E7-18 cell lines in normoxia and coverslip-induced hypoxia. (E) Variance
of nuclear dry mass density in HaCaT parental and HaCaT E5/E6/E7-18 cell lines in normoxia and coverslip-
induced hypoxia. For Figs. 4B, C, D and E, three independent experiments were conducted. Mean ± SD is shown
for each cell line. For all experiments, a one-way analysis of variance (ANOVA) test was conducted to assess
statistical significance among groups along with Tukey’s HSD test for intra-group comparison (*) P < 0.05, (**)
P < 0.01, (***) P < 0.001, (****) p < 0.0001. Figure 4. Quantitative phase imaging of HaCaT parental and HaCaT E5/E6/E7-18 cell lines. (A) Representative
quantitative phase images of HaCaT parental and HaCaT E5/E6/E7-18 cell lines in normoxia and coverslip-
induced hypoxia. Scale bar: 10 μm. Discussion (B) Nuclear volume in HaCaT parental and HaCaT E5/E6/E7-18 cell lines
in normoxia and coverslip-induced hypoxia. (C) Nuclear dry mass density (g/dL) in HaCaT parental and
HaCaT E5/E6/E7-18 cell lines in normoxia and coverslip-induced hypoxia. (D) Nuclear dry mass (picograms)
in HaCaT parental and HaCaT E5/E6/E7-18 cell lines in normoxia and coverslip-induced hypoxia. (E) Variance
of nuclear dry mass density in HaCaT parental and HaCaT E5/E6/E7-18 cell lines in normoxia and coverslip-
induced hypoxia. For Figs. 4B, C, D and E, three independent experiments were conducted. Mean ± SD is shown
for each cell line. For all experiments, a one-way analysis of variance (ANOVA) test was conducted to assess
statistical significance among groups along with Tukey’s HSD test for intra-group comparison (*) P < 0.05, (**)
P < 0.01, (***) P < 0.001, (****) p < 0.0001. normal cells results in DNA damage and genomic instability21, one of the hallmarks of HPV-induced carcino-
genesis. Hypoxia has been associated with DNA damage as well, as for instance moderate hypoxia can cause
indirectly DNA damage in the form of replication stress, as evidenced by S-phase arrest, ATR activation and
phosphorylation of H2AX-Ser139 (γ-H2AX), p53-Ser15, Chk1- Ser345, and Chk2-Thr6835,36. In the present
study, we evaluated DNA damage by assessing γH2AX nuclear accumulation and by the comet assay. γH2AX
nuclear accumulation was markedly increased by hypoxia, but we did not observe differences between HaCaT
E5/E6/E7-18 cells and HaCaT parental cells in normoxia. On the other hand, the DNA damage index measured
by the alkaline comet assay increased in hypoxia for HaCaT E5/E6/E7-18 cells, but not for HaCaT parental cells. The γH2AX immunoassay is a sensitive technique to detect DNA double-strand breaks37, while the alkaline
comet assay does not discriminate between alkali-labile sites and single or double-strand breaks38,39. Therefore,
these results suggest, firstly, that the hypoxia condition is the main responsible for inducing DNA double-strand
breaks and that HPV-18 viral oncogenes alone had no significant implications in this type of DNA damage. On
the other hand, the combination of HPV oncogenes and hypoxia increases the DNA damage detected by the
alkaline comet assay in HaCaT E5/E6/E7-18 cells, which, taken together with the evidence of increased oxida-
tive stress in hypoxia in these cells, suggests that hypoxia and HPV oncogenes combine to induce DNA damage
consisting of single and double strand breaks and alkali-labile sites via higher oxidative damage. Discussion normal cells results in DNA damage and genomic instability21, one of the hallmarks of HPV-induced carcino-
genesis. Hypoxia has been associated with DNA damage as well, as for instance moderate hypoxia can cause
indirectly DNA damage in the form of replication stress, as evidenced by S-phase arrest, ATR activation and
phosphorylation of H2AX-Ser139 (γ-H2AX), p53-Ser15, Chk1- Ser345, and Chk2-Thr6835,36. In the present
study, we evaluated DNA damage by assessing γH2AX nuclear accumulation and by the comet assay. γH2AX
nuclear accumulation was markedly increased by hypoxia, but we did not observe differences between HaCaT
E5/E6/E7-18 cells and HaCaT parental cells in normoxia. On the other hand, the DNA damage index measured
by the alkaline comet assay increased in hypoxia for HaCaT E5/E6/E7-18 cells, but not for HaCaT parental cells. The γH2AX immunoassay is a sensitive technique to detect DNA double-strand breaks37, while the alkaline
comet assay does not discriminate between alkali-labile sites and single or double-strand breaks38,39. Therefore,
these results suggest, firstly, that the hypoxia condition is the main responsible for inducing DNA double-strand
breaks and that HPV-18 viral oncogenes alone had no significant implications in this type of DNA damage. On
the other hand, the combination of HPV oncogenes and hypoxia increases the DNA damage detected by the
alkaline comet assay in HaCaT E5/E6/E7-18 cells, which, taken together with the evidence of increased oxida-
tive stress in hypoxia in these cells, suggests that hypoxia and HPV oncogenes combine to induce DNA damage
consisting of single and double strand breaks and alkali-labile sites via higher oxidative damage. https://doi.org/10.1038/s41598-023-44880-3 Scientific Reports | (2023) 13:17734 | www.nature.com/scientificreports/ Figure 5. Nuclear foci quantitative image analysis (A) Segmentation of nuclear foci of a representative image
of HaCaT E5/E6/E7-18 cells. a) Hoechst 33342 greyscale image. b) Threshold segmentation. c) StarDist 2D
segmentation. Note that using this method the result is more precise than using only threshold, for example in
the shape of the pink nucleus. d) Nuclear profile recognition and numeration. Note that on edge nucleus were
excluded and only complete profiles were segmented. e) Find maxima single point tool for foci analysis. Isolated
single points outside the nucleus were not considered. f) Combination of D and E for foci measurement. (B)
Quantification of nuclear foci. Discussion For this analysis Mann–Whitney test and one-way analysis of variance ANOVA
test was conducted to assess statistical significance among groups (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****)
p < 0.0001. Figure 5. Nuclear foci quantitative image analysis (A) Segmentation of nuclear foci of a representative image
of HaCaT E5/E6/E7-18 cells. a) Hoechst 33342 greyscale image. b) Threshold segmentation. c) StarDist 2D
segmentation. Note that using this method the result is more precise than using only threshold, for example in
the shape of the pink nucleus. d) Nuclear profile recognition and numeration. Note that on edge nucleus were
excluded and only complete profiles were segmented. e) Find maxima single point tool for foci analysis. Isolated
single points outside the nucleus were not considered. f) Combination of D and E for foci measurement. (B)
Quantification of nuclear foci. For this analysis Mann–Whitney test and one-way analysis of variance ANOVA
test was conducted to assess statistical significance among groups (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****)
p < 0.0001. Moreover, it is important to consider that early HPV infection produces DNA damage and upregulates DNA
damage response genes, as shown for instance in a model of HPV infection using organotypic raft cultures of
human cervical keratinocytes40. The combination of HPV infection and a hypoxic microenvironment could
therefore result in increased selective pressure on the DNA damage repair capability of cells, leading to a cellular
phenotype tolerant of higher DNA damage burdens and eventually to the emergence of resistant and aggressive
tumors. Genetic damage can cause profound alterations in nuclear architecture41. In this study, we used quantitative
phase imaging to assess nuclear dry mass distribution as an indirect way to evaluate changes in nuclear archi-
tecture caused by hypoxia and HPV oncogenes. Our results indicate that nuclear dry mass density increases
significantly with hypoxia, whereas nuclear volume tends to decrease, although not significantly. This would
imply that the increase in nuclear dry mass density observed in hypoxia is mainly due to a decrease in nuclear
volume and not an increase in nuclear dry mass. This appears to be the case for HaCaT parental cells, which do
not show an increase in nuclear dry mass in hypoxia, whereas nuclear dry mass in HaCaT E5/E6/E7-18 cells
increases in hypoxia compared to normoxia and compared to HaCaT parental cells in hypoxia. www.nature.com/scientificreports/ redistribution in hypoxia, and therefore supporting the notion that hypoxia results in profound rearrangements
of nuclear components. As future perspectives of this work, it will be important to identify if these foci contain
components of the DNA damage repair machinery, and in particular which DNA damage repair pathways are
activated in response to the combination of hypoxia and HPV oncogenes.i redistribution in hypoxia, and therefore supporting the notion that hypoxia results in profound rearrangements
of nuclear components. As future perspectives of this work, it will be important to identify if these foci contain
components of the DNA damage repair machinery, and in particular which DNA damage repair pathways are
activated in response to the combination of hypoxia and HPV oncogenes.i p
yp
g
In conclusion, this is the first report that studies the contributions of hypoxia and the three viral oncogenes
of HPV-18 to oxidative stress and DNA damage, and our results suggest that the interplay between microenvi-
ronmental hypoxia and HPV oncogenes is likely to cause increased oxidative stress and DNA damage, as well
as an altered nuclear architecture, therefore propelling forward the process of HPV induced carcinogenesis. Nitric oxide‑releasing activity g
In order to measure the ·NO production in our cells, we used the Griess reaction as we described previously45. This assay consists in measuring the nitrate/nitrite content in the cell culture medium. We performed this assay
in normoxic and hypoxic conditions previously described. For the latter condition, cells were cultured under
coverslips for 24 h, and after this period, cell supernatants were collected and analyzed. Briefly, 50 µl of culture
medium were transferred to a new multiwell plate and incubated first with 1% sulfanilamide solution and then
with 0.1% N-1-naphtilethylenrdiamine dihydrochloride protected from light. In parallel, a reference curve with
NaNO2 was performed at serial dilutions between 0 and 100 µmol/l in 50 µl of culture medium. The absorbance
was measured at 540 nm using a microplate spectrophotometer (Varioskan Flash Microplate spectrophotometer;
Thermo Fisher, Vantaa, Finland). Induction of hypoxia yp
In order to induce hypoxia to HaCaT parental and HaCaT E5/E6/E7-18 cells, we used the variant of coverslip-
induced hypoxia method previously described 23. Briefly, this assay consists in culturing cells in the 10 mm radius
wells of glass bottom dishes of 35 mm diameter (Cellvis, CA, USA) at increasing distances from an oxygen source. For this, after the cells were adhered to the wells, they were covered for 24 h with square acrylic coverslips of
24 mm of width and 2 mm of thickness with a square hole in the middle. In this way, an oxygen gradient is gener-
ated, with cells in the periphery of the chamber located farthest from the coverslip hole (the sole oxygen source). γH2AX immunocytochemistryh The DNA damage was evaluated by quantification of γH2AX nuclear immunofluorescence. For this, HaCaT
parental and HaCaT E5/E6/E7-18 cells were seeded in a 24-well plate at density of 2 × 105 per well and incubated
for 24 h in a humidified 5% CO2 atmosphere at 37 °C. For the hypoxia condition, cells were cultured under
coverslips for 24 h before the experiment. After that, culture medium was removed and cells were washed once
with PBS. Then, cells were fixed for 15 min in 4% paraformaldehyde, rinsed in PBS three times, 5 min each,
and permeabilized with blocking buffer (0.05% Triton X-100, 1% BSA in PBS) for 45 min at room temperature. Afterwards, permeabilized cells were incubated with the anti-γH2AX antibody diluted in blocking buffer (1/500,
ab26350; Abcam, Cambridge, Massachusetts, USA) overnight at 4 °C. Finally, cells were washed with PBS three
times for 5 min each, and incubated for 45 min with the secondary antibody (1:1000, goat anti-mouse conjugated
with Alexa488; Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA), and with DAPI (300 nmol/l;
Invitrogen) to stain nuclei. Finally, cells were visualized using a Zeiss LSM800 confocal microscope. The γH2AX
nuclear fluorescence was quantified related to DAPI signal for each condition, using the ImageJ software v. 1.43n4. Materials and methods
Cell lines Spontaneously immortalized human keratinocyte (HaCaT) cells were purchased from Banco de Células do Rio
de Janeiro (BCRJ), Brazil (batch number 001071, certificate of analysis provided by the supplier) and maintained
in Dulbecco’s Modified Eagle’s Medium (DMEM) low glucose medium (Capricorn, Ebsdorfergrund, Germany)
supplemented with 10% fetal bovine serum (FBS) (Gibco, Massachusetts, USA). HaCaT cells were transduced
with three viral oncogenes (E5/E6/E7-18) of HPV-18 using lentiviral packaging systems. For this, HaCaT E5/E6/
E7 HPV-18 cells were previously prepared by co-infection with a retroviral vector carrying the MSCV-N-puro-
18E5 plasmid (Addgene # 37,882, MA, USA) and with a pLXSN retroviral vector that contained cloned both E6/
E7 of HPV-18 genes that was kindly provided by Dra, Sichero from Instituto do Câncer do Estado de São Paulo. The preparation and characterization of these cell lines was detailed in Hochmann et al.22. Intracellular ROS production p
We evaluated the intracellular ROS production in live HaCaT parental and HaCaT E5/E6/E7-18 cells using
ROS Detection Reagent Deep Red (Sigma, MO, USA, 1:1000 dilution from stock solution). Once cells reached
a confluence of 60 per cent approximately, they were incubated with the reagent for one hour at 37 °C following
manufacturer instructions. Then cells were visualized by confocal microscopy using a Zeiss LSM800 confocal
microscope (Zeiss, Oberkochen, Germany) equipped with a Plan Apo N 63X oil NA 1.4 lens. Quantification of
ROS intensity was evaluated using Image J software v. 1.43n4. Discussion yp
p
p
p
yp
A previous study has shown that nuclear dry mass is similar in SW480 and SW620 cell lines (derived from the
same patient before and after metastatic spread), but that nuclear volume is smaller in metastatic SW620 cells42,
suggesting that nuclear dry mass tends to be more conserved than nuclear volume. However, another study found
that nuclear dry mass increases after ionizing radiation, in parallel with an increase in fluorescence intensity of
γH2AX nuclear foci43, suggesting that an increase in nuclear dry mass might be related to the mobilization of
DNA repair machinery. The increase in nuclear dry mass in HaCaT E5/E6/E7-18 cells in hypoxia might therefore
be related to an increased DNA repair response, which could be caused in turn by the increased DNA damage
observed in this condition using the comet assay. We also observed a marked increase in nuclear dry mass density variance in hypoxia, indicating that the het-
erogeneity of the nuclear dry mass distribution increases in this condition, which implies that marked changes
in nuclear architecture are taking place during hypoxia. Moreover, increased variance in dry mass distribution is
linked to a decrease in cell stiffness44, whereas DNA damage has been shown to decrease nuclear stiffness44. There-
fore, the marked increase in the variance of nuclear dry mass density that we observe in hypoxia would indicate
that nuclear stiffness decreases in this condition, which could be related in turn to the increase in DNA damage
observed. In summary, we have observed that both hypoxia and HPV oncogenes alter key physical nuclear
parameters, which is likely related to the contributions of both factors to increased DNA damage. Moreover, as
shown by Hoechst 33342 staining of live cells, hypoxia increases the number of nuclear foci, indicating chromatin Scientific Reports | (2023) 13:17734 | https://doi.org/10.1038/s41598-023-44880-3 www.nature.com/scientificreports/ Quantitative phase imaging and nuclear dry mass density calculation HaCaT parental and E5/E6/E7-18 cells were imaged in normoxia or under thin glass coverslips to induce hypoxia
using the quantitative phase microscope Nanolive 3D Cell Explorer-Fluo, which produces z-stacks of 96 slices
with a depth of field of 30 μm. Cells were imaged for 24 h, and the refractive index distribution inside cell nuclei
in normoxia and after 24 h under coverslips was obtained from the best focused slice, as each pixel in the quan-
titative phase images corresponds to a refractive index value. Next, refractive index values were converted to dry
mass density values according to the equation46: Drymassdensity
g/dL
= 665. RI
RIH2O
−1 where RI is refractive index and RIH2O is the refractive index of water, equal to 1.33. Nuclei were segmented manu-
ally from quantitative phase images, and the average nuclear dry mass density and its variance were calculated
using Python scripts. 30 nuclei were analyzed for each condition, and their volume was calculated from their areas
in the slices in focus from the z-stacks. From the nuclear volume, and the nuclear dry mass density measured in
the best focused slice, the estimated nuclear dry mass (expressed in picograms) was obtained. Expression level of eNOS and SOD2h The relative intensities of eNOS and SOD2 proteins were evaluated using the Human XL Oncology Array Kit
(R&D Systems, MN, USA, ARY026), and Human Cell Stress Array Kit (R&D Systems, MN, USA, ARY018)
respectively following manufacturer instructions. Protein levels were quantified using the ImageQuant TL soft-
ware (GE Healthcare, Buckinghamshire, UK), and the individual Western blot membranes were normalized
according to the pixel densities of the 6 reference spots. Nuclear foci quantitative image analysis q
g
y
HaCaT parental and E5/E6/E7-18 live cells were stained with Hoechst 33342 (Sigma, MO, USA, final concen-
tration 1 μg/ml) for 30 min either in normoxia or after 24 h under coverslips. Images were obtained with the
fluorescence module of the Nanolive 3D Cell Explorer-Fluo and were analysed in order to determine the pres-
ence of nuclear foci. Normalization of the images was performed using Enhance Contrast tool of ImageJ, with
0,35% saturated pixels. Nuclear profiles were segmented with color threshold or ImageJ/Fiji StarDist 2D with
the default parameters of the plugin47. Afterwards, overlapping or adjacent nuclei were separated with the macro
Separate Labels48, and on edge nucleus were excluded. Nuclear foci were determined by Find Maxima ImageJ
tool, which locates the maxima intensity in an image and, as a result, creates a binary mask of one segmented
particle per maximum. Finally, both images were combined in order to measure the number of foci per nucleus. www.nature.com/scientificreports/ were trypsinized and neutralized with culture medium. Then, cells were centrifuged for 5 min at 1200 rpm and
the pellet was resuspended in 10 µl of PBS. The cell suspension (20 µl) mixed with 80 µl of 0.75% low melting
point agarose (LMP) (Gibco #15,517–014) was extended on 1.0% NMP-agarose (Invitrogen #15,510–019) pre-
coated slide. A coverslip was added and the agarose was allowed to set for 10 min at 4 °C. Then, the coverslip was
removed and the cells were lysed by immersion of the slide in cold lysis buffer solution (pH 10, 2.5 mol/l NaCl,
100 mmol/l Na2EDTA, 10 mmol/l Tris–HCl, 1% Triton X-100) and 10% DMSO for 1 h at 4 °C. Electrophoresis
was conducted in alkaline buffer (3 mol/l NaOH, 100 mmol/l EDTA, pH 13) at 25 V for 20 min in a cold unit
at 4 °C with a distance between electrodes of 34 cms. Following neutralization, cells were stained with DAPI
(6 µg/ml; Invitrogen) for 10 min. Two slides per condition were analyzed in each experiment and 100 cells per
slide were scored. Damage was quantified as the damage index, which represents damage in individual cells
DNA damage scores vary from 1 to 5, with 1 being the lower damage and 5 the higher damage. The Damage
Index is calculated based on 100 cells, with the following equation: 5
1n.α where n is the cell number and α is
the damage score. Statistical analysis y
Statistical analysis and graphical presentation were performed using GraphPad Prism version 8.0.1 software
(GraphPad Software Inc., San Diego, CA, USA). All experiments were performed in triplicate, and data were
presented as the mean ± standard deviation (SD), with the exception of the comet assay, in which two experiments
were performed induplicate. For all statistical analysis, One-Way unpaired ANOVA statistical test followed by
Tukey’s HSD post-hoc test was conducted with the exception of data retrieved from ROS quantification, which
was analyzed using Student`s Test in order to compare only two means, and data obtained from protein profile
analysis which was analyzed using two-way analysis of variance (ANOVA) test in order to assess statistical sig-
nificance among groups along with Dunnett post-hoc test for intra-group comparison. Statistical significance was
determined at p < 0.05. Descriptive statistics, Mann–Whitney test and ANOVA was also performed for nuclear
foci quantitative image analysis. DNA Damage measurement by comet assay g
y
y
In order to evaluate the DNA damage generated in the hypoxic microenvironment in both HaCaT parental and
HaCaTE5/E6/E7-18 cells, we used the alkaline comet assay. For this, HaCaT cells were plated in wells of 35 mm
glass bottom dishes at densities of 2 × 105 per well and incubated for 24 h in a humidified 5% CO2 atmosphere
at 37 °C. For the hypoxia condition, cells were previously cultured under coverslips for 24 h. After that, cells https://doi.org/10.1038/s41598-023-44880-3 Scientific Reports | (2023) 13:17734 | www.nature.com/scientificreports/ References References
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This work was supported partially by Programa de Desarrollo de las Ciencias Básicas (PEDECIBA) and Agencia
Nacional de Investigación e Innovación (ANII). Acknowledgements g
We are grateful to Comisión Sectorial de Investigación Científica (CSIC), Programa de Desarrollo de las Cien-
cias Básicas (PEDECIBA) and Agencia Nacional de Investigación e Innovación (ANII). We also grateful to Dra. Laura Sichero for her assistance in the preparation of cell lines containing HPV-18 oncogenes, and to Rosana
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6. Phillips, K. G., Jacques, S. L. & McCarty, O. J. Competing interests h p
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The authors declare no competing interests. p
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The authors declare no competing interests. Additional information
Supplementary Information The online version contains supplementary material available at https://doi.org/
10.1038/s41598-023-44880-3. Additional information
Supplementary Information The online version contains supplementary material available at https://doi.org/
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https://doi.org/10.53930/27892182.dialogos.5.50 1 Mestre em Ciências da Religião pela Universidade Federal de Sergipe. Advogado e presidente
da Comissão de Liberdade Religiosa da OAB/SE. Email: advdouglascosta@gmail.com.
https://doi.org/10.53930/27892182.dialogos.5.50 eduCação patrimonial Como base de preservação Douglas Lima da Costa1 Resumo: O presente artigo objetiva conduzir à reflexão sobre o papel que desempe-
nha o Patrimônio Histórico Cultural como componente simbólico representativo
do poder e da identidade da memória coletiva das comunidades e do local em que
constroem sua história. Pontua a contribuição oferecida pela Nova História Cultural
como elemento de registro para a identificação e a preservação da memória, sobretudo,
das classes populares e dos locais onde constroem sua cultura bem como, analisa a
proposta de Educação Patrimonial contida no Guia Básico de Educação Patrimonial
organizado pelo IPHAN – Instituto do Patrimônio Histórico e Artístico Nacional. Palavras-chave: Patrimônio Histórico Cultural; Memória; Educação Patrimonial. Douglas Lima da Costa Mestre em Ciências da Religião pela Universidade Federal de
Sergipe. Advogado e presidente da Comissão de Liberdade
Religiosa da OAB/SE. e-mail: advdouglascosta@gmail.com Submetido: 11 de março de 2020
Aceito: 15 de agosto de 2020
Publicado: 17 de novembro de 2020 Submetido: 11 de março de 2020
Aceito: 15 de agosto de 2020
Publicado: 17 de novembro de 2020 Diálogos | Volume 05 | 2020 heritage eduCation as a preservation base Abstract: This article aims to reflect on the role played by Cultural Historical
Heritage as a symbolic component representative of the power and identity of
the collective memory of the communities and the place where they build their
history. It points out the contribution offered by the New Cultural History as an
element of registration for the identification and preservation of memory, above
all, of the popular classes and the places where they construct their culture, as well
as analyses the proposal of Education Patrimonial contained in the Basic Guide
of Patrimonial Education organized by IPHAN – Institute of National Historical
and Artistic Heritage. Keywords: Cultural Historical; Heritage Memory; Patrimonial Education. Em seu significado mais primitivo, a palavra patrimônio tem origem atre-
lada ao termo grego pater, que significa “pai” ou “paterno”, ou seja, tal qual um
pai transmite ao filho seus bens, essa foi a ideia que se relacionou ao patrimônio
como legado ao povo. O Patrimônio Histórico e Cultural é o conjunto de bens
materiais e imateriais arraigados de valor simbólico para uma dada comunidade,
diz respeito a memória que foi valorizada e materializada ao longo do tempo. 1 Mestre em Ciências da Religião pela Universidade Federal de Sergipe. Advogado e presidente
da Comissão de Liberdade Religiosa da OAB/SE. Email: advdouglascosta@gmail.com. https://doi.org/10.53930/27892182.dialogos.5.50 Diálogos | Volume 05 | 2020 Diálogos | Volume 05 | 2020 | 129 Douglas Lima da Costa Douglas Lima da Costa Essa noção de patrimônio ganhou força no século XIX, quando a
Revolução Francesa ressaltou a necessidade de elencar monumentos que refu-
tassem o esquecimento do passado. Nesse período a preservação do passado
colocava-se presa a uma noção de “melhoria”, “evolução” e “progresso”, pro-
messas da Revolução. As sociedades sempre deixam marcas no lugar onde se estabelecem que
identificam a sua história, materializam sua identidade, tradições e seus cos-
tumes. Como afirma Susana Gastal (2006, p. 101) nestes ambientes estão “as
marcas do local construídas no tempo”. Neste sentido, entende-se que todos
os lugares trazem traços peculiares do modo de ver e viver da população que
habita ou habitou o local. Para Gastal (2006, p. 101), “o Lugar seria o Locus,
no tempo e no espaço, do acúmulo de experiência em forma de história e de
tradição, a segurança da identidade”. Figura 1: A liberdade guiando o povo de Eugène Delacroix
Fonte: <https://chelios.jusbrasil.com.br/artigos/464544307/os-ide-
ais-da-revolucao-francesa-e-o-direito-moderno>. Acessado em: 20 jul. 2018. Figura 1: A liberdade guiando o povo de Eugène Delacroix Fonte: <https://chelios.jusbrasil.com.br/artigos/464544307/os-ide-
ais-da-revolucao-francesa-e-o-direito-moderno>. Acessado em: 20 jul. 2018. Fonte: <https://chelios.jusbrasil.com.br/artigos/464544307/os-ide-
ais-da-revolucao-francesa-e-o-direito-moderno>. Acessado em: 20 jul. 2018. Diálogos | Volume 05 | 2020
130 | 130 | Educação patrimonial como base de preservação Douglas Lima da Costa Douglas Lima da Costa Portanto, o objeto “fala sempre de um lugar, seja ele qual for, porque
está ligado à experiência dos sujeitos com e no mundo, posto que ele representa
uma porção significativa da paisagem vivida” (Silveira e Lima Filho, 2005, p. 40). Os autores acrescem que: É nesse sentido que é possível falar numa memória que
impregna e restitui “a alma nas coisas”, referida a uma paisagem
(inter) subjetiva onde o objeto (re) situa o sujeito no mundo
vivido mediante o trabalho da memória, ou ainda, é da força e
dinâmica da memória coletiva que o objeto, enquanto expressão
da materialidade da cultura de um grupo social remete à
elasticidade da memória como forma de fortalecer os vínculos
com o lugar, considerando as tensões próprias do esquecimento. (Silveira e Lima Filho, 2005, p. 39). É nesse sentido que é possível falar numa memória que
impregna e restitui “a alma nas coisas”, referida a uma paisagem
(inter) subjetiva onde o objeto (re) situa o sujeito no mundo
vivido mediante o trabalho da memória, ou ainda, é da força e
dinâmica da memória coletiva que o objeto, enquanto expressão
da materialidade da cultura de um grupo social remete à
elasticidade da memória como forma de fortalecer os vínculos
com o lugar, considerando as tensões próprias do esquecimento. (Silveira e Lima Filho, 2005, p. 39). É importante ainda tratar, brevemente, sobre o desgaste sofrido pela
memória com o passar do tempo. Diehl (2002, p. 118) nomeia este processo
de “corrosão temporal” da memória. Para Diehl, quanto mais distante do
fato, ou da época, ou do contexto tomado como objeto de investigação, mais
desgastada a memória estará. A memória “vai perdendo força, capacidade
explicativa, capacidade de informar, tornar-se transparente, sem pontos de
referência substantiva para manter suas funções (...)” (p. 118). Dihel (2002,
p. 119) aduz então, que a memória seja “refrescada constantemente” e que
seja grafada, narrada ou tornada uma fonte histórica ou ainda que seja trans-
formada em Patrimônio Histórico Cultural da coletividade. Mas o que deve ser visto como memória a fim de haver a preocu-
pação de ser guardada? Chartier (1991) acredita que existem esquemas
geradores das classificações e das percepções, que atuam como verdadeiras
instituições sociais criadoras do mundo social ou das representações coleti-
vas. Para o autor em comento nem sempre a representação é o representado
e o signo é o significado. Bourdieu (1989, p. Educação patrimonial como base de preservação Já Bartholo (2005, p. 19) afirma que “o lugar é produzido na relação
com o espaço construído socialmente, mediante uma rede de significados
e sentidos que são historicamente e culturalmente tecidos”. Astor Antônio
Diehl (2002, p. 112) entende a construção da memória dos lugares “como
um processo dinâmico da própria rememorização” do passado histórico. Para
o autor, essa reconstrução histórica produz um novo sentido e significação da
história de modo a possibilitar os grupos perceberem futuro no seu passado. Assim, as comunidades, enquanto oriundas de um processo de materia-
lização e reunião de um passado, constituem verdadeiras fontes documentais
da história do local. Para Diehl (2002, p. 116), a memória “constitui-se de um
saber, formando tradições, caminhos – como canais de comunicação entre di-
mensões temporais –, ao invés de rastros e restos como no caso da lembrança”. Em continuidade, o autor afirma que a formalização do passado deva
cumprir também o papel de identificação cultural dos indivíduos já que “o
ato de rememorar produz sentido e significação através da ressubjetivação do
sujeito e a repoetização do passado, produzindo uma nova estética do passado”
(p. 114). Assim, a memória torna-se fator de identidade e valorização da cultu-
ra dos mesmos. Conforme Andrade (2008, p. 570) estes lugares de memória
“são verdadeiros patrimônios culturais, projetados simbolicamente e podem
estar atrelados a um passado vivo que ainda marca presença e reforça os traços
identitários do lugar". Além de ser arquivos da memória coletiva de um povo, os lugares tam-
bém representam a memória coletiva de uma comunidade que pode ser reco-
nhecida também em objetos, festas, músicas, danças, práticas alternativas de
medicina, técnicas, culinária e diversas outras representações que estão tomadas
de significação das diversas formas de viver que constituem as culturas dos
povos. Referindo-se sobre este patrimônio, apesar de não ser como afirma
Fonseca (2005), feitos de “pedra e cal” também são memórias que podem
interligar diálogos entre as gerações. O objeto, afirma Debary (2010, pp. 38-39), “guarda consigo uma história que é retomada com ele” e é um teste-
munho que funciona “como um transmissor de história” que é reativado com
as lembranças que ele traz ao indivíduo ou sociedade. | 131 Diálogos | Volume 05 | 2020 Educação patrimonial como base de preservação O poder se vê por toda parte e é necessário saber descobri-lo
onde ele se deixa ver menos, onde ele é completamente ignorado,
portanto, reconhecido: o poder simbólico é, com efeito, esse
poder invisível o qual só pode ser exercido com a cumplicidade
daqueles que querem saber que lhe estão sujeitos ou mesmo que
o exercem. (Bourdieu, 1989, pp. 7-8). O poder se vê por toda parte e é necessário saber descobri-lo
onde ele se deixa ver menos, onde ele é completamente ignorado,
portanto, reconhecido: o poder simbólico é, com efeito, esse
poder invisível o qual só pode ser exercido com a cumplicidade
daqueles que querem saber que lhe estão sujeitos ou mesmo que
o exercem. (Bourdieu, 1989, pp. 7-8). Para Langer um monumento “não é simplesmente o que restou do tem-
po, é o que deve permanecer na História” (1997, p. 121) porque nele a sociedade
pode reconhecer-se nele e os poderes nele constituídos, revelados. Aliado ao conceito de patrimônio histórico está o de patrimônio cultural. Segundo o artigo 216.º da Constituição brasileira, o patrimônio cultural repre-
senta os bens: “(...) de natureza material e imaterial, tomados individualmente
ou em conjunto, portadores de referência à identidade, à ação, à memória dos
diferentes grupos formadores da sociedade brasileira”. Canani (2005, p. 3) afirma que o termo patrimônio “pode ser entendido
como um conjunto de bens, materiais ou não, direitos, ações, posse e tudo o
mais que pertença a uma pessoa e seja suscetível de apreciação econômica”. Desse modo, o termo patrimônio cultural está relacionado a um bem paternal,
de tamanha valia que justifica sua herança. O roteiro histórico da construção do conceito Patrimônio Cultural no
Brasil está vinculado ao pertencimento do patrimônio como um bem. O decreto-lei
que Getúlio Vargas assinou em 1937 utiliza este vocábulo. O decreto-lei nº 25,
de 30 de novembro de 1937, além de criar o Serviço do Patrimônio Histórico
e Artístico Nacional (SPHAN), definiu que patrimônio é o “conjunto de bens
móveis e imóveis de interesse público” que possuam “excepcional valor arqueo-
lógico ou etnográfico, bibliográfico ou artístico”. No Brasil, este patrimônio histórico é gerido pelo Instituto do Patrimônio
Histórico e Artístico Nacional (Iphan), o qual está atualmente vinculado ao
Ministério do Turismo, sendo anteriormente vinculado ao extinto Ministério
da Cultura que fora transformado em Secretaria da Cultura pela Lei 8.028 de
12 de abril de 19902. 2 Em 2019, a estrutura da Cultura foi incluída no Ministério da Cidadania por meio do
Decreto Nº 9.674, de 2 de janeiro. O Ministério da Cidadania ainda incorporou as pastas
de Desenvolvimento Social, Esporte, e parte da Secretaria Nacional de Políticas sobre
Drogas. Disponível em http://cultura.gov.br/acesso-a-informacao/institucional/historico/
Acesso 19 maio. 2020. Douglas Lima da Costa 12) aplica a expressão poder simbólico como sen-
do aquele que é capaz de construir uma realidade. O autor acredita que “o
campo da produção simbólica é um microcosmos da luta simbólica entre as
classes” e que ele é uma forma transformada, irreconhecível, transfigurada
e legitimada, das outras formas de poder. E mais: Diálogos | Volume 05 | 2020 132 | Educação patrimonial como base de preservação Ele foi criado em 1937 pela Lei n.º 378 no governo de 2 Em 2019, a estrutura da Cultura foi incluída no Ministério da Cidadania por meio do
Decreto Nº 9.674, de 2 de janeiro. O Ministério da Cidadania ainda incorporou as pastas
de Desenvolvimento Social, Esporte, e parte da Secretaria Nacional de Políticas sobre
Drogas. Disponível em http://cultura.gov.br/acesso-a-informacao/institucional/historico/
Acesso 19 maio. 2020. Diálogos | Volume 05 | 2020 | 133 Douglas Lima da Costa Douglas Lima da Costa Getúlio Vargas. Ribeiro (2005, p. 52) afirma que, no Brasil “a atribuição de
valores aos bens segue a tradição européia, em que os patrimônios nacionais
são constituídos a partir das categorias de história da arte”. E acrescenta: “na
prática, as ações são direcionadas para a proteção da cultura da elite”. Getúlio Vargas. Ribeiro (2005, p. 52) afirma que, no Brasil “a atribuição de
valores aos bens segue a tradição européia, em que os patrimônios nacionais
são constituídos a partir das categorias de história da arte”. E acrescenta: “na
prática, as ações são direcionadas para a proteção da cultura da elite”. Nesse sentido, com relação à preservação, quando os bens são tomba-
dos pelo órgão responsável, significa que possuem estimado valor histórico e
cultural. Essa intervenção tem como objetivo preservar o patrimônio, uma
vez que depois do tombamento eles não podem ser demolidos ou reformados. Abaixo, segue imagem da Praça São Francisco que é uma praça localizada
no centro histórico de São Cristóvão, no estado de Sergipe. A Praça foi fundada
junto com a cidade em 1607, e seu entorno possui edificações construídas entre
os séculos XVII e XIX. Foi protegida em nível estadual e nacional e designada
Patrimônio da Humanidade em 1º de agosto de 2010 pela Organização das
Nações Unidas para a Educação, a Ciência e a Cultura – UNESCO. Todo mês
de agosto são feitas manifestações para que a população possa se sentir inserida
nesse complexo patrimonial e sinta o interesse de protegê-lo como algo de sua
identidade. Normalmente a praça é palco de várias manifestações artísticas e
culturais como uma forma de apropriação do local. Figura 2: Praça São Francisco, São Cristovão - Sergipe
Fonte:
< ht t p://e x pre s s aoserg ipa n a .c om.br/10 -ben s- de-serg i-
pe-que-orgulham-o-povo-sergipano/>. Acessado em: 20 jul. 2018. Figura 2: Praça São Francisco, São Cristovão - Sergipe Fonte:
< ht t p://e x pre s s aoserg ipa n a .c om.br/10 -ben s- de-serg i-
pe-que-orgulham-o-povo-sergipano/>. Acessado em: 20 jul. 2018. Diálogos | Volume 05 | 2020
134 | 134 | Educação patrimonial como base de preservação Educação patrimonial como base de preservação Não obstante, o compromisso de preservação não diz respeito apenas
ao ente público, Lemos (1981, p. 29) em seu livro sobre “O que é Patrimônio
Histórico” sugere que preservemos para “garantir a compreensão de nossa
memória social preservando o que for significativo dentro de nosso vasto
repertório de elementos componentes do Patrimônio Cultural”. E acrescenta Assim, preservar não é só guardar uma coisa, um objeto,
uma construção, um miolo histórico de uma grande cidade
velha. Preservar também é gravar depoimentos, sons, músicas
populares e eruditas. Preservar é manter vivos, mesmo que
alterados, usos e costumes populares. È fazer, também,
levantamentos, levantamentos de qualquer natureza, de sítios
variados, de cidades, de bairros, de quarteirões significativos
dentro do contexto urbano. É fazer levantamentos de
construções, especialmente aquelas sabidamente da especulação
imobiliária. (Lemos, 1981, p. 29). Portanto, uma vez ampliada à concepção de patrimônio através das
possibilidades de preservação a partir da nova Constituição de 1988 resta o
desafio de estabelecer uma nova política e consciência de como preservar, um
caminho apontado é a participação da comunidade na tomada de decisões com
relação à preservação do patrimônio cultural, sensibilizando-as inicialmente a
fim do inventário participativo, conforme orientado pela cartilha do IPHAN,
esse trabalho possibilita uma identificação, reconhecimento e autovalorização
dos cidadãos como artífices da identidade social. Assim, a construção de uma identidade, ligada ao patrimônio cultural
necessita de um importante cuidado “ao modo como esse objeto tem sido
construído e ideologicamente elaborado por determinados sujeitos sociais, que
têm tido, no Brasil, o monopólio dessa construção” (Fonseca, 2005, p. 28). Fonseca ainda considera o valor do patrimônio nacional, mas tem se
tornado pesado e mudo: Pesado, não só por sua monumentalidade, pela solidez dos
materiais e pelo lugar que ocupa no espaço público. Pesado
porque mudo, na medida em que, ao funcionar apenas como
símbolo abstrato e distante da nacionalidade, em que um grupo
muito reduzido se reconhece, e referido a valores estranhos ao
imaginário da grande maioria da população brasileira, o ônus de
sua proteção e conservação acaba sendo considerado como um
fardo por mentes mais pragmáticas. (Fonseca, 2005, pp. 26-27). Pesado, não só por sua monumentalidade, pela solidez dos
materiais e pelo lugar que ocupa no espaço público. Educação patrimonial como base de preservação Pesado
porque mudo, na medida em que, ao funcionar apenas como
símbolo abstrato e distante da nacionalidade, em que um grupo
muito reduzido se reconhece, e referido a valores estranhos ao
imaginário da grande maioria da população brasileira, o ônus de
sua proteção e conservação acaba sendo considerado como um
fardo por mentes mais pragmáticas. (Fonseca, 2005, pp. 26-27). | 135 Diálogos | Volume 05 | 2020 Diálogos | Volume 05 | 2020 Douglas Lima da Costa Douglas Lima da Costa Neste contexto surge a Educação Patrimonial como um instrumento
pedagógico eficaz e capaz de efetivar a identificação, a valorização e a preser-
vação do Patrimônio Histórico e Cultural dos lugares de memória. Assunção (2003, p. 55) entende que “o patrimônio cultural constitui
uma herança histórica, deixada pelas gerações anteriores, que cabe a todos
preservar para que seja transmitida às gerações vindouras”. A autora acredita
que por aproximar o indivíduo com o patrimônio, a Educação Patrimonial Contribui para a formação de um cidadão consciente dos seus
direitos e deveres, que compreenderá a importância da preservação
dos bens culturais para a preservação da memória e da identidade
de um povo ou nação e da necessidade da ação de proteger e
escolher seus bens patrimoniais. (Assunção, 2003, p. 51). Segundo Horta (1999) a proposta de Educação Patrimonial surgiu efe-
tivamente e foi organizada em termos conceituais e práticos no Brasil, a partir
de 1983, quando no Museu Imperial de Petrópolis, RJ, ocorreu o I Seminário
de Educação Patrimonial. Para a autora, a Educação Patrimonial, além de ser
uma meta da educação contemporânea e dos órgãos responsáveis pela preser-
vação patrimonial, torna-se, [...] um instrumento de “alfabetização cultural” que possibilita
ao indivíduo fazer a leitura do mundo que o rodeia, levando-o a
compreensão do universo sóciocultural e da trajetória histórico-
temporal em que está inserido. Este processo leva ao reforço
da auto-estima dos indivíduos e comunidades e à valorização
da cultura brasileira, compreendida como múltipla e plural. (Horta, 1999, p. 6). Neste viés, o IPHAN elaborou um Guia Básico de Educação Patrimonial,
que auxilia todas as unidades do país no desenvolvimento de atividades que
contribuem com o trabalho educacional em museus, monumentos e sítios
históricos, trabalhando com conceitos ligados ao patrimônio além de sugerir
metodologias para o desenvolvimento das mesmas. As bases conceituais e práticas da metodologia da Educação
Patrimonial objetiva orientar, em especial os professores e profissionais
responsáveis pela preservação da cultura, em como preservar os bens cul-
turais e do meio ambiente histórico em que estão inseridos considerando
a Educação Patrimonial como “um processo permanente e sistemático de 136 | Diálogos | Volume 05 | 2020 Educação patrimonial como base de preservação Educação patrimonial como base de preservação trabalho educacional” (Horta, 1999, p. 6), a partir da observação direta
dos bens culturais. Este é o ponto que dá centralidade a todo o trabalho de
Educação Patrimonial sugerido pelo IPHAN. Figura 3: Slogan
IPHAN – principal autarq
sobre patrimônio no Brasil. Fonte: <http://infon
com.br/noticias/livrarias-po
rao-vender-livros-do-iphan
Acessado em: 20 jul. 20 Figura 3: Slogan do
IPHAN – principal autarquia
sobre patrimônio no Brasil. Figura 3: Slogan do
IPHAN – principal autarquia
sobre patrimônio no Brasil. Fonte: <http://infonet. com.br/noticias/livrarias-pode-
rao-vender-livros-do-iphan/>. Acessado em: 20 jul. 2018. Fonte: <http://infonet. com.br/noticias/livrarias-pode-
rao-vender-livros-do-iphan/>. Acessado em: 20 jul. 2018. A partir da concepção de que todo e qualquer objeto cultural é uma fonte
primária de conhecimento, o IPHAN estruturou uma metodologia específica que
possibilita a percepção e a compreensão dos fatos e dos fenômenos sociais estudados
para que os professores se apropriem dos objetos culturais “na sala de aula ou nos
próprios locais onde são encontrados, como peças chave no desenvolvimento dos
currículos e não simplesmente como mera ilustração das aulas” (Horta, 1999, p. 9). Assim, o Patrimônio Cultural da comunidade ganha vida através da
valorização da história do seu espaço além de provocar nos alunos sentimentos
de curiosidade essencial para que adquiram habilidades e conceitos relaciona-
dos ao Patrimônio Cultural. Essa aproximação, segundo Assunção (2003, p. 51) desperta quatro atitudes: a observação e a reflexão sobre o bem cultural;
a manifestação das impressões sobre o mesmo; a capacidade de pesquisa e
discussão sobre os resultados e a apropriação de um novo significado do bem
para cada um que participa da proposta. Assunção (2003, p. 51) entende que os projetos de Educação Patrimonial
desenvolvidos nas comunidades devam procurar “envolver o indivíduo e a
comunidade como agentes ativos, na preservação sustentável e na gestão do
patrimônio cultural”. Assim, é indispensável que a comunidade participe de
todas as etapas do processo de seu amadurecimento cultural na área da preser-
vação de seus bens patrimoniais. Diálogos | Volume 05 | 2020 | 137 Educação patrimonial como base de preservação Portanto, o Patrimônio Cultural deve ser visto como um conjunto de
coisas da invenção do homem para o próprio homem sejam elas para facilitar
a vivência em grupo e presença nos espaços. É ainda, resultante de construções
da coletividade através dos tempos e um determinado espaço, esse vínculo
direto com a memória e a construção de uma identidade deve ser tratada
como material a ser preservado. Para isso, a educação patrimonial cumpre o
papel aproximador e sensibilizador. Em um movimento de continuidade, a
preservação e a conservação dependem da participação de todas as pessoas,
independente do grupo social e do papel que exerçam. Assim sendo, enxergar-se nos elementos compostos do passado e no
passado, gerará no indivíduo o senso de comprometimento em olhar o preté-
rito como elo de compreeensão do presente e respeito para com a história e o
povo que a constrói. Douglas Lima da Costa Douglas Lima da Costa A utilização da metodologia do Guia Básico de Educação Patrimonial
sugerida pelo IPHAN nos programas curriculares do sistema educacional
contribue para o diálogo e enriquecimento de dois segmentos que quase nun-
ca estão conectados: os órgãos responsáveis pela preservação do patrimônio
histórico e cultural e as comunidades que o produziram. O patrimônio cultural tem que ser considerado um instrumento
valioso das experiências humanas, mas a participação popular
não pode mais ser ignorada pelo poder público. Aos indivíduos
que são excluídos desse processo, a educação patrimonial deve
ser uma prática de participação social. E essas práticas políticas
que envolvem a experiência da sociabilidade, exigem de todos
os envolvidos uma compreensão do uso social do espaço de
convívio entre os indivíduos que compõem àquela comunidade. (Certeau, 1996, p. 233). Figura 4: Cartilha
de Educação Patrimonial Desse modo, seria relevante que a
educação patrimonial estivesse presente
nos currículos escolares, nas ações edu-
cacionais, nos mais variados espaços e
de outras instituições e atividades de
educação informal, de forma interdis-
ciplinar. Fonte: < https://issuu.com/letiz/
docs/educa_o_web>. Acessado em:
20 jul. 2018. Assim, é necessário entender o pa-
pel que o Patrimônio Histórico Cultural
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identidade de cada indivíduo e da coleti-
vidade que constrói num mesmo local a
sua história. Este é também um desafio
que a escola deveria imprimir. Dessa forma, os bens culturais
adquiririam a conotação de bem públi-
co, ou seja, que pertence ao povo, e não
ao Estado ou a uma restrita parcela da
sociedade, e a preservação ganharia um
novo olhar da comunidade. Fonte: < https://issuu.com/letiz/
docs/educa_o_web>. Acessado em:
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nio imaterial de Iguatu, Andaraí, BA. Passos Revista de Turismo y Patrimonio
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a proteção do patrimônio histórico e artístico nacional. Disponível
em: http://www.planalto.gov.br/ccivil_03/Decreto-Lei/Del0025.htm. Acesso em: 23 jul. 2018. Brasil. (1988). Constituição da República Federativa do Brasil de 1988. Disponível
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Acesso em: 23 jul. 2018. Brasil. (1990). Lei nº 8.028 de 12 de abril de 1990. Dispõe sobre a organiza- Diálogos | Volume 05 | 2020 | 139 Douglas Lima da Costa ção da Presidência da República e dos Ministérios, e dá outras providências. Disponível: http://www.planalto.gov.br/ccivil_03/leis/l8028.htm Acesso em:
19 maio. 2020. ção da Presidência da República e dos Ministérios, e dá outras providências. Disponível: http://www.planalto.gov.br/ccivil_03/leis/l8028.htm Acesso em:
19 maio. 2020. Brasil. (2019). Decreto nº 9.674, de 2 de janeiro de 2019. Disponível:
http://www.planalto.gov.br/ccivil_03/_ato2019-2022/2019/decreto/D9674.htm
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grafias. Revista Memória em Rede, Pelotas, v. 2, n. 3, ago.-nov. 2010. Disponível
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tidade e representação). Bauru: EDUSC. Fonseca, M. C. L. (2005). O patrimônio em processo. Rio de Janeiro: UFRJ /
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espaço e visualidade na pós-modernidade. Papirus Editora. Disponível em:
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do Patrimônio Histórico e Artístico Nacional, Museu Imperial. Langer, J. (1997). Mitos arqueológicos e poder. Clio – Série Arqueológica
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Paulo: Annablume.
Silveira, F. L. A., & Lima Filho M. F. (2005). Por uma antropologia do
objeto documental: entre a “alma nas coisas” e a coisificação do objeto.
Horizontes Antropológicos. Porto Alegre, ano 11, n. 23, jan/jun, pp. 37-50.
https://doi.org/10.1590/S0104-71832005000100003 reFerênciAS A. C. (1981). O que é patrimônio histórico. São Paulo: Editora
Brasiliense S.A. Diálogos | Volume 05 | 2020 140 | Educação patrimonial como base de preservação Diálogos | Volume 05 | 2020
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DADOS TÉCNICOS E EDITORIAIS
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Revista da ANPOLL
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n. 32, p. 1-208, Brasília-DF, Jan./ Jul. 2012 ISSN 1414-7564
Revista da 32
Linguística: linguagem, história e acontecimento
Associação Nacional de Pós-Graduação
e Pesquisa em Letras e Linguística
Associaçao Nacional de Pós-Graduação e Pesquisa em Letras e Linguística n. 32, p. 1-208, Brasília-DF, Jan./ Jul. 2012 ISSN 1414-7564 Revista da Reitor: Roberto de Souza Salles
Vice-Reitor: Sidney Luiz de Matos Mello
Universidade Federal Fluminense Reitor: Roberto de Souza Salles
Vice-Reitor: Sidney Luiz de Matos Mello Comissão Editorial
Ida Maria Santos Ferreira Alves (UFF - 2010/12)
Mariangela Rios de Oliveira (UFF - 2010/12)
Maria Antonieta Jordão de Oliveira Borba (UERJ - 2010/12)
Lucia Teixeira (UFF - 2010/12) Audemaro Goulart (PUC/MG - 2008/10)
Fábio Alves (UFMG - 2008/10)
André Luís Gomes (UnB - 2006/08)
Maria Cristina Faria Dalacorte (UFG - 2006/08) Conselho Editorial
Almuth Grésillon (ITEM - França)
Aryon Dall’ Igna Rodrigues (UnB)
Carlos Reis (Univ. de Coimbra - Portugal)
Diana Luz Pessoa de Barros (MACKENZIE - USP)
Edna Maria Fernandes do Nascimento (UNIFRAN)
Eduardo Guimarães (UNICAMP)
Eni Pulcinelli Orlandi (UNICAMP - Univ. do Vale do Sapucaí)
Fernando Cristóvão (Univ. de Lisboa - Portugal)
Freda Indursky (UFRGS)
Francisco da Silva Borba (UNESP - CNPq)
Francisco Gomes de Matos (UFPE) Jacqueline-Authier-Revuz (Univ. Paris II/CNRS - França)
John Gledson (Univ. de Liverpool - Inglaterra)
Laura Padilha (UFF)
Leci Barbisan (PURS)
Margarida T. Petter (USP)
Maria Célia Leonel (UNESP - Araraquara)
Milton Azevedo (Univ. de Berkeley - EUA)
Philippe Léon M. G. Willemar (NAP/USP - SP)
Pierre Rivas (Univ. de Paris 10 - França)
Sylvain Aurox (CNRS - França)
Teresa Cristina Cerdeira da Silva (UFRJ) Conselho da ANPOLL
Estudos Literários:
Titulares: José Luiz Jobim de Salles Fonseca (UERJ - 2010/14), Eurídice Figueiredo (UFF - 2010/14),
Rogério da Silva Lima (UnB - 2008/12), Ivete Lara Camargo Walty (PUC/MG - 2008/12)
Suplentes: Luiz Roberto Velloso Cairo (UFRJ - 2010/14), Antônia Torreão Herrero (UFBA - 2010/14),
Maria Zaira Turchi (UFG - 2008/12), Lúcia Rebello (UFRGS - 2008/12) Endereço Editorial:
Universidade Federal Fluminense;
Instituto de Letras - Bloco C;
Campus Gragoatá - São Domingos
Niterói - RJ; CEP 24.210-200. Proibida a reprodução parcial ou integral desta obra, por qualquer meio eletrônico ou mecânico, inclusive por processo xerográfico, ou sem permissão do editor
(Lei n. 9.610 de 19/2/1998). Os conceitos emitidos em artigos assinados são de absoluta e exclusiva responsabilidade de seus autores. Associação Nacional de Pós-Graduação
e Pesquisa em Letras e Linguística Associação Nacional de Pós-Graduação
e Pesquisa em Letras e Linguística CIP-BRASIL. CATALOGAÇÃO-NA-FONTE
SINDICATO NACIONAL DOS EDITORES DE LIVROS, RJ CIP-BRASIL. CATALOGAÇÃO-NA-FONTE
SINDICATO NACIONAL DOS EDITORES DE LIVROS, RJ R349
Revista da ANPOLL / Associação Nacional de Pós-Graduação e Pesquisa em Letras e Linguística. - 1(1994)- . -
- Brasília, DF : ANPOLL, 1994-. v. :
Semestral
Descrição baseada no: v. 18, N.32 (2012)
Tema especial: Linguística: linguagem, história e acontecimento
Organizador: André Luís Gomes
ISSN 1414-7564
1. Linguística. 2. Linguagem e línguas. 3. Linguística histórica. 4. Linguística - Periódicos. I. Gomes, André
Luís, 1963-. II. Associação Nacional de Pós-Graduação e Pesquisa em Letras e Linguística (Brasil). III. Título:
Linguística: linguagem, história e acontecimento. 12-3743. CDD: 410
CDU: 81’1
06.06.12
15.06.12
036125 R349
Revista da ANPOLL / Associação Nacional de Pós-Graduação e Pesquisa em Letras e Linguística. - 1(1994)- . -. - Brasília, DF : ANPOLL, 1994-. v. : Semestral
Descrição baseada no: v. 18, N.32 (2012)
Tema especial: Linguística: linguagem, história e acontecimento
Organizador: André Luís Gomes
ISSN 1414-7564 Descrição baseada no: v. 18, N.32 (2012)
Tema especial: Linguística: linguagem, história e acontecimento
Organizador: André Luís Gomes
ISSN 1414-7564
1. Linguística. 2. Linguagem e línguas. 3. Linguística histórica. 4. Linguística - Periódicos. I. Gomes, André
Luís, 1963-. II. Associação Nacional de Pós-Graduação e Pesquisa em Letras e Linguística (Brasil). III. Título:
Linguística: linguagem, história e acontecimento. 12-3743. CDD: 410
CDU: 81’1
06.06.12
15.06.12
036125 Organização deste número: Mariangela Rios de Oliveira e André Luís Gomes
Pareceristas deste número:
Beatriz Feres dos Santos: beatrizferes@yahoo.com.br, Bethania Sampaio Mariani: bmariani@uol.com.br,
Décio Rocha: rochadm@uol.com.br, Edila Vianna: edila@openlink.com.br, Eduardo Kenedy: edu.kenedy@gmail.com,
Fernando Afonso de Almeida: fealfal@gmail.com, Fernando Muniz: fernandomuniz@id.uff.br,
Jussara Abraçado: almeidajussara@hotmail.com, Lívia Lindóia Paes Barreto: liviapaesbarreto@yahoo.com.br,
Lygia Gonçalves Trouche: lymt@terra.com.br, Maria Del Carmen Daher: deldaher@terra.com.br,
Maria Elizabeth Chaves de Mello: bethcmello@gmail.com, Maurício Beck: pardalbeck@gmail.com,
Mônica Savedra: msavedra@faperj.br, Patrícia Ferreira Ribeiro: patleitura@gmail.com,
Paulo Antonio Pinheiro Correa: papicorrea@gmail.com, Renata Mancini: renata.mancini@gmail.com,
Ricardo Stavola Cavaliere: cavaliere@oi.com.br, Rosane Santos Mauro Monnerat: rosanemmonnerat@gmail.com,
Silmara Cristina Dela da Silva: silmaradela@gmail.com, Silvia Maria de Sousa: smsousa2002@yahoo.com.br,
Solange Coelho Vereza: svereza@uol.com.br, Vanda Maria Cardozo de Menezes: vcmenezes@terra.com.br,
Victoria Wilson: victoriawilson@superig.com.br, Xoán Lagares Diez: xlagares@gmail.com
Projeto gráfico e capa: Grupodesign
Revisão: Ana Cristina Vilela
Produção gráfica: Sermograf
Todos os direitos reservados
Universidade Federal Fluminense
Instituto de Letras
Campus Gragoatá - São Domingos - Niterói - RJ
CEP: 24.210-200
Fone: +55 (21) 2629-2601
egl@vm.uff.br Organização deste número: Mariangela Rios de Oliveira e André Luís Gomes
Pareceristas deste número:
Beatriz Feres dos Santos: beatrizferes@yahoo.com.br, Bethania Sampaio Mariani: bmariani@uol.com.br,
Décio Rocha: rochadm@uol.com.br, Edila Vianna: edila@openlink.com.br, Eduardo Kenedy: edu.kenedy@gmail.com,
Fernando Afonso de Almeida: fealfal@gmail.com, Fernando Muniz: fernandomuniz@id.uff.br,
Jussara Abraçado: almeidajussara@hotmail.com, Lívia Lindóia Paes Barreto: liviapaesbarreto@yahoo.com.br,
Lygia Gonçalves Trouche: lymt@terra.com.br, Maria Del Carmen Daher: deldaher@terra.com.br,
Maria Elizabeth Chaves de Mello: bethcmello@gmail.com, Maurício Beck: pardalbeck@gmail.com,
Mônica Savedra: msavedra@faperj.br, Patrícia Ferreira Ribeiro: patleitura@gmail.com,
Paulo Antonio Pinheiro Correa: papicorrea@gmail.com, Renata Mancini: renata.mancini@gmail.com,
Ricardo Stavola Cavaliere: cavaliere@oi.com.br, Rosane Santos Mauro Monnerat: rosanemmonnerat@gmail.com,
Silmara Cristina Dela da Silva: silmaradela@gmail.com, Silvia Maria de Sousa: smsousa2002@yahoo.com.br,
Solange Coelho Vereza: svereza@uol.com.br, Vanda Maria Cardozo de Menezes: vcmenezes@terra.com.br,
Victoria Wilson: victoriawilson@superig.com.br, Xoán Lagares Diez: xlagares@gmail.com
Projeto gráfico e capa: Grupodesign
Revisão: Ana Cristina Vilela
Produção gráfica: Sermograf
Todos os direitos reservados
Universidade Federal Fluminense
Instituto de Letras
Campus Gragoatá - São Domingos - Niterói - RJ
CEP: 24.210-200
Fone: +55 (21) 2629-2601
egl@vm.uff.br
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https://openalex.org/W3129646928
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https://link.springer.com/content/pdf/10.1007/s00265-021-02996-4.pdf
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Individual behavioural traits not social context affects learning about novel objects in archerfish
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Behavioral ecology and sociobiology
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https://doi.org/10.1007/s00265-021-02996-4
Behavioral Ecology and Sociobiology (2021) 75: 58 https://doi.org/10.1007/s00265-021-02996-4
Behavioral Ecology and Sociobiology (2021) 75: 58 ORIGINAL ARTICLE Abstract Learning can enable rapid behavioural responses to changing conditions but can depend on the social context and behavioural
phenotype of the individual. Learning rates have been linked to consistent individual differences in behavioural traits, especially
in situations which require engaging with novelty, but the social environment can also play an important role. The presence of
others can modulate the effects of individual behavioural traits and afford access to social information that can reduce the need for
‘risky’ asocial learning. Most studies of social effects on learning are focused on more social species; however, such factors can
be important even for less-social animals, including non-grouping or facultatively social species which may still derive benefit
from social conditions. Using archerfish, Toxotes chatareus, which exhibit high levels of intra-specific competition and do not
show a strong preference for grouping, we explored the effect of social contexts on learning. Individually housed fish were
assayed in an ‘open-field’ test and then trained to criterion in a task where fish learnt to shoot a novel cue for a food reward—with
a conspecific neighbour visible either during training, outside of training or never (full, partial or no visible presence). Time to
learn to shoot the novel cue differed across individuals but not across social context. This suggests that social context does not
have a strong effect on learning in this non-obligatory social species; instead, it further highlights the importance that inter-
individual variation in behavioural traits can have on learning. Significance statement Some individuals learn faster than others. Many factors can affect an animal’s learning rate—for example, its behav-
ioural phenotype may make it more or less likely to engage with novel objects. The social environment can play a big
role too—affecting learning directly and modifying the effects of an individual’s traits. Effects of social context on
learning mostly come from highly social species, but recent research has focused on less-social animals. Archerfish
display high intra-specific competition, and our study suggests that social context has no strong effect on their learning
to shoot novel objects for rewards. Our results may have some relevance for social enrichment and welfare of this
increasingly studied species, suggesting there are no negative effects of short- to medium-term isolation of this species—
at least with regards to behavioural performance and learning tasks. Keywords Social context . Inter-individual differences . Social learning . Toxotes . Exploratory tendency . Learning ndividual behavioural traits not social context affects learning
about novel objects in archerfish Nick A. R. Jones1
& Helen C. Spence-Jones1
& Mike Webster1
& Luke Rendell1 Received: 25 November 2020 /Revised: 9 February 2021 /Accepted: 12 February 2021
# The Author(s) 2021
/ Published online: 22 February 20 1
Centre for Social Learning and Cognitive Evolution, School of
Biology, University of St Andrews, Sir Harold Mitchell Building, St
Andrews, Fife KY16 9TH, UK * Nick A. R. Jones
narj@st-andrews.ac.uk Introduction 2016; Pearish et al. 2019; Tucker and Suski 2019); however,
the importance of social context may depend on the species. Studies exploring the effects of social context on learning
have focused on relatively few highly social species, and there
is current debate about the evolution of social learning on
social and non-social species (Heyes 2012; Reader 2016). We were interested in exploring whether social context would
facilitate learning in a species not generally considered social
but capable of receiving social information, and whether this
could overcome pre-existing variation in relevant traits such
as exploratory tendency. Specifically, we tested the effect of
different social context on the rate of learning an association
between shooting novel objects and obtaining a food reward
in archerfish, Toxotes chatareus. Famous for their ability to
‘shoot’ down prey (Bekoff and Dorr 1976; Dill 1977; Schuster
2007), archerfish exhibit low sociality with no grouping pref-
erence (Timmermans and Maris 2000), possibly linked to their
highly competitive foraging behaviour (Rischawy et al. 2015)
and tendency for intra-specific kleptoparasitism (Davis and
Dill 2012). Previous work has suggested that they use social
information (Schuster et al. 2006) and alter their shooting
behaviour in response to a conspecific in a neighbouring tank
(Jones et al. 2018). However, little else is known about their
social behaviour and social effects on learning. Most research
has focused on understanding the mechanisms underlying
their shooting abilities (Timmermans and Vossen 2000;
Gerullis and Schuster 2014; Reinel and Schuster 2018) and
related abilities (Ben-Tov et al. 2018; Newport and Schuster
2020) including image search (Temple et al. 2010; Rischawy
and Schuster 2013; Ben-Tov et al. 2015) and associated neu-
robiology (Schlegel and Schuster 2008; Ben-Simon et al. 2012; Machnik et al. 2018a, b). Archerfish have been shown
to exhibit consistent individual differences in latency to
shoot—with consequences for their performance in learning
and discrimination trials (Jones et al. 2020): fish with low
latencies were quicker to learn to shoot targets for reward. The effects of these individual differences have only been
explored in fish in solitary conditions, and no social element
has been tested. Our main question in this study was whether
archerfish learn to shoot a novel object faster when housed in
solitary conditions or with a visible conspecific. We included
a variation of the open-field assay to determine whether indi-
vidual variation in learning rates was also associated with
individual differences in exploratory tendency. Introduction Communicated by I. Hamilton Learning enables rapid responses to change (Shettleworth
1972, 2009), but in order to learn about a novel object, an
animal must first engage with it. Exploring a potential new
food source can provide obvious foraging benefits but may
expose an individual to potential risk from predation or nox-
ious substances (Lima and Dill 1990). The social environment * Nick A. R. Jones
narj@st-andrews.ac.uk 58 Page 2 of 11 Behav Ecol Sociobiol (2021) 75: 58 can play a large role in an individual’s motivation to engage
with novelty and subsequent learning opportunities (Boogert
et al. 2006; Mirville et al. 2016; Langley et al. 2018; Kent et al. 2019; Hansen et al. 2020). Individuals can be more likely to
approach novel foods with conspecifics present (Gómez-
Laplaza and Morgan 1986; Sherwin 2003). For example, ca-
puchin monkeys (Cebus apella) in social conditions had
higher levels of engagement with and consumption of novel
foods than solitary individuals (Visalberghi and Fragaszy
1995). Being in a group also affords learning opportunities
through social learning mechanisms (Coussi-Korbel and
Fragaszy 1995; Hoppitt and Laland 2013; Ward and
Webster 2016) such as social facilitation (Zentall and Hogan
1976; Dindo et al. 2009). The social environment can also
enhance learning effects through the ‘social buffering of
stress’ (Smith et al. 1998; DeVries et al. 2003; Allen et al. 2009; Crane et al. 2018; Culbert et al. 2019), for example
cichlids (Cichlasoma paranaense) in groups showed lower
stress and faster learning rates than isolated individuals
(Brandão et al. 2015). These social benefits are not limited
to obligate social species. The solitary octopus (Octopus
vulgaris) (Fiorito and Scotto 1992) and several non-
grouping species of fish (Webster and Laland 2017), includ-
ing solitary sharks (Vila Pouca and Brown 2019; Vila Pouca
et al. 2020), use social learning. However, the effects of the
social environment on learning can be complex. Individuals exhibiting specific ‘coping styles’ (Koolhaas et al. 1999) can perform differently in cognition studies (Zidar et al. 2017; Mazza et al. 2019). Several studies have shown that
‘proactive’ individuals, with low cortisol levels and high levels
of exploratory activity, learn faster than ‘reactive’ individuals
which tend to take longer to approach novel objects (Mesquita
et al. 2015; Bensky et al. 2017; Raoult et al. 2017). These
individual differences in behavioural traits can be moderated
by social environment (Webster and Ward 2011; Jolles et al. Subjects We tested 19 archerfish, T. chatareus, 12 ± 0.5 cm (total
length), from animals housed at the University of St. Andrews. All fish were wild-caught as juveniles from a fresh-
water population, sourced from an accredited ornamental fish
supplier. Fish were of unknown sex as archerfish are sexually
monomorphic. The fish had been kept in their stock tanks for a
minimum of 6 months prior to use in the study and had not
been trained to shoot or exposed to experimental tanks in that
time. Stock tanks (180 × 45 cm and 35 cm deep) held at
temperatures between 25 and 26°C under a 12:12 h light:dark
cycle, had environmental enrichment in the form of 3-cm-
deep gravel substrate and several plastic plants. Water quality
parameters (pH, nitrite, ammonia and nitrate concentrations)
were measured weekly, and levels were kept within a range
appropriate for archerfish (Newport et al. 2013; Jones et al. 2018), using external canister filters and regular water
changes. Each of four stock tanks held between 5 and 8 fish, with a
total of 27 fish. We aimed to test groups of familiar fish so we
selected 21 fish for the experiment to ensure all groups of fish
came from the same stock tank. At the time of the experiment,
the fish were estimated to be 18–24 months old, based on their
size and date of arrival from the retailer; however, this is an
estimate as the correlation between age and growth rate in
captivity is not known for this species. The fish were fed daily
with an alternating mixture of commercial fish food (Tetra
Cichlid Sticks) and freeze-dried bloodworms. When moving
fish into the experimental setup, fish were always captured
from the same tank. Fish were measured as they were captured
from the stock tank—using a ruler taped to the side of the tank,
netted fish were gently pressed against the side of the tank to
record their length before being moved to the experimental
tank. All the fish that were tested were used only once in this
experiment. General procedure Fish were tested in groups of three; each fish in a tested trio
came from the same stock tank and had been kept together for
at least 6 months prior to the experiment to ensure they were
familiar with each other. Each fish was moved from the stock
tank and introduced into one of three experimental tanks
which had been randomly assigned to one of the social treat-
ments. Each fish was then given a full day to acclimate to the
experimental tank. After 24 h, the video recordings were taken
for the behavioural assay. At the time of introduction and until
after the behavioural assay, all barriers were kept in place such
that all fish were in the same solitary conditions with no other
fish visible to them. Introduction Learning performance can depend on the social dynamics
(Nicol and Pope 1999; Soma and Hasegawa 2004; Boogert
et al. 2006; Grampp et al. 2019), behavioural traits (An et al. 2011; González-Bernal et al. 2014; Krueger et al. 2014), sex
(Snijders et al. 2019) and level of familiarity (Guillette et al. 2016) of the individuals involved. In some species, individ-
uals show no benefit from the presence of others when learn-
ing, for example the facultatively social tree skinks, Egernia
striolata, (Riley et al. 2017, 2018). The presence of conspe-
cifics can even worsen learning performance (Zajonc et al. 1969; Webster and Laland 2012; Fei et al. 2019). Notwithstanding social factors, individual variation can also
have significant effects on learning. Many of the commonly
measured behavioural traits and syndromes relate to explorato-
ry tendency and associated neophobia (aversion to novelty
(Greenberg and Mettke-Hofmann 2001; Mettke-Hofmann
2017)), and these traits can affect learning, as well as cognitive
performance more generally (Sih and Del Giudice 2012;
Greggor et al. 2015; Morand-Ferron et al. 2016; Boogert
et al. 2018; Dougherty and Guillette 2018). Several studies
have shown that individuals which exhibit consistently high
levels of exploratory tendency tend to show lower levels of
neophobia and engage with/learn-to-associate cues with food
rewards more quickly than individuals with low activity levels
(An et al. 2011; Guenther et al. 2014; Guillette et al. 2015;
Reader 2015; Mazza et al. 2018). Differences in response to
stress and physiology may drive variation in learning. Behav Ecol Sociobiol (2021) 75: 58 Page 3 of 11 58 Page 3 of 11 58 Experimental setup Experimental tanks (55 × 55 cm and 45 cm deep) were each
set up with an immersion heater (kept at 24.5 ± 0.5°C), a small
internal filter (Eheim Biopower 200 Internal Filter), a 1-cm-
deep gravel bottom, and plastic plants (positioned to provide
structure and refuge but allow a clear view of neighbouring
tanks). We used two replicate setups of three tanks each to run
up two groups of fish at a time. In each setup, three tanks were
set up side by side with a 0.5-cm gap between them, using the
same setup as in Jones et al. (2018) (see Fig. 1). A 3-mm-thick
black opaque plastic barrier could be easily inserted between
each tank to block or allow vision between tanks. The barriers
were used to create different social conditions detailed below,
by controlling the visibility of the side tanks. Methods Between 46 and 48 h after being moved into experimental
tanks, the barriers were re-positioned in accordance with the
social treatment assigned to each tank. Fish were then given
another period of ~24 h acclimation to the conditions of their
respective social treatment before training began. During the
training period, the fish were trained to learn to shoot at a
novel target to obtain a food reward. Archerfish will readily
shoot novel targets and can learn to associate shooting a target
with receiving food rewards (Newport et al. 2013; Karoubi
et al. 2017). Each fish was given a single training session
per day until it reached the training criterion: 10 on-target
shots a day for two consecutive days. In each session, an
initially novel target (a five-armed star-shaped cut from black
plastic, max diameter 2cm) was placed on the glass 15 cm
above the water level of the tank. A training session lasted
45 min, or until a fish had shot at the target 10 times. Shots
were easily visible as they left water droplets on the underside
of the transparent platform on which the target was placed,
signalling where the shot had impacted. A shot was consid-
ered on-target if it was within 2 cm of the target; successful
shots were rewarded with food, and the target was removed
and repositioned on the glass for a subsequent trial. We con-
tinued training for all fish in each set until all three of the fish
had reached the criterion or until the 50th session. Fish were
given a maximum of 50 sessions to reach the criterion. Behavioural assay We scored fish in their experimental tanks using a simple
‘open-field’ assay. The ‘open-field test’ exposes animals to a
novel area and allows measurements such as amount of activ-
ity and time spent in the open as a measure of exploratory
behaviour, where animals that spend more time in the open
are considered to have greater exploratory behaviour (Burns
2008). This assay is widely used in fishes (Webster et al. 2007;
Chapman et al. 2011; Conrad et al. 2011; Lucon-Xiccato et al. 2020). For this assay, each fish was recorded for 10 min, and 58 Page 4 of 11 Behav Ecol Sociobiol (2021) 75: 58 Fig. 1 Experimental set-up, from a top-down view as recorded by the camera above the tanks. The different experimental treatments and level of social
context was manipulated by use of the opaque barriers. Solo treatments were always set up in one of the two end tanks Fig. 1 Experimental set-up, from a top-down view as recorded by the camera above the tanks. The different experimental treatments and level of social
context was manipulated by use of the opaque barriers. Solo treatments were always set up in one of the two end tanks ‘partial’ treatment, fish had partial social exposure: they were
able to see a neighbouring fish when given opportunities to
shoot the presented target. However, they could not observe
when the neighbour was being trained. Fish in the partial
treatment were therefore not exposed to social information
about the presented target and associated food reward by other
fish, but they may have been impacted by the presence of
another fish. This presence could have had either positive
impacts on learning rates through social buffering of stress,
or, more likely, negative impacts due to the presence of po-
tential competitors per (Simon et al. 2011; Jones et al. 2018). Fish in the ‘social’ treatment were exposed to full social con-
ditions and had visible contact with a neighbouring fish at all
times (including during the neighbour’s training sessions),
except when presented with the target itself. A visible neigh-
bour fish may act as a potential distraction when shooting, as
shown by Jones et al. (2018), so the social condition was
designed to afford social information about the target while
reducing the chance of distraction. The treatments were
pseudo-randomly assigned to each tank prior to transferring
fish into them. Behavioural assay For each trio of fish tested, the solo treatment
was randomly assigned to one of the two end tanks: the social
and partial treatments were then randomly allocated a tank
from the two remaining. the proportion of time spent in the open, where the fish was
more than 1 body length away from the plant shelter or corners
of the tank, was recorded. We used a 24-h acclimation period
prior to conducting the assay, informed by our previous work
with this species in a similar setup (Jones et al. 2018); while a
single measurement per individual is not ideal for a labile trait
(Biro and Stamps 2010), suitable acclimation periods can pro-
vide more robust estimates of individual behavioural traits
(Biro 2012), as demonstrated by high estimates of repeatabil-
ity in guppy activity levels following acclimation (O’Neill
et al. 2018). To minimize observer bias, blinded methods were
used; specifically, a hypothesis naïve scorer was used to score
videos of the behavioural assays. We initially planned on testing 21 fish, but the social con-
text of two fish (in a solo and partial treatment) may have been
compromised as there was a gap in the barrier between the two
tanks for at least one session. We noticed this on day 4 of
testing this group. These two fish were excluded from the data
and were not tested further except to provide a social context
for the fish in the social treatment. Of the remaining 19 fish
tested, one—in the social treatment—was unable to hit the
target or shoot within 2 cm of it. This fish showed no obvious
injury or morphological difference from other fish but was
unable to reach training criterion for this reason. All other fish
were able to shoot and hit the target. Experimental treatments (social context) All analysis was conducted using R base package (R Core
Team 2019) and specific packages as detailed below. The opaque barriers were employed to block visible contact
between fish in neighbouring tanks. Fish in the ‘solo’ treat-
ment were left in isolation, with no visible neighbour. In the In order to determine whether the different treatments affect-
ed learning rates, we conducted a survival analysis using a Cox Behav Ecol Sociobiol (2021) 75: 58 Page 5 of 11 58 were more exploratory) were faster learners, taking fewer ses-
sions on average to reach the criterion. proportional hazards regression model, as per similar studies
exploring time to reach the criterion in learning tasks (Bensky
and Bell 2018). We used this approach as six of the 19 fish
failed to reach criterion in the given 50 sessions. The numbers
of sessions required to reach training criterion were used as the
dependent variable. The social treatment (solo, partial, or so-
cial) and score from the behavioural assay (the proportion of
time spent in the open) were the two independent variables
included in the models. We fitted a model using the coxph
function in the ‘survival’ package in R (Therneau 2019), with
the variables detailed above, where survival equated to failing
to reach the learning criterion. We included an interaction be-
tween the two independent variables, as we expected that the
effect of social treatment may depend on the behavioural traits
of individual fish. This analysis allowed us to explore the ef-
fects of the independent variables of interest on the probability
of reaching learning criterion using all the available data. The link between individual behavioural traits and learning
rates is apparent in an increasing number of species (Dougherty
and Guillette 2018; Smit and van Oers 2019); our results com-
plement earlier findings showing that inter-individual differ-
ences in latency to shoot relate to differences in learning in
archerfish (Jones et al. 2020). Our inability to detect any effect
of social context on learning rates is perhaps more interesting
given that archerfish were affected by the presence of others in a
previous experiment with similar conditions and setup: visible
conspecifics in a neighbouring tank increased the latency to
shoot and behaviour of shooting fish (Jones et al. 2018). Results There was no effect of social context on the probability of
reaching the learning criterion (Table 1; Fig. 2); however,
the probability of an individual reaching criterion did depend
on the proportion of time that the individual spent in the open
in the open-field assay. Time spent in the open during the
initial behavioural assay is significantly associated with time
to learn: greater time spent in the open, our measure of explor-
atory tendency, is associated with increased learning rates
(Table 1; Fig. 3). Experimental treatments (social context) It
certainly raises more questions: is there no effect of social con-
text on learning, or are these effects more subtle and therefore
masked by the individual differences? Our limited sample size
prevents us from being able to address this issue. Effects of
social environment on learning can vary with behavioural phe-
notype where more exploratory individuals tend (relative to less
exploratory individuals) to be less sensitive to external stimuli
(Guillette et al. 2011; Kelly et al. 2020) and derive less benefit
from social environment (Barou Dagues et al. 2020). The lack
of significant effect in our study may therefore be a result of
exploratory fish that are more likely to engage in risk-taking
behaviour but less likely to use social information. Five fish that
did not reach training criterion in the allotted time (but could
shoot), and it is possible that these individuals may have been
more affected by different social contexts. Given the large var-
iation between individuals we found, future studies with a great-
er sample size will be needed to detect any social effects in less
exploratory, potentially more socially sensitive fish, or at least
be more confident that there is no social effect on learning to
shoot a novel target in this species. Our results, however, do
correspond with other studies, across several different species,
which have shown that social context does not always have a
strong effect on learning performance, at least not when there
are strong inter-individual differences in behavioural traits
(Seferta et al. 2001; Krueger et al. 2014; Riley et al. 2017, 2018). Discussion Black lines indicate median number of
sessions to reach criterion for each treatment; coloured dots represent
data points for individual fish. White points indicate fish which failed to
reach criterion within 50 sessions had access to social information regarding the novel objects and
associated food rewards. Black lines indicate median number of
sessions to reach criterion for each treatment; coloured dots represent
data points for individual fish. White points indicate fish which failed to
reach criterion within 50 sessions Fig. 2 Effect of different social contexts on time taken to reach training
criterion. The treatments relate to level of exposure to a visible
conspecific: solo (no social exposure); partial (visual social contact
except during training of neighbours); social (full visual social contact
including during training of neighbours). Only fish with ‘social’ exposure constrain learning (Shettleworth 1972). In addition to showing
no preference for shoaling with conspecifics as juveniles
(Timmermans and Maris 2000), archerfish are highly compet-
itive predators (Rischawy et al. 2015). Intra-specific competi-
tion can be a major driver of individual differences in behav-
iour, with stronger competition potentially leading to more
defined inter-individual differences (Bolnick et al. 2003;
Svanbäck and Bolnick 2007; Laskowski and Bell 2013) and
archerfish like other predators show strong inter-individual
variation in latency to respond to and attack prey (Szopa-
Comley et al. 2020a, b). These individual differences may
override effects of the social environment, particularly in so-
cial species—as shown in threespine sticklebacks
(Gasterosteus aculeatus) where, individual differences were suppressed in certain social contexts, but bolder individuals
were more likely to feed first in social conditions (McDonald
et al. 2016). Our experimental design limited our ability to
detect more subtle effects or interactions of this nature. Archerfish do respond to the presence of other fish by increas-
ing their latency to shoot (Jones et al. 2018) and exhibit lower
shooting accuracy in the presence of others (Simon et al. 2011). This may have impacted training results of fish in the
partial social condition, the only condition in which subject
fish were exposed to a visible conspecific when given oppor-
tunities to shoot. The presence of other fish may have
inhibited learning through distraction by the conspecific (as
demonstrated in zebrafish Dario rerio; Ayoub et al. 2019) or
inhibition from potential conspecific competition. Fig. 3 Relationship between the
proportion of time spent in the
open and number of sessions
taken to reach training criterion. Discussion The social environment did not influence the associative
learning rates of the archerfish in this experiment. Instead,
learning rates were related to behavioural traits linked to ex-
ploratory tendency—specifically, time spent in the open in an
open-field test. Fish which spent more time in the open (i.e., Despite the potential benefits of social learning, public in-
formation use appears to be species-specific (Webster et al. 2019), and individual biases can have a greater effect on learn-
ing than social information even when it is employed (Szabo
et al. 2017). Even in species known to use social information,
social learning may be dependent upon an individual having
some experience with novel foraging opportunities, as recent-
ly shown in American crows (Corvus brachyrhynchos)
(Pendergraft et al. 2020) and stickleback species (Webster
and Laland 2018). While it is likely that archerfish do use
social information, given previous findings suggesting social
learning in this species (Schuster et al. 2006), many factors
may impact the use of social leaning or our ability to detect it. Especially, as both social and asocial learning may depend on
the same basic mechanisms (Heyes 2012) and factors that Table 1
Summary output of survival model
Coef (β)
SE (coef)
z
Pr(>|z|)
Social treatment
2.411
1.735
1.389
0.1648
Solo treatment
1.181
1.422
0.83
0.4063
Open-field score
11.14
5.154
2.162
0.0307
Social: open-field score
−10.77
6.292
−1.711
0.0871
Social: open-field score
2.911
4.466
0.652
0.5146
Positive coefficients are associated with shorter time to reach learning
criterion. Significant coefficients (p < 0.05) are highlighted in bold. Likelihood ratio test = 16.61 on 5 df, p = 0.005, concordance = 0.724 Table 1
Summary output of survival model Positive coefficients are associated with shorter time to reach learning
criterion. Significant coefficients (p < 0.05) are highlighted in bold. Likelihood ratio test = 16.61 on 5 df, p = 0.005, concordance = 0.724 58 Page 6 of 11 Behav Ecol Sociobiol (2021) 75: 58 Fig. 2 Effect of different social contexts on time taken to reach training
criterion. The treatments relate to level of exposure to a visible
conspecific: solo (no social exposure); partial (visual social contact
except during training of neighbours); social (full visual social contact
including during training of neighbours). Only fish with ‘social’ exposure
had access to social information regarding the novel objects and
associated food rewards. Declarations Ethics approval
This research was approved by the University of St
Andrews Animal Welfare and Ethics Committee (AWEC). No procedures
required U.K. Home Office licensing. All tanks were enriched with gravel
and plastic plants for cover. All fish were retained in the laboratory after the
study period to be used in future projects. Handling was kept to a mini-
mum, and when fish had to be moved between tanks, they were caught
using two large hand nets to reduce the likelihood of extended capture
periods. In the experimental tanks, fish were kept singly in a volume of
at least 125 l. Archerfish are not considered a social species. There are no
published studies on the effects or preference for any social context and or
isolation in this species, but several previous studies have maintained ar-
cherfish in isolation with no reported ill effects or perceived likelihood of
stress (Gabay et al. 2013; Gerullis and Schuster 2014; Newport et al. 2014,
2018). During our study, we closely monitored each fish, specifically for
signs of reduced feeding rate, responsiveness, stereotypic behaviour and
colour changes. We observed few instances of these signs: all of the in-
stances of short-term colour change occurred immediately post-transfer
between tanks and reduced feeding rate for the first 12 h post-transfer. Consent for publication
All authors consent to publication of this
article. Conflict of interest
The authors declare no conflict of interest. Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing, adap-
tation, distribution and reproduction in any medium or format, as long as
you give appropriate credit to the original author(s) and the source, pro-
vide 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/. Archerfish are not a highly social species but do modify
their shooting behaviour in response to the presence of con-
specifics. Declarations The lack of variation in learning rates with social
context in this study suggests that learning about what to shoot
and the decision to take a shot may not be affected equally by
social factors. Other non-grouping species may similarly be
less likely to show strong effects of social context on learning
behaviour, even when their behaviour can be affected by so-
cial context in other situations. Discussion Each point represents a single fish
that reached criterion (n = 13) and
is coloured according to the social
treatment they were exposed to. The predicted response and
estimated standard error from a
linear model are given by the
black line and grey shading,
respectively Behav Ecol Sociobiol (2021) 75: 58 Page 7 of 11 58 One area where our results may be useful is in informing
future studies, particularly with regard to acclimation, train-
ing and related ethical and welfare concerns. Archerfish, like
other animals perhaps especially other wild caught
animals—which may be slower or less likely to engage with
novelobjectsthancaptive-bredorurbanindividuals(Gajdon
et al. 2004; Jarjour et al. 2020)—can require extended pe-
riods to acclimate and train (Archard and Braithwaite 2010;
Patton and Braithwaite 2015). Acclimation can play an im-
portant role in the expression of behaviours and performance
in behavioural assays (O’Neill et al. 2018), and sufficient
acclimationmayhelpreducetheissuesinherentinmeasuring
cognition in the laboratory (Webster and Rutz 2020). Cognition studies may be particularly susceptible to factors
affecting stress and environmental conditions which can af-
fect measures (Pritchard et al. 2016), and this is especially
important when exploring consistent individual variation
(Strand et al. 2010; Rowe and Healy 2014; Griffin et al. 2015; White et al. 2017; Boogert et al. 2018; da Silva et al. 2020). There have been no studies of archerfish welfare, and
further work into social and environmental enrichment is
required, but our study suggests that keeping archerfish
alone may not be detrimental to their welfare in the short
term. Learning rates are one measurable aspect of animal
welfare and benefit of enrichment (Strand et al. 2010) and
the lack of visible conspecifics had no negative effects on
learning rates in our study, for the fish that completed train-
ing.However,italsosuggeststhattherelativelylongtraining
and acclimation periods required for learning in this species
may not be easily reduced by including a visible conspecific. This may be important for other species where social effects
are relatively unstudied, and where they are likely to be in-
creasingly used as a model species. Funding This study was funded by the Fisheries Society of the British
Isles (studentship to NARJ). Funding This study was funded by the Fisheries Society of the British
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Genetic risk, incident stroke, and the benefits of adhering to a healthy lifestyle: cohort study of 306 473 UK Biobank participants
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BMJ. British medical journal
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ABSTRACT
Objective For numbered affiliations see
end of article. In this cohort study, genetic and lifestyle factors were
independently associated with incident stroke. These
results emphasise the benefit of entire populations
adhering to a healthy lifestyle, independent of genetic
risk. To evaluate the associations of a polygenic risk score
and healthy lifestyle with incident stroke. Introduction Stroke is one of the leading reasons for disability and
death worldwide.1 It is a complex disease, caused by
both genetic and environmental factors, including diet
and lifestyle.2 Results on 3 December 2018 by guest. Protected by copyright. ttp://www.bmj.com/ During a median follow-up of 7.1 years (2 138 443
person years), 2077 incident strokes (1541 ischaemic
stroke, 287 intracerebral haemorrhage, and 249
subarachnoid haemorrhage) were ascertained. The
risk of incident stroke was 35% higher among those
at high genetic risk (top third of polygenic score)
compared with those at low genetic risk (bottom
third): hazard ratio 1.35 (95% confidence interval
1.21 to 1.50), P=3.9×10−8. Unfavourable lifestyle (0 or
1 healthy lifestyle factors) was associated with a 66%
increased risk of stroke compared with a favourable
lifestyle (3 or 4 healthy lifestyle factors): 1.66 (1.45
to 1.89), P=1.19×10−13. The association with lifestyle
was independent of genetic risk stratums. on 3 December 2018 by guest. Protected by copyright. m/ We investigated whether a weighted genetic risk
score based on the genome wide association results
for stroke in MEGASTROKE is associated with incident
stroke in a large population based cohort (UK Biobank). We also investigated whether adherence to a healthy
lifestyle influences this association. What is already known on this topic
Stroke is a complex disease caused by both genetic and environmental factors,
including diet and lifestyle
Whether adhering to a healthy lifestyle could attenuate the effect of genetic
background on risk of incident stroke is currently unknown
What this study adds
Genetic and lifestyle factors were independently associated with risk of incident
stroke
An unfavourable lifestyle profile was associated with increased risk of stroke
across all genetic risk stratums
These findings highlight the potential of lifestyle interventions to reduce risk of
stroke across entire populations, even in those at high genetic risk of stroke uest. Protected by copyright. RESEARCH RESEARCH on 3 December 2018 by guest. Protected by copyright. http://www.bmj.com/
BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from Genetic risk, incident stroke, and the benefits of adhering
to a healthy lifestyle: cohort study of 306 473 UK Biobank
participants Loes CA Rutten-Jacobs,1,2 Susanna C Larsson,3 Rainer Malik,4 Kristiina Rannikmäe,5,6
Cathie L Sudlow,5,6,7 Martin Dichgans,4,8,9 Hugh S Markus,2 Matthew Traylor2 Conclusion Main outcome measurei Early evidence supporting a role for genetics in risk
of stroke came from twin studies and family history
studies.3 4 Further evidence has emerged from genome
wide association studies. MEGASTROKE, a large meta-
analysis of genome wide association studies tripled
the number of loci robustly associated with stroke
risk.5i Hazard ratios for a first stroke, estimated using
Cox regression. A polygenic risk score of 90 single
nucleotide polymorphisms previously associated
with stroke was constructed at P<1×10−5 to test for
an association with incident stroke. Adherence to a
healthy lifestyle was determined on the basis of four
factors: non-smoker, healthy diet, body mass index
<30 kg/m2, and regular physical exercise. Lifestyle is an important modifiable risk factor for
stroke. Clear evidence shows that adhering to a healthy
lifestyle, including not smoking, reducing the risk of
diabetes, regular physical activity, and a healthy diet,
decreases the risk of stroke substantially.6 7 It might
be hypothesised that adhering to a healthy lifestyle
could attenuate the effect of genetics on stroke risk. A
previous study in coronary artery disease—a condition
closely related to ischaemic stroke, found a statistically
significant interplay between genetic and lifestyle risk
factors in the risk of coronary artery disease.8 Study population Stroke is a complex disease caused by both genetic and environmental factors,
including diet and lifestyle UK Biobank is a prospective study that recruited
500 000 community dwelling participants, aged 40-
69 years, from across the United Kingdom between
2006 and 2010 (www.ukbiobank.ac.uk).9 Participants
attended one of 22 assessment centres across England,
Scotland, and Wales. The study collects extensive
data from questionnaires, interviews, health records,
physical measures, biological samples, and imaging. Main outcomes and exposures of interest in the current
study include imputed genetic data, incident stroke,
and lifestyle (smoking, diet, body mass index, and
physical activity). These findings highlight the potential of lifestyle interventions to reduce risk of
stroke across entire populations, even in those at high genetic risk of stroke the bmj | BMJ 2018;363:k4168 | doi: 10.1136/bmj.k4168 1 RESEARCH RESEARCH on 3 December 2018 by guest. Protected by copyright. http://www.bmj.com/
BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from In the present study we included all people who
were classified as white British (all who self identified
as white British, followed by the exclusion of ethnic
outliers identified by principal components analysis
on the genotype data), without a history of stroke or
myocardial infarction on the basis of self report or
medical records, or both, and with complete data on
lifestyle. people) and the UK Biobank Axiom array, to genotype
about 805 426 markers with good genome wide
coverage. Phasing was performed using SHAPEIT3
and imputation using IMPUTE4.12 15 Two reference
panels were used for imputation; the Haplotype
Reference Consortium reference panel (39 131 578
autosomal single nucleotide polymorphisms, SNPs)
and a merged UK10K and 1000 Genomes Phase 3
panel.14 Imputed genotypes were available for 488 369
participants in this study.12 From the resulting dataset,
we excluded those who self reported ancestry other
than white British, related people (second degree
or greater: kinship coefficient ≥0.884), people with
high levels of heterozygosity and missingness (>5%),
and people whose reported sex was inconsistent
with sex inferred from the genetic data. The UK
Biobank core team centrally performed a check for
excessive heterozygosity.13 Extreme heterozygosity or
high rates of missingness, or both, can be indicators
of poor sample quality due to, for example, DNA
contamination. UK Biobank provided a list of samples
with unusually high heterozygosity and we excluded
those samples according to its recommendations. Study population To evaluate a mismatch in sex self reported sex was
compared with sex inferred from the genetic data
(based on relative intensity of markers on the Y and
X chromosomes). This sex mismatch evaluation was
centrally performed by the UK Biobank core team and
is described in detail elsewhere.12 This evaluation
can be used as a way to detect sample mishandling or
other kinds of clerical error. However, in a dataset of
this size, some such mismatches would be expected
owing to transgender people or instances of real (but
rare) genetic variation, such as aneuploidies in sex
chromosomes. on 3 December 2018 by guest. P
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BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from Polygenic risk score derived from MEGASTROKE
2 We derived three sets of independent (r2<0.05 or
1000 Kb apart) SNPs associated with any stroke in
people of European ancestry in MEGASTROKE (see
supplemental text S2) at P<5×10−8, P<1×10−6, and
P<1×10−5 using an LD clumping procedure employed
using plink v1.90b3.45.5 16 For each individual in
the UK Biobank sample we calculated quantitative
aggregate risk scores, defined as the sum of the
number of risk alleles present at each locus weighted
by the log of the odds ratio for that locus estimated
from the MEGASTROKE sample using the plink
“–score” command. We took into account the occurrence of myocardial
infarction during the study as this could potentially
result in lifestyle changes during follow-up that
affect the risk of stroke. The occurrence of myocardial
infarction was defined according to the UK Biobank
algorithmic definition (see supplemental table S2). Details of the myocardial infarction algorithm have
been described previously and are available on the UK
Biobank website (www.ukbiobank.ac.uk).11 We excluded people from the analysis who self
reported stroke or myocardial infarction. The three polygenic scores were tested for an
association with incident any stroke, and for further
analyses we used the polygenic risk score most
statistically significantly associated with incident
stroke. Supplemental table S3 lists the SNPs included
in the MEGASTROKE risk score (all stroke, P<1×10−5). We repeated the previous steps while restricting
to ischaemic stroke in those of European ancestry
to create a genetic risk score for ischaemic stroke
(P<1×10−5). The three polygenic scores were tested for an
association with incident any stroke, and for further
analyses we used the polygenic risk score most
statistically significantly associated with incident
stroke. Supplemental table S3 lists the SNPs included
in the MEGASTROKE risk score (all stroke, P<1×10−5). on 3 December 2018 by guest.
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168 on 24 October 2018. Downloaded from The
UK
Biobank
participants
completed
a
questionnaire on their usual diet pattern. In this
analysis, a healthy diet was determined according to
the increased consumption of fruit, vegetables, and fish
and the decreased consumption of processed meats
and red meats. We defined a healthy diet as adherence
to at least two of the healthy food items. Supplemental
text S1 and table S1 provide additional details on the
specific questions asked and the construction of a
healthy diet score. Moderate physical activity was defined as at least
150 minutes of moderate intensity activity weekly or
75 minutes of vigorous activity weekly. Incident stroke Incident stroke in UK Biobank was based on medical
history and linkage to data on hospital admissions
and mortality. We used the stroke variables provided
by UK Biobank, which were created by combining
information from these different data sources (see
supplemental table S2). Details of the algorithms used
to combine the data from different sources to identify
stroke have been described previously and are available
on the UK Biobank website (www.ukbiobank.ac.uk). We subtyped stroke as ischaemic stroke, intracerebral
haemorrhage, or subarachnoid haemorrhage. In this analysis we only included SNPs imputed from
the Haplotype Reference Consortium panel. Healthy lifestyle factorsi We defined four healthy lifestyle factors on the basis of
the American Heart Association guidelines: no current
smoking, healthy diet, body mass index <30 kg/m2,
and moderate physical activity two or more times
weekly.10 Statistical analysisi fatal or non-fatal strokes were reported as first incident
vascular event or death, of which 1541 were ischaemic
stroke, 287 intracerebral haemorrhage, and 249
subarachnoid haemorrhage. Furthermore, 3436 cases
of fatal or non-fatal myocardial infarction and 6646
deaths due to other causes than stroke or myocardial
infarction were reported as first incident vascular event
or death. We defined genetic risk in thirds: “low risk” (lowest
third of genetic risk score), “intermediate risk” (second
third), “high risk” (highest third). Lifestyle was
recorded as “favourable” (three or four healthy lifestyle
factors), “intermediate” (two healthy lifestyle factors),
“unfavourable” (no or one healthy lifestyle factor). To test the association of genetic and lifestyle factors
with incident stroke we used Cox proportional hazards
models. The duration of follow-up was calculated as
time between the baseline assessment and the first
event of either stroke, myocardial infarction, death,
or 1 March 2016, which was the end of follow-up
for the current data release. Participants who had a
myocardial infarction or died before a stroke occurred
were censored at the time of the respective event. Polygenic risk scores containing independent SNPs
(on basis of linkage disequilibrium patterns) derived
from MEGASTROKE at three different significance
thresholds were tested for association with incident
stroke in UK Biobank (see supplemental figure S1). The
three polygenic risk scores were associated with risk of
incident stroke, but the genetic risk score including
all SNPs associated with stroke in MEGASTROKE
at P<1×10−5 (90 SNPs, see supplemental table S3)
showed the strongest association and was therefore
selected for subsequent analyses. The polygenic risk
approximated a normal distribution (see supplemental
figure S2). We repeated the previous steps for the ischaemic
stroke genetic risk score and assessed the association
between this score and incident ischaemic stroke. In this analysis we censored participants with a
diagnosis of intracerebral haemorrhage, subarachnoid
haemorrhage, or myocardial infarction or who died
before an ischaemic stroke occurred at the time of the
respective event. In Cox proportional hazards analysis, the risk of
incident stroke was higher for those with intermediate
(hazard ratio 1.20, 95% confidence interval 1.08
to 1.34) and high genetic risk scores (1.35, 1.21 to
1.50) compared with those with a low genetic risk
score (fig 2). We tested available cardiometabolic
risk factors for an association with the genetic risk
score, adjusting for the first 10 principal components
of ancestry, genotyping batch, age, and sex. Statistical analysisi The
genetic risk score was significantly associated with
systolic blood pressure (P=1.5×10−15), diastolic blood
pressure (P=1.1×10−7), use of lipid lowering drugs
(P=7.5×10−13), and diabetes (7.6×10−4), but not with
body mass index (P=0.18). Cox
proportional
hazards
models
included
adjustment for age and sex for the lifestyle score
models. For the models including the genetic score
we additionally adjusted for the first 10 principal
components of ancestry and genotyping batch. Model
discrimination was evaluated with the concordance (c)
statistic. We included an interaction term in the regression
model to test for statistical interaction between the
lifestyle and genetic risk score. To obtain cumulative incidence for lifestyle
and genetic risk stratums we used competing risk
regression; the cumulative incidence function. We
compared the hazard ratios for the genetic and lifestyle
score in the risk of stroke derived from Cox proportional
hazards models with the subdistribution hazard ratios,
calculated using proportional subdistribution hazards
regression models.17 Total UK Biobank participants
Excluded
Missingness >5%
Sex mismatch
Excessive heterozygosity
199
298
528
1025
Excluded
History of stroke
History of myocardial infarction
Self report only of incident myocardial infarction or stroke
Any missing lifestyle information
5821
8469
60
56 217
70 567
502 619
Genetic data
488 369
Unrelated White British
378 065
Passed genetic quality control
377 040
Total included in analysis
306 473
Fig 1 | Flow of participants through study R software version 3.4.2 was used for the Cox
proportional hazards regression (package “survival”)
and proportional subdistribution hazards regression
(package “cmprsk”). Patient and public involvement The development of the research question or outcome
measures was not informed by patients’ priorities,
experience, or preferences. No patients were involved
in the design and conduct of the present study. There
are no plans to disseminate the results to study
participants. Genetic data We used the June 2017 release of the imputed genetic
data from UK Biobank (downloaded 3 June 2017). Details of the design of the arrays, sample processing,
and stringent quality control have been described
in detail elsewhere12 and summarised previously.13
Briefly, we used two closely related arrays from
Affymetrix, the UK BiLEVE Axiom array (9.9% of We repeated the previous steps while restricting
to ischaemic stroke in those of European ancestry
to create a genetic risk score for ischaemic stroke
(P<1×10−5). doi: 10.1136/bmj.k4168 | BMJ 2018;363:k4168 | the bmj RESEARCH on 3 December 2018 by guest. Protected by copyright. http://www.bmj.com/
BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from Statistical analysisi on 3 December 2018 by guest. Protected b
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BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from on 3 December 2018 by guest. Protected by copyright. http://www.bmj.com/
MJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from genetic risk score (c statistic 0.69 (SE) 0.01), lifestyle
score (0.69 (SE 0.01)), and the combined genetic and
lifestyle score (0.70 (SE 0.01)). The cumulative risk of stroke increased with age. Blood pressure, diabetes, and the use of lipid lowering
drugs did not seem to influence the association
between genetic risk of stroke and incident stroke (see
supplemental table S4). Similarly, the risk of stroke
was increased in those with an unfavourable (hazard
ratio 1.66, 95% confidence interval 1.45 to 1.89) and
intermediate (1.27, 1.16 to 1.40) lifestyle compared
with those with a favourable lifestyle (fig 2).i We repeated the analysis to test the associations
of the genetic and lifestyle scores with incident
stroke while restricting only to ischaemic stroke for
both genetic risk score and outcome. In addition, we
compared the results derived from the Cox proportional
hazards model with those derived from the competing
risk proportional subdistribution hazards model. Results did not change substantially when restricting
to ischaemic stroke or when using the subdistribution
hazards model compared with the original analyses
(see supplementary tables S7-S9). Supplemental figure S3 shows the distribution
of thirds of genetic risk and lifestyle scores. The
genetic risk score was not associated with any of the
single healthy lifestyle factors: odds ratio 0.98 (95%
confidence interval 0.93 to 1.04) for body mass index
score, 1.04 (0.99 to 1.09) for diet score, 0.97 (0.88
to 1.07) for smoking score, and 0.98 (0.94 to 1.03)
for exercise score. Furthermore, the association of
genetic risk with incident stroke was unchanged after
adjustment for lifestyle (see supplemental table S5). Likewise, the association of lifestyle with incident
stroke was essentially unchanged after adjustment for
the genetic risk score (see supplemental table S5). Among individual components of the lifestyle score,
smoking and body mass index ≥30 kg/m2 contributed
most to the risk of incident stroke (table 3). For all
lifestyle factors, the effects were similar across genetic
risk stratums.f As the effect of smoking was about twice as strong
as the other individual lifestyle scores (table 3). We
repeated the analysis of the risk of incident stroke for
combined genetic risk and lifestyle profile in which
smoking was counted twice, and this resulted in slightly
increased point estimates in the unfavourable versus
favourable lifestyle categories (see supplementary
table S10). Results Complete data for the present analysis were available
for 306 473 participants in the UK Biobank Study
(fig 1). Table 1 shows the baseline characteristics of the
study population. During a total of 2 138 443 person
years (median follow-up 7.1 years), 2077 incident Fig 1 | Flow of participants through study the bmj | BMJ 2018;363:k4168 | doi: 10.1136/bmj.k4168 3 RESEARCH Table 1 | Characteristics of participants at baseline. Values are numbers (participants) unless stated otherwise
Characteristic
All (n=306 473)
Incident stroke (n=2077)
No stroke (n=304 396)
Mean (SD) age (years)
56.7 (7.9)
61.2 (6.8)
56.6 (7.9)
Men
136 654 (44.6)
1184 (57.0)
135 470 (44.5)
Mean (SD) systolic blood pressure (mm Hg)
138 (18)
146 (21)
138 (19)
Mean (SD) diastolic blood pressure (mm Hg)
82 (10)
85 (11)
82 (10)
Mean (SD) body mass index
27.2 (4.7)
28.0 (4.8)
27.2 (4.7)
Diabetes
12 927 (4.2)
209 (10.1)
1865 (4.2)
Use of lipid lowering drugs
44 785 (14.7)
497 (24.1)
44 288 (14.6)
Use of antihypertensives
61 218 (20.0)
697 (33.6)
60 521 (19.9)
Healthy lifestyle factors:
No current smoking
286 352 (93.4)
1822 (87.7)
284 530 (93.5)
Body mass index <30
236 326 (77.1)
1489 (71.7)
588 (77.1)
Regular moderate physical activity
181 234 (59.1)
1203 (57.9)
180 031 (59.1)
Healthy diet
139 328 (45.5)
884 (42.6)
138 444 (45.5)
Healthy lifestyle score:
Favourable (3 or 4 healthy lifestyle factors)
191 003 (62.3)
1157 (55.7)
189 846 (62.4)
Intermediate (2 healthy lifestyle factors)
86 710 (28.3)
652 (31.4)
86 058 (28.3)
Unfavourable (0 or 1 healthy lifestyle factor)
28 760 (9.4)
268 (12.9)
28 492 (9.4)
Genetic risk category:
Low
101 977 (33.3)
589 (28.4)
101 388 (33.3)
Intermediate
102 300 (33.4)
703 (33.8)
101 597 (33.4)
High
102 196 (33.3)
785 (37.8)
101 411 (33.3) Discussion We investigated the association between genetic risk of
stroke, lifestyle, and incident risk of stroke in 306 473
people within the population based UK Biobank
study. Risk of incident stroke was 35% higher among
those at high genetic risk compared with those at low
genetic risk, and these associations were independent
of lifestyle profile. Furthermore, an unfavourable
lifestyle was associated with a 66% increased risk of
incident stroke compared with a favourable lifestyle,
and this increased risk was present within any genetic
risk category. A high genetic risk combined with an
unfavourable lifestyle profile was associated with a
more than twofold increased risk of stroke compared
with a low genetic risk and a favourable lifestyle. The present study provides further support that
common genetic variants are implicated in the
development of stroke. Our findings showing that a
polygenic risk score is associated with incident stroke
is in line with both clinical and population based
studies.18-21 Cumulative risk of stroke (%) The genetic risk score was also associated with
blood pressure and use of lipid lowering drugs, which
suggest that the effect of the genetic variants on risk
of incident stroke might at least in part be mediated
by vascular risk factors. However, adjusting for those
factors did not change the effect size of association
between genetic risk and incident stroke, which
emphasises that other mechanisms than those that
involve the traditional cardiovascular risk factors are
likely important. In the MEGASTROKE genome wide
association analysis of stroke, only about half of the
identified loci shared genetic variation with related
vascular traits, including blood pressure and lipid
levels, which support that the genetic risk might act
through additional mechanisms.5 on 3 December 2018 by guest. Protected by copyright. http://www.bmj.com/ Fig 2 | Standardised risk of incident stroke according
to genetic risk and lifestyle profile. on 3 December 2018 by guest. Protected b
http://www.bmj.com/
BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from Table 2 shows the risk of incident stroke for
combined genetic risk and lifestyle profiles. An additive
effect was found for genetic risk and lifestyle on risk
of incident stroke. Within each genetic risk stratum
there was an increase in strength of association with
decreasing number of favourable life style factors (see
supplemental table S6). The highest risk of incident
stroke was observed in participants with a high
genetic risk and an unfavourable lifestyle: hazard
ratio 2.30 (95% confidence interval 1.84 to 2.87);
see supplemental figure S4. The test for statistical
interaction between lifestyle score and genetic risk
score in relation to incident stroke was not significant
(P=0.57) compared with participants with low genetic
risk and favourable lifestyle. guest. Protected by copyright. The associations of genetic risk score with incident
stroke were consistent in men and women (interaction
P=0.70, supplemental figure S5). However, across
all genetic risk stratums the absolute risk of incident
stroke was lower in women than in men.i A statistically significant interaction on the
multiplicative scale was found between sex and
lifestyle profile in the risk of incident stroke
(interaction P=0.01, supplemental figure S6). For men
there was an increase in association with decreasing
number of healthy lifestyle factors: hazard ratio 1.20
(95% confidence interval 1.05 to 1.36) and 1.82 (1.55 Model discrimination was similar between the
main Cox proportional hazards models including the doi: 10.1136/bmj.k4168 | BMJ 2018;363:k4168 | the bmj RESEARCH on 3 December 2018 by guest. Protected by copyright. http://www.bmj.com/
BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. on 3 December 2018 by guest. Protected b
http://www.bmj.com/
BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from Downloaded from Genetic risk
No at risk
High
Intermediate
Low
Age (years)
Cumulative risk of stroke (%)
0
2
3
5
4
1
50
55
60
65
70
75
16 693
16 718
16 628
19 885
20 106
19 895
23 927
24 020
23 608
31 663
31 569
31 823
24 740
25 312
25 044
4906
4957
5185
High; hazard ratio 1.35 (1.21 to 1.50)
Intermediate; hazard ratio 1.20 (1.08 to 1.34)
Low (reference)
Lifestyle
No at risk
Unfavourable
Intermediate
Favourable
Age (years)
Cumulative risk of stroke (%)
0
2
3
5
4
1
50
55
45
45
60
65
70
75
80
80
5087
14 762
30 190
6427
17 920
35 539
7387
20 904
43 264
8327
26 033
60 695
5716
19 635
49 403
989
3843
10 216
Unfavourable; hazard ratio 1.66 (1.45 to 1.89)
Intermediate; hazard ratio 1.27 (1.16 to 1.40)
Favourable (reference)
Fig 2 | Standardised risk of incident stroke according
to genetic risk and lifestyle profile. Cox proportional
hazards models were adjusted for age and sex, and
the genetic risk models included additionally the first
10 principal components of ancestry and genotyping
batch to 2.15) for intermediate and unfavourable lifestyle
versus favourable lifestyle, respectively. However,
among women there was no difference between
intermediate
and
unfavourable
lifestyle
versus
favourable lifestyle: 1.39 (1.21 to 1.61) and 1.36 (1.08
to 1.72), respectively. Genetic risk
No at risk
Age (years)
Cumulative risk of stroke (%)
0
2
3
5
4
1
50
55
60
65
70
75
High; hazard ratio 1.35 (1.21 to 1.50)
Intermediate; hazard ratio 1.20 (1.08 to 1.34)
Low (reference)
45
80 Discussion Cox proportional
hazards models were adjusted for age and sex, and
the genetic risk models included additionally the first
10 principal components of ancestry and genotyping
batch The reduction of stroke risk by adhering to a
healthy lifestyle has been well reported.7 22-25 The Table 2 | Relative and absolute risk of incident stroke according to genetic and lifestyle profiles
Genetic risk
Lifestyle
Favourable
Intermediate
Unfavourable
Low
Hazard ratio* (95% CI)
1 (reference)
1.36 (1.14 to 1.63), P=7.3×10−04
1.84 (1.44 to 2.35), P=8.0×10−07
8 year cumulative incidence† (%) (95% CI)
0.54 (0.47 to 0.60)
0.74 (0.63 to 0.85)
0.95 (0.74 to 1.17)
Intermediate
Hazard ratio* (95% CI)
1.26 (1.09 to 1.46), P=0.002
1.62 (1.37 to 1.92), P=3.2×10−08
1.85 (1.46 to 2.37), P=5.4×10−07
8 year cumulative incidence† (%) (95% CI)
0.67 (0.60 to 0.74)
0.82 (0.71 to 0.93)
0.92 (0.72 to 1.12)
High
Hazard ratio* (95% CI)
1.44 (1.25 to 1.66), P=7.0×10−07
1.70 (1.44 to 2.01), P=8.1×10−10
2.30 (1.84 to 2.87), P=3.3×10−13
8 year cumulative incidence† (%) (95% CI)
0.78 (0.70 to 0.86)
0.91 (0.78 to 1.04)
1.11 (0.89 to 1.33)
*Calculated using Cox proportional hazards models, adjusted for age, sex, first 10 principal components of ancestry, and genotyping batch. †Calculated using the cumulative incidence function as implemented in the “cmprsk” R package. Table 2 | Relative and absolute risk of incident stroke according to genetic and lifestyle profiles
Lifestyle the bmj | BMJ 2018;363:k4168 | doi: 10.1136/bmj.k4168 5 RESEARCH RESEARCH on 3 December 2018 by guest. Protected by copyright. http://www.bmj.com/
BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Discussion Downloaded from Table 3 | Multivariable Cox regression analysis of age, sex, and lifestyle factors in relation to risk of stroke, stratified by
genetic risk
Lifestyle factors
Low genetic risk
Moderate genetic risk
High genetic risk
Hazard ratio (95% CI) P value
Hazard ratio (95% CI) P value
Hazard ratio (95% CI) P value
Age, per year
1.08 (1.07 to 1.09)
<2×10−16
1.09 (1.08 to 1.10)
<2×10−16
1.11 (1.10 to 1.12)
<2×10−16
Male sex
1.47 (1.25 to 1.74)
4.6×10−06
1.65 (1.42 to 1.93)
1.2×10−10
1.56 (1.35 to 1.80)
1.4×10−09
Current smoking
2.35 (1.84 to 3.01)
7.3×10−12
2.81 (2.27 to 3.48)
<2×10−16
1.87 (1.48 to 2.37)
1.5×10−07
Body mass index ≥30
1.43 (1.20 to 1.71)
8.0×10−05
1.38 (1.17 to 1.63)
1.2×10−04
1.19 (1.02 to 1.40)
0.03
No regular moderate
physical activity
1.09 (0.92 to 1.28)
0.32
0.99 (0.85 to 1.63)
0.91
1.09 (0.94 to 1.26)
0.24
Unhealthy diet
1.10 (0.93 to 1.30)
0.27
1.11 (0.77 to 1.05)
0.16
1.21 (1.05 to 1.40)
0.01 | Multivariable Cox regression analysis of age, sex, and lifestyle factors in relation to risk of stroke, stratified by
risk lifestyle in detail. Furthermore, to derive a genetic
risk score for stroke, we used MEGASTROKE, which is
currently the largest genome wide association study
of stroke.5 Another distinctive feature of this analysis
compared with a previous study is that we also included
single nucleotide polymorphisms (SNPs) associated
with stroke at a subthreshold level of significance
(P<1×10−5).19 This concurs with a previous study
examining the predictive utility of genetic risk scores
for incident coronary heart disease, which showed
that the best performance was achieved by including
SNPs that did not necessarily reach the genome wide
statistical significance threshold in previous genome
wide association studies.32 lifestyle in detail. Comparison with previous studies Therefore, our
results may not be generalisable to populations with
distinct ancestry. Future studies are needed that test
these relations in more diverse populations. Conclusion In the present prospective population based cohort
study of 306 473 people we found that genetic and
lifestyle factors were independently associated with
risk of incident stroke. These findings highlight the
potential of lifestyle interventions to reduce risk of
stroke across entire populations, even in those at high
genetic risk of stroke. Other possibilities for the increased relative risk
include potential differences in duration of exposure to
unfavourable lifestyle factors. Future studies are needed
to evaluate the effect of the duration of exposure to an
unfavourable lifestyle profile on the risk of stroke. Comparison with previous studies Across all categories of genetic risk and lifestyle, the risk
of incident stroke was higher in men than women. This
is an expected finding given the previously consistently
shown higher incidence of stroke in men compared
with women at the age of most of the UK Biobank
participants.26 Our results suggested that the relative
risk of incident stroke associated with high genetic
risk versus low genetic risk was similar in men and
women. A family history and genome wide association
studies suggested that genetic susceptibility to stroke
is somewhat stronger in women than in men.27 28 The
methodological differences of those previous studies
and the current study might explain the different
conclusions. In the current study, only genetic variants
associated with stroke in MEGASTROKE at P<1×10−5
were considered, whereas the other studies evaluated
all genome wide variants within the study population
or family history, which also includes environmental
effects. Our study has several limitations. Firstly, behavioural
changes before or after the baseline examinations
might have had an effect on the risk estimates. We tried
to reduce the effect of behavioural changes that could
be related to vascular disease by excluding all those
with a history of stroke and by censoring those at the
time a myocardial infarction occurred. Secondly, this
analysis focused on a narrow range of lifestyle factors,
based on the American Heart Association guidelines.10 Expanding the range of lifestyle factors (ie, stress, sleep,
alcohol and drug use) and more detailed assessment of
diet and physical activity would be of interest for future
studies. Thirdly, in the current study we only evaluated
stroke of any cause. The effects of lifestyle and genetic
variants might differ according to the cause of stroke,
although some genetic risk variants and vascular risk
factors are shared between two or more causal factors.5 Considerable
evidence
from
previous
epidemiological studies also suggests differences
in risk factors that are associated with stroke in
men compared with women. Women have a higher
prevalence of hypertension, whereas men have a
higher prevalence of heart disease, diabetes, and
unhealthy lifestyle behaviours, including smoking,
obesity, and alcohol use.29-31 In the present study we
found a higher relative risk of stroke associated with
an unfavourable lifestyle in men than women (82% v
36% increased risk, respectively). mber 2018 by guest. Protected by copyright. Finally, in the present study we restricted our
analysis to people of European descent. Strengths and limitations of this study The major strengths of the current study include the
large sample size of UK Biobank participants, which
enabled study of the combination of genetic risk and AUTHOR AFFILIATIONS Discussion Furthermore, to derive a genetic
risk score for stroke, we used MEGASTROKE, which is
currently the largest genome wide association study
of stroke.5 Another distinctive feature of this analysis
compared with a previous study is that we also included
single nucleotide polymorphisms (SNPs) associated
with stroke at a subthreshold level of significance
(P<1×10−5).19 This concurs with a previous study
examining the predictive utility of genetic risk scores
for incident coronary heart disease, which showed
that the best performance was achieved by including
SNPs that did not necessarily reach the genome wide
statistical significance threshold in previous genome
wide association studies.32 risk reduction associated with adherence to a healthy
lifestyle in the present study was similar across all
stratums of genetic risk, which emphasises the benefit
for entire populations of adhering to a healthy lifestyle,
independent of genetic risk. Among the lifestyle factors,
the most statistically significant associations were
observed for smoking and body mass index ≥30 kg/m2. on 3 December 2018 by guest. Protected b
http://www.bmj.com/
BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from N Engl J
Med 2016;375:2349-58. doi:10.1056/NEJMoa1605086 9
Sudlow C, Gallacher J, Allen N, et al. UK biobank: an open access
resource for identifying the causes of a wide range of complex
diseases of middle and old age. PLoS Med 2015;12:e1001779. doi:10.1371/journal.pmed.1001779 9
Sudlow C, Gallacher J, Allen N, et al. UK biobank: an open access
resource for identifying the causes of a wide range of complex
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adjudication: UK Biobank; 2017. https://biobank.ctsu.ox.ac.uk/
crystal/docs/alg_outcome_mi.pdf [accessed 1 July 2017]. ft
k
l
d Competing interests: All authors have completed the ICMJE uniform
disclosure form at www.icmje.org/coi_disclosure.pdf and declare: no
support from any organisation for the submitted work; HSM has been
paid for delivering educational presentations for AstraZeneca; no
other financial relationships with any organisations that might have
an interest in the submitted work in the previous three years; no other
relationships or activities that could appear to have influenced the
submitted work. 12 Bycroft C, Freeman C, Petkova D, et al. Genome-wide genetic
data on ~500 000 UK Biobank participants. BioRxiv
2017 20-07-2017. 13 Rutten-Jacobs LCA, Tozer DJ, Duering M, et al. Genetic Study of
White Matter Integrity in UK Biobank (N=8448) and the Overlap
With Stroke, Depression, and Dementia. Stroke 2018;49:1340-7. doi:10.1161/STROKEAHA.118.020811 14 McCarthy S, Das S, Kretzschmar W, et al, Haplotype Reference
Consortium. A reference panel of 64,976 haplotypes for genotype
imputation. Nat Genet 2016;48:1279-83. doi:10.1038/ng.3643 Ethical approval: UK Biobank received ethical approval from the
research ethics committee (reference 13/NW/0382). All participants
provided informed consent to participate. The present analyses were
conducted under UK Biobank application number 19463. 15 O’Connell J, Sharp K, Shrine N, et al. Haplotype estimation for
biobank-scale data sets. Nat Genet 2016;48:817-20. doi:10.1038/ng.3583 Data sharing: The genetic and phenotypic UK Biobank data are
available on application to the UK Biobank (www.ukbiobank.ac.uk/). on 3 December 2018 by guest. Protected b
http://www.bmj.com/
BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from Healthy diet and lifestyle and
risk of stroke in a prospective cohort of women. Neurology 2014;83:1699-704. doi:10.1212/
WNL.0000000000000954 7
Larsson SC, Akesson A, Wolk A. Healthy diet and lifestyle and
risk of stroke in a prospective cohort of women. Neurology 2014;83:1699-704. doi:10.1212/
WNL.0000000000000954 Funding: This work was in part supported by a British Heart
Foundation programme grant (RG/16/4/32218). LCAR-J was
supported by a British Heart Foundation immediate research
fellowship (FS/15/61/31626). CLS is chief scientist for UK Biobank. The main sources of funding for CLS’s salary are UK Biobank and the
Scottish funding Council. MD received funding from the European
Union’s Horizon 2020 research and innovation programme under
grant agreement No 666881 (SVDs@target) and from the DFG
through the CRC 1123 (B3) and the Munich Cluster for Systems
Neurology (EXC 1010 SyNergy). HSM is supported by a National
Institute for Health Research (NIHR) senior investigator award, and his
work is supported by the Cambridge Universities NIHR Comprehensive
Biomedical Research Centre. The funding sources had no role in the
design or conduct of the study; collection, management, analysis, and
interpretation of the data; or preparation, review, or approval of the
manuscript. The MEGASTROKE project received funding from sources
specified at www.megastroke.org/acknowledgments.html. Funding: This work was in part supported by a British Heart
Foundation programme grant (RG/16/4/32218). LCAR-J was
supported by a British Heart Foundation immediate research
fellowship (FS/15/61/31626). CLS is chief scientist for UK Biobank. The main sources of funding for CLS’s salary are UK Biobank and the
Scottish funding Council. MD received funding from the European
Union’s Horizon 2020 research and innovation programme under
grant agreement No 666881 (SVDs@target) and from the DFG
through the CRC 1123 (B3) and the Munich Cluster for Systems
Neurology (EXC 1010 SyNergy). HSM is supported by a National
Institute for Health Research (NIHR) senior investigator award, and his
work is supported by the Cambridge Universities NIHR Comprehensive
Biomedical Research Centre. The funding sources had no role in the
design or conduct of the study; collection, management, analysis, and
interpretation of the data; or preparation, review, or approval of the
manuscript. The MEGASTROKE project received funding from sources
specified at www.megastroke.org/acknowledgments.html. 8
Khera AV, Emdin CA, Drake I, et al. Genetic Risk, Adherence
to a Healthy Lifestyle, and Coronary Disease. N Engl J
Med 2016;375:2349-58. doi:10.1056/NEJMoa1605086 8
Khera AV, Emdin CA, Drake I, et al. Genetic Risk, Adherence
to a Healthy Lifestyle, and Coronary Disease. on 3 December 2018 by guest. Protected b
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Flossmann E, Schulz UG, Rothwell PM. Systematic review
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for Heart and Aging Research in Genomic Epidemiology
(CHARGE) Consortium, International Genomics of Blood Pressure
(iGEN-BP) Consortium, INVENT Consortium, STARNET, BioBank
Japan Cooperative Hospital Group, COMPASS Consortium,EPIC-CVD
Consortium, EPIC-InterAct Consortium, International Stroke Genetics
Consortium (ISGC), METASTROKE Consortium, Neurology Working
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s41588-018-0058-3 g
7Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK
8 Edinburgh, Edinburgh, UK
8 9German Center for Neurodegenerative Diseases (DZNE), Munich,
Germany Contributors: LCAR-J and MT conceived the study. LCAR-J and MT
wrote the first and successive drafts of the manuscript. LCAR-J and MT
analysed the data. SCL contributed to study conception and design. All authors revised the manuscript for important intellectual content. LCAR-J and MT had full access to the data and take responsibility for
the integrity of the data and the accuracy of the data analysis. LCAR-J
is the guarantor. The corresponding author attests that all listed
authors meet authorship criteria and that no others meeting the
criteria have been omitted. Contributors: LCAR-J and MT conceived the study. LCAR-J and MT
wrote the first and successive drafts of the manuscript. LCAR-J and MT
analysed the data. SCL contributed to study conception and design. All authors revised the manuscript for important intellectual content. LCAR-J and MT had full access to the data and take responsibility for
the integrity of the data and the accuracy of the data analysis. LCAR-J
is the guarantor. The corresponding author attests that all listed
authors meet authorship criteria and that no others meeting the
criteria have been omitted. 6
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disease and all-cause mortality in middle-aged and elderly
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Epidemiol 2018;33:831-45. doi:10.1007/s10654-018-0374-z p
7
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http://www.bmj.com/
BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from Summary statistics from the MEGASTROKE meta-analysis of genome
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from www.megastroke.org. 16 Purcell S, Neale B, Todd-Brown K, et al. PLINK: a tool set for
whole-genome association and population-based linkage analyses. Am J Hum Genet 2007;81:559-75. doi:10.1086/519795 17 Fine JP, Gray RJ. A Proportional Hazards Model for the Subdistribution
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of a Competing Risk. J Am Stat Assoc 1999;94:496-509
doi:10.1080/01621459.1999.10474144. Transparency: The lead author (LCAR-J) affirms that the manuscript
is an honest, accurate, and transparent account of the study
being reported; that no important aspects of the study have been
omitted; that discrepancies from the study as planned have been
explained, and that the paper conforms to transparency policy of
the International Committee of Medical Journal Editors uniform
requirement for manuscripts submitted to biomedical journals. 18 Fava C, Sjögren M, Olsson S, et al. A genetic risk score for
hypertension associates with the risk of ischemic stroke in a
Swedish case-control study. Eur J Hum Genet 2015;23:969-74. doi:10.1038/ejhg.2014.212 explained, and that the paper conforms to transparency policy of
the International Committee of Medical Journal Editors uniform
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STROKEAHA.113.003044f This is an Open Access article distributed in accordance with the
terms of the Creative Commons Attribution (CC BY 4.0) license, which
permits others to distribute, remix, adapt and build upon this work,
for commercial use, provided the original work is properly cited. See:
http://creativecommons.org/licenses/by/4.0/. 20 Tada H, Shiffman D, Smith JG, et al. Twelve-single nucleotide
polymorphism genetic risk score identifies individuals at increased
risk for future atrial fibrillation and stroke. Stroke 2014;45:2856-62. doi:10.1161/STROKEAHA.114.006072 21 Traylor M, Rutten-Jacobs LC, Thijs V, et al. Genetic Associations
With White Matter Hyperintensities Confer Risk of Lacunar Stroke. Stroke 2016;47:1174-9. doi:10.1161/STROKEAHA.115.011625 21 Traylor M, Rutten-Jacobs LC, Thijs V, et al. Genetic Associations
With White Matter Hyperintensities Confer Risk of Lacunar Stroke. Stroke 2016;47:1174-9. doi:10.1161/STROKEAHA.115.011625 1
GBD 2013 Mortality and Causes of Death Collaborators. Global, regional, and national age-sex specific all-cause
and cause-specific mortality for 240 causes of death,
1990-2013: a systematic analysis for the Global Burden
of Disease Study 2013. Lancet 2015;385:117-71. the bmj | BMJ 2018;363:k4168 | doi: 10.1136/bmj.k4168 AUTHOR AFFILIATIONS 1German Center for Neurodegenerative diseases (DZNE), Population
Health Sciences, Sigmund-Freud-Strasse 27, 53127 Bonn, Germany 1German Center for Neurodegenerative diseases (DZNE), Population
Health Sciences, Sigmund-Freud-Strasse 27, 53127 Bonn, Germany doi: 10.1136/bmj.k4168 | BMJ 2018;363:k4168 | the bmj RESEARCH on 3 December 2018 by guest. Protected by copyright. http://www.bmj.com/
BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from 2Department of Clinical Neurosciences, Stroke Research Group,
University of Cambridge, UK
3Unit of Nutritional Epidemiology, Institute of Environmental
Medicine, Karolinska Institutet, Stockholm, Sweden
4Institute for Stroke and Dementia Research (ISD), University
Hospital, LMU Munich, Munich, Germany
5Centre for Medical Informatics, Usher Institute of Population Health
Sciences and Informatics, University of Edinburgh, Edinburgh, UK
6Centre for Clinical Brain Sciences, University of Edinburgh,
Edinburgh, UK
7Institute of Genetics and Molecular Medicine, University of
Edinburgh, Edinburgh, UK
8Munich Cluster for Systems Neurology (SyNergy), Munich,
Germany
9German Center for Neurodegenerative Diseases (DZNE), Munich,
Germany 2Department of Clinical Neurosciences, Stroke Research Group,
University of Cambridge, UK
3Unit of Nutritional Epidemiology, Institute of Environmental
Medicine, Karolinska Institutet, Stockholm, Sweden
4Institute for Stroke and Dementia Research (ISD), University
Hospital, LMU Munich, Munich, Germany
5Centre for Medical Informatics, Usher Institute of Population Health
Sciences and Informatics, University of Edinburgh, Edinburgh, UK
6Centre for Clinical Brain Sciences, University of Edinburgh,
Edinburgh, UK
7Institute of Genetics and Molecular Medicine, University of
Edinburgh, Edinburgh, UK
8Munich Cluster for Systems Neurology (SyNergy), Munich,
Germany
9German Center for Neurodegenerative Diseases (DZNE), Munich,
Germany 2
Boehme AK, Esenwa C, Elkind MS. Stroke Risk Factors,
Genetics, and Prevention. Circ Res 2017;120:472-95. doi:10.1161/CIRCRESAHA.116.308398 2
Boehme AK, Esenwa C, Elkind MS. Stroke Risk Factors,
Genetics, and Prevention. Circ Res 2017;120:472-95. doi:10.1161/CIRCRESAHA.116.308398 1
GBD 2013 Mortality and Causes of Death Collaborators.
Global, regional, and national age-sex specific all-cause
and cause-specific mortality for 240 causes of death,
1990-2013: a systematic analysis for the Global Burden
of Disease Study 2013. Lancet 2015;385:117-71.
doi:10.1016/S0140-6736(14)61682-2 No commercial reuse: See rights and reprints http://www.bmj.com/permissions on 3 December 2018 by guest. Protected b
http://www.bmj.com/
BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from doi:10.1016/S0140-6736(14)61682-2 1
GBD 2013 Mortality and Causes of Death Collaborators. Global, regional, and national age-sex specific all-cause
and cause-specific mortality for 240 causes of death,
1990-2013: a systematic analysis for the Global Burden
of Disease Study 2013. Lancet 2015;385:117-71. doi:10.1016/S0140-6736(14)61682-2 22 Chiuve SE, Rexrode KM, Spiegelman D, Logroscino G,
Manson JE, Rimm EB. Primary prevention of stroke by
healthy lifestyle. Circulation 2008;118:947-54. doi:10.1161/CIRCULATIONAHA.108.781062 22 Chiuve SE, Rexrode KM, Spiegelman D, Logroscino G,
Manson JE, Rimm EB. Primary prevention of stroke by
healthy lifestyle. Circulation 2008;118:947-54. doi:10.1161/CIRCULATIONAHA.108.781062 the bmj | BMJ 2018;363:k4168 | doi: 10.1136/bmj.k4168 RESEARCH on 3 December 2018 by guest. Protected by copyright. http://www.bmj.com/
BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from 23 Larsson SC, Åkesson A, Wolk A. Primary prevention of stroke by a
healthy lifestyle in a high-risk group. Neurology 2015;84:2224-8. doi:10.1212/WNL.0000000000001637
24 Zhang Y, Tuomilehto J, Jousilahti P, Wang Y, Antikainen R, Hu G. Lifestyle factors on the risks of ischemic and hemorrhagic
stroke. Arch Intern Med 2011;171:1811-8. doi:10.1001/
archinternmed.2011.443
25 Myint PK, Luben RN, Wareham NJ, Bingham SA, Khaw KT. Combined effect of health behaviours and risk of first ever
stroke in 20,040 men and women over 11 years’ follow-up in
Norfolk cohort of European Prospective Investigation of Cancer
(EPIC Norfolk): prospective population study. BMJ 2009;338:b349. doi:10.1136/bmj.b349
26 Seshadri S, Wolf PA. Lifetime risk of stroke and dementia:
current concepts, and estimates from the Framingham
Study. Lancet Neurol 2007;6:1106-14. doi:10.1016/S1474-4422(07)70291-0
27 Touzé E, Rothwell PM. Sex differences in heritability of
ischemic stroke: a systematic review and meta-analysis. Stroke 2008;39:16-23. doi:10.1161/STROKEAHA.107.484618
28 Traylor M, Rutten-Jacobs LC, Holliday EG, et al. Differences in
Common Genetic Predisposition to Ischemic Stroke by Age and Sex. Stroke 2015;46:3042-7. doi:10.1161/STROKEAHA.115.009816 29 Andersen KK, Andersen ZJ, Olsen TS. Age- and gender-specific
prevalence of cardiovascular risk factors in 40,102 patients
with first-ever ischemic stroke: a Nationwide Danish Study. Stroke 2010;41:2768-74. doi:10.1161/STROKEAHA.110.595785f 23 Larsson SC, Åkesson A, Wolk A. Primary prevention of stroke by a
healthy lifestyle in a high-risk group. Neurology 2015;84:2224-8. doi:10.1212/WNL.0000000000001637 23 Larsson SC, Åkesson A, Wolk A. Primary prevention of stroke by a
healthy lifestyle in a high-risk group. Neurology 2015;84:2224-8. doi:10.1212/WNL.0000000000001637 24 Zhang Y, Tuomilehto J, Jousilahti P, Wang Y, Antikainen R, Hu G. Lifestyle factors on the risks of ischemic and hemorrhagic
stroke. Arch Intern Med 2011;171:1811-8. doi:10.1001/
archinternmed.2011.443 30 Reeves MJ, Bushnell CD, Howard G, et al. on 3 December 2018 by guest. Protected b
http://www.bmj.com/
BMJ: first published as 10.1136/bmj.k4168 on 24 October 2018. Downloaded from Sex differences
in stroke: epidemiology, clinical presentation, medical
care, and outcomes. Lancet Neurol 2008;7:915-26. doi:10.1016/S1474-4422(08)70193-5 25 Myint PK, Luben RN, Wareham NJ, Bingham SA, Khaw KT. Combined effect of health behaviours and risk of first ever
stroke in 20,040 men and women over 11 years’ follow-up in
Norfolk cohort of European Prospective Investigation of Cancer
(EPIC Norfolk): prospective population study. BMJ 2009;338:b349. doi:10.1136/bmj.b349 26 Seshadri S, Wolf PA. Lifetime risk of stroke and dementia:
current concepts, and estimates from the Framingham
Study. Lancet Neurol 2007;6:1106-14. doi:10.1016/S1474-4422(07)70291-0f 27 Touzé E, Rothwell PM. Sex differences in heritability of
ischemic stroke: a systematic review and meta-analysis. Stroke 2008;39:16-23. doi:10.1161/STROKEAHA.107.484618f Supplementary information: additional material
Supplementary table: table S3 showing SNPs
included in MEGASTROKE risk score Supplementary information: additional material
Supplementary table: table S3 showing SNPs
included in MEGASTROKE risk score 28 Traylor M, Rutten-Jacobs LC, Holliday EG, et al. Differences in
Common Genetic Predisposition to Ischemic Stroke by Age and Sex. Stroke 2015;46:3042-7. doi:10.1161/STROKEAHA.115.009816 No commercial reuse: See rights and reprints http://www.bmj.com/permissions Subscribe: http://www.bmj.com/subscribe
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EDUCAÇÃO EM DIREITOS HUMANOS NO ESTADO DE SÃO PAULO
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Revista Espaço do Currículo
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cc-by
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EDUCAÇÃO EM DIREITOS HUMANOS
NO ESTADO DE SÃO PAULO
EDUCATION IN HUMAN RIGHTS IN
THE STATE OF SÃO PAULO
EDUCACIÓN EN DERECHOS
HUMANOS EN EL ESTADO DE SÃO
PAULO Revista Espaço do Currículo
ISSN 1983-1579
Doi: 10.15687/rec.v16i1.63713
http://periodicos.ufpb.br/ojs2/index.php Resumo: Este artigo tem o objetivo de
contribuir com as permanentes e recentes
discussões acerca da educação em direitos
humanos e apresenta uma análise do Plano
Estadual do Estado de São Paulo de Educação
em Direitos Humanos, as questões relacionadas
aos Direitos Humanos vêm ganhando destaque
em diversos setores da sociedade brasileira, seja
de forma declarada, seja na sua negação. Tal
debate, portanto, não pode ficar de fora da
educação formal, reconhecendo o espaço
escolar como um local privilegiado para que se
possam realizar momentos de reflexão em
torno das concepções e práticas, sem esquecer
que a instituição escolar e seu público, estão
inseridos em um determinado contexto social,
político, econômico e cultural. A política pública
desenvolvida para a promoção da educação em
direitos humanos para os estudantes de São
Paulo. Trata-se de uma pesquisa de natureza
qualitativa, alicerçada na Pesquisa bibliográfica. Neste contexto, busca-se através da leitura de
documentos sobre direitos humanos, e análise
das
ações
públicas
desenvolvidas
para
implementação da Educação em Direitos
Humanos no Currículo Paulista. Priscila Lourenço Soares Santos
Licenciada em História
Mestranda em Ciências Humanas pela
Universidade Santo Amaro e Professora
de História na Educação Básica de São
Paulo, Brasil. E-mail:
priscilalourenco@prof.educacao.sp.gov.br
Orcid: https://orcid.org/0000-0001-5346-
9732 Palavras-chave: Políticas Públicas. Educação. Currículo. Diretos Humanos. Palavras-chave: Políticas Públicas. Educação. Currículo. Diretos Humanos. REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 1
Recebido em: 15/07/2022
Alterações recebidas em: 22/09/2022
Aceito em: 25/09/2022
Publicação em: 31/01/2023
Como citar este artigo:
SANTOS P. L. S. EDUCAÇÃO EM DIREITOS
HUMANOS NO ESTADO DE SÃO PAULO. Revista Espaço do Currículo, v. 16, n. 1, p. 1-
13,
2023. ISSN2177-2886. DOI:
https://doi.org/10.15687/rec.v16i1.63713. Recebido em: 15/07/2022
Alterações recebidas em: 22/09/2022
Aceito em: 25/09/2022
Publicação em: 31/01/2023 REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 1 SANTOS P. L. S. Abstract: This article aims to contribute to the ongoing and recent discussions about human rights
education and presents an analysis of the State Plan of the State of São Paulo for Human Rights
Education. Brazilian society, either in a declared way or in its denial. Keywords: Public Policies. Education. Resume. Human Rights. Resumem: Este artículo tiene como objetivo contribuir a las discusiones permanentes y recientes sobre
la educación en derechos humanos y presenta un análisis del Plan Estatal del Estado de São Paulo para la
Educación en Derechos Humanos, las cuestiones relacionadas con los Derechos Humanos vienen
ganando protagonismo en diversos sectores de la sociedad brasileña. , ya sea de forma declarada o en su
negación. Tal debate, por lo tanto, no puede quedar fuera de la educación formal, reconociendo el
espacio escolar como un lugar privilegiado para momentos de reflexión sobre concepciones y prácticas,
sin olvidar que la institución escolar y sus públicos están insertos en un determinado contexto social,
político, económico. y contexto cultural. La política pública desarrollada para promover la educación en
derechos humanos de los estudiantes de São Paulo. Se trata de una investigación cualitativa, basada en
una investigación bibliográfica. En este contexto, se busca a través de la lectura de documentos sobre
derechos humanos y análisis de acciones públicas desarrolladas para la implementación de la Educación
en Derechos Humanos en el Currículo de São Paulo. Palabras clave: Políticas públicas. Educación. Reanudar. Derechos humanos. EDUCAÇÃO EM DIREITOS HUMANOS
NO ESTADO DE SÃO PAULO
EDUCATION IN HUMAN RIGHTS IN
THE STATE OF SÃO PAULO
EDUCACIÓN EN DERECHOS
HUMANOS EN EL ESTADO DE SÃO
PAULO Such a debate, therefore, cannot be
left out of formal education, recognizing the school space as a privileged place so that moments of
reflection can be carried out around the concepts and practices, without forgetting that the school
institution and its public are inserted in a given social, political, economic and cultural context. The public
policy developed to promote human rights education for students in São Paulo. This is a qualitative
research, based on bibliographic research. In this context, it is sought through the reading of documents
on human rights, and analysis of public actions developed for the implementation of Education in Human
Rights in the Paulista Curriculum. Keywords: Public Policies. Education. Resume. Human Rights. ou ela possam vir a sofrer. Os Direitos Humanos nascem justamente das preocupações em relação às formas autoritárias e
arbitrárias de tolher a dignidade, a liberdade e as oportunidades de igualdade dos seres humanos em sua
convivência social. Lembrando que o fato histórico que desencadeia tal discussão são os Estados
nazifascistas, sob o pano de fundo dos acontecimentos da II Guerra Mundial (1939 – 1945), um dos
conflitos armados mais devastadores da liberdade e dignidade que o Ocidente vivenciou em sua história,
com um saldo de mais de 30 milhões de mortos, considerando estimativas muito otimistas. Cremos,
portanto, que os Direitos Humanos não são assuntos marginais e nos negamos a acreditar que a sua
defesa e promoção sejam inofensivas e sem resultados práticos. Olhando para o tempo presente, podemos elencar pelo menos duas razões que nos levam a tal
consideração. A primeira é a de que, a partir da Declaração de 1948, atualizadas pela Declaração de Viena
de 1993, os Direitos Humanos se tornam em um instrumento legal, corroborado internacionalmente,
permitindo ações efetivas na sua defesa e promoção, como sanções comerciais e condenações junto à
Corte Internacional de Justiça. A segunda razão se deve ao fato de que ao adotar os Direitos Humanos
como a referência de ação do Estado, nos ancoramos sob questões que perpassaram por um amplo
debate ao nível internacional, ratificados por um documento que conta com mais de 190 países
signatários. Ou seja, a construção de seus conceitos e as suas práticas estão assente em discussões que
procuram ir além das especificidades culturais de cada sociedade ou nação, porém, afirmando suas
autodeterminações e colocando o aspecto da tolerância como um dos principais pilares que os
constituem. Nesse bojo, compreendendo as principais questões que perpassaram e perpassam a constituição
do conceito de Direitos Humanos, ficará claro que a perspectiva de inclusão dos Direitos Humanos nos
currículos escolares não deveres se restringir à criação de mais uma disciplina. Mas sim que eles devem
ser um referencial de ação da estrutura escolar, englobando a educação tanto de alunos quanto de
professores, funcionários e equipe gestora, ou seja, todos os agentes envolvidos no processo de ensino
e aprendizagem. 1. HISTÓRICO Em nossa análise para este artigo, partimos do questionamento da centralidade do Estado de São
Paulo no estabelecimento das políticas curriculares para o desenvolvimento da Educação em Direitos
Humanos. As questões relacionadas aos Direitos Humanos vêm ganhando destaque em diversos setores da
sociedade brasileira, seja de forma declarada, seja na sua negação. Tal debate, portanto, não pode ficar
de fora da educação formal, reconhecendo o espaço escolar como um local privilegiado para se poderem
realizar momentos de reflexão em torno das concepções e práticas, sem esquecer que a instituição
escolar e seu público, estão inseridos em um determinado contexto social, político, econômico e cultural. Quando vamos falar a respeito de valores entre os seres humanos, todo cuidado é pouco para que
não os tornemos absolutos. Caso façamos isso, corremos o risco de naturalizar e tornar inerentes
características humanas que, na conformação das estruturas das sociedades, são frutos de constructos
sociais e dependem de complexas relações intersubjetivas e coletivas, aspectos que variam no tempo e
no espaço. Nesse sentido, compreendemos que os Direitos Humanos englobam os valores humanos, pois
defendem a garantia da dignidade humana contra qualquer forma de opressão, para que o sujeito
portador de direitos possa usufruir de sua liberdade em igualdade de condições e oportunidades na sua
atuação em sociedade, sempre na busca de uma convivência pacífica e harmônica. É somente na garantia
desses direitos que podemos exigir do cidadão e da cidadã uma atuação consciente e responsável,
possibilitando um sujeito que saiba defender os seus direitos, pois reconhece que é somente a partir de
sua garantia que se efetivará as suas próprias condições de liberdade. Tais preceitos dos Direitos Humanos estão embasados em dois documentos: a Declaração
Universal dos Direitos Humanos, aprovada pela ONU em 1948, e a Declaração da Conferência Mundial de
Direitos Humanos, aprovada em 1993, em Viena. Documentos dos quais o Brasil é um dos signatários. Assim, mais do que uma carta de intenções, as declarações são instrumentos jurídicos que visam garantir
a defesa e a promoção dos direitos do cidadão ou da cidadã, contra as violações e arbitrariedades que ele REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 2 2. DIREITOS HUMANOS EM SÃO PAULO Nesta fase do estudo, foi realizada uma primeira busca de informações sobre estado de São Paulo,
concernentes em relação aos Direitos Humanos oficialmente. Para desenvolver a análise, além de contar
a documentação oficial e leitura de bibliografia. Assim também irei apresentar ações desenvolvidas pelo
estado de São Paulo sobre a temática, para estabelecer uma linha de trabalho com a Educação em Direitos
Humanos. Importante ressaltar que a questão das discussões sobre Direitos Humanos ainda está muito no
início mesmo com todas as legislações vigentes no Brasil, sendo necessário este trabalho de construção
de uma política pública como diz: Daí a importância educativa, preventiva, inibidora e transformadora das
políticas públicas de direitos humanos, as quais se encontram em estado de
germinação, desenvolvimento, implementação e diagnóstico por parte do
Estado brasileiro, em diversos âmbitos de atuação, através dos governos federal,
estaduais e municipais. (BITTAR, 2018, P. 16) No âmbito da Secretaria da Justiça e da Defesa da Cidadania foi instituído o Observatório de Direitos
Humanos, para monitorar as violações, denunciadas pela sociedade civil, na abrangência estadual. Ou
seja, criou-se um instrumento de controle da sociedade civil sobre o Estado, para ele poder possa garantir
cada vez mais a defesa dos direitos de cidadania. Com relação à Secretaria de Estado de Educação se mostra preocupado com as ações a serem
desenvolvidas, buscando um plano de ação no sistema de ensino que reforce o caráter transversal e
interdisciplinar, em que os Direitos Humanos sejam um eixo estruturador, tendo como objetivo a
construção de valores democráticos voltados para a cultura dos Direitos Humanos. Segundo Candau a educação escolar não pode ser pensando e construída com uma perspectiva
meramente instrumental. A educação escolar não pode ser reduzida a um produto que se negocia na lógica
do mercado; nem ter como referência quase que exclusivamente a aquisição de
determinados “conteúdos”, por mais socialmente reconhecidos que sejam. Deve ter como horizonte a construção de uma cidadania participativa, a
formação de sujeitos de direito, o desenvolvimento da vocação humana de todas
as pessoas nela implicadas. (CANDAU, 2012, p. 721) Assim da mesma forma devemos buscar a compreensão sobre educação em Direitos Humanos nas
escolas em seus currículos e nas políticas públicas que estão sendo construídas para o desenvolvimento
dela. ou ela possam vir a sofrer. Para desenvolver a análise
mbém irei apresentar ações des
ma linha de trabalho com a Educa
s sobre Direitos Humanos aind
, sendo necessário este trabalho
preventiva, inibidora e trans
umanos, as quais se encontram
implementação e diagnóstico
mbitos de atuação, através dos go
2018, P. 16) SANTOS P. L. S. direitos humanos, a educação por meio dos direitos humanos e a educação para os direitos humanos”. (ROSENDO, 2018, p. 470). Assim é importante este olhar para educação formal e a não formal dos
estudantes, pois estão em processo de formação com cidadão. Para esta pesquisa realizei uma análise
dos documentos presentes no site da Secretaria de Educação do Estado de São Paulo, e selecionei livros
que trabalham com a temática Educação em Direitos Humanos com base a formação formal dos
estudantes da educação básica. ou ela possam vir a sofrer. Contudo, vale lembrar que as questões colocadas sob a rubrica de Educação em Direitos Humanos
não se restringem apenas à educação formal, inclui também a educação não formal, a educação dos
agentes de justiça, segurança e a educação e mídia, conforme colocado no Plano Nacional de Educação
em Direitos Humanos, de 2003. A proposição da Educação de Direitos Humanos, assim, tem uma abrangência que ultrapassa
questões curriculares das escolas e se compromete com todos os níveis da vida social, sendo, então, uma
preocupação de todos os cidadãos e cidadãs interessados numa convivência justa e igualitária, quer seja
na sua defesa na sua promoção. Segundo Haddad a DUDH estipula que a educação deve ter como objetivo ampliar a personalidade
humana e fortalecer os direitos de todos. Em outras palavras, não basta que todos tenham acesso à
educação. Também é necessário que essa educação promova a individualidade de cada pessoa, sua
personalidade e fortaleça seus direitos declarados pela DUDH, assim ele afirma: A educação é um elemento fundamental para a realização dessa vocação humana não apenas a
educação escolar, mas a educação no seu sentido amplo, a educação pensa da num sistema geral, que
implica na educação escolar, mas que não se basta nela, porque o processo educativo começa como
nascimento extermina apenas no momento da morte do ser humano. (HADDAD, 2004, p.1) Mais uma vez destacamos que não é apenas a partir de documentos e decretos que se transformam
pensamentos e atitudes, no entanto, eles são instrumentos mais incisivos de cobrança perante os
poderes constituídos, na medida em que a sociedade civil participa de forma ativa em suas elaborações,
ao menos é o que se espera de seu envolvimento nesses processos. É de suma importância, assim, que a
sociedade civil se organize e cobre do Estado que suas ações estejam baseadas em princípios que
respeitem a igualdade de oportunidades e o respeito à diferença, que constitui essa nossa sociedade tão
plural e diversificada. Segundo Rosendo “A educação e formação em direitos humanos incluem: a educação sobre os REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 3 S. manos e a educação para os dire
har para educação formal e a
dadão. Para esta pesquisa real
ação do Estado de São Paulo, e
Humanos com base a forma
usca de informações sobre estad
nte. 2. DIREITOS HUMANOS EM SÃO PAULO O que propomos aqui são análises dos documentos nacionais e demais literatura sobre a EDH, não
podendo ser considerado como um manual, mas sim como ponto de partida para que profissionais da
educação envolvidos no projeto de Escola de Tempo Integral entendam o significado desta ação e
cumpram com êxito o seu papel de ministrar oficinas eletivas sobre esta temática para a construção de
um conhecimento para a rede pública de ensino paulista. Assim, trata-se de uma tarefa e missão pretensiosas e temos a plena consciência de que a sociedade
brasileira exige muito mais do que apenas uma séria discussão em torno dos tantos problemas
enfrentados em nosso dia a dia. Contudo, sabemos também que não conseguiremos efetivar tais medidas e políticas públicas se a
sociedade não se envolver e abraçar os esforços que encaminhamos para essas questões, não apenas no
auxílio para implementar políticas públicas que sejam eficazes, mas, sobretudo, que a sociedade civil
possa ser os nossos olhos e ouvidos, cobrando e controlando quaisquer desvios que observarem na
administração de recursos e ações. Esse também é um papel que o cidadão brasileiro deve começar a exercer em sua cidadania, o de
fiscalizar as ações desenvolvidas pelo Estado, no intuito de que ele seja efetivamente direcionado para o
cumprimento de seu papel, o de garantir uma qualidade de vida e dignidade à sua população. No entendimento de Silva (2016), a educação é dedicada a isso. A emancipação fundamenta-se no
processo de conscientização e responsabilização das pessoas diante de condições de opressão, domínio,
exclusão social e política, com o objetivo de que as pessoas demonstrem sua raiva e intolerância diante
do desrespeito ao próximo e da valorização da vida em sua plenitude. Na mesma medida, a educação paulista vem contribuindo para que se aprofundem na educação
básica, junto à sociedade civil, os debates concernentes às questões relacionadas aos Direitos Humanos
que vêm ganhando destaque em diversos setores da sociedade brasileira, seja de forma declarada, seja
na sua negação. Tal debate, portanto, não pode ficar de fora da educação formal, reconhecendo o espaço
escolar como um local privilegiado para se poderem realizar momentos de reflexão em torno das
concepções e práticas, sem esquecer que a instituição escolar e seu público, estão inseridos em um
determinado contexto social, político, econômico e cultural. 2. DIREITOS HUMANOS EM SÃO PAULO Esta análise busca sintetizar todas as ideias aqui colocadas, procurando refletir e dialogar com os
documentos paulistas e ações desenvolvidas pela secretaria de educação, o Currículo Paulista e seus
materiais de apoio, podemos expandi-las para todas Ciências Humanas Aplicadas para os professores e
estudantes, assim uma cultura que efetive uma sociedade democrática, em que o respeito e a tolerância
sejam os princípios que norteiam a convivência social estão presentes no material do Currículo em Ação. Certamente de que isso se dá das mais diversificadas maneiras, e não se efetivará apenas discutindo
tais temas em sala de aula, mas sim procurando incutir formas práticas, em que os valores dos Direitos
Humanos se conectem de forma intrínseca ao cotidiano vivido dos profissionais das escolas estaduais de
São Paulo. REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 4 O tema Educação em Direitos Humanos (EDH) se configura como um tema transversal, mas
consideramos importante que todo o currículo esteja integrado a essa proposta. E que toda a escola e
professores possam contribuir para a construção de uma educação que perceba os indivíduos como
sujeitos de direitos, colocando a dignidade da pessoa humana no centro de referência de todas as ações
desencadeadas por estes atores. Há uma clara necessidade do Brasil em avançar na EDH. Dentre os agentes que podem empenhar
para este propósito está a educação básica. Dessa forma, as unidades de ensino poderão contribuir para
valorização da diversidade, para o respeito ao outro, para conscientização e ampliação da cidadania. Há
uma compreensão de que a prática da EDH ultrapassa até mesmo os muros da escola. Sabemos dos
desafios a serem enfrentados, mas sabemos da importância da incorporação da EDH nas políticas
educacionais e principalmente no cotidiano das instituições escolares. Umas das prerrogativas para o desenvolvimento da EDH na educação básica será relacionamento
da escola com a comunidade local, reconhecendo suas fragilidades, debatendo e buscando soluções para
os problemas comuns que afetam, não somente os estudantes, mas suas famílias e o contexto no qual
está inserido. Entendemos que os processos educacionais voltados para os direitos humanos deverão
contribuir para a desnaturalização em relação a situações que desumanizam os sujeitos, que retiram a
cidadania, que violam os direitos fundamentais do cidadão. 2. DIREITOS HUMANOS EM SÃO PAULO O atual Currículo Paulista do Estado de São Paulo está em consonância com a perspectiva da EDH,
sendo que desde o Ensino Fundamental dos Anos Finais até a última série do Ensino Médio, em todas as
disciplinas, os professores estão trabalhando os seus aspectos institucionais, históricos, práticos e
aspectos teóricos e conceituais do tema. Em relação ao Ensino Médio, destacamos a área de Ciências
Humanas, que possui maior ênfase em relação à apresentação de temas importantes para a EDH. Nunca podemos esquecer que currículo, escolhas de culturalmente limitadas pelo tempo escolar e
a capacidade do estudante estão presentes nesta instituição. O primeiro problema que deve ser
enfrentado pelos agentes que lidam com essa tarefa é baseado na esfera política e social, onde deve-se
reconhecer que todos são levados em conta por sua personalidade, pois nem todos os estudantes leem
tudo ou o mesmo conteúdo. Portanto, é preciso pensar nos objetivos a serem alcançados, ou seja, o que,
como, como e por que ensinar, quais valores devem ser transmitidos, quais métodos devem ser utilizados
na seleção. Nestas questões, estarão os fundamentos da especificação curricular. Ao analisar o Currículo de Ciências Humanas (Filosofia Geografia, História e Sociologia),
encontramos os temas como: democracia e justiça social, o estado, ética, racismo, meio ambiente,
construção da identidade, gênero, migração e Diversidade: O estrangeiro do ponto de vista sociológico,
o significado de ser cidadão ontem e hoje, os direitos civis, políticos, sociais e humanos, as diferentes
formas de violência, a desigualdade social e a própria desumanização do outro. Encontramos pontualmente, no Currículo de História, ao longo do Ensino Fundamental dos Anos
Finais e Ensino Médio, os estudos sobre o Iluminismo, Revolução Francesa, Golpe e Ditadura Militar,
remetem a discussões sobre Direitos Humanos e sua violação. Desse modo, são de conhecimento dos
educandos da rede de ensino do Estado de São Paulo, conteúdos curriculares referentes ao tema Direitos
Humanos presentes para ser desenvolvidos com os estudantes durante a educação formal. Muito importante a presenças dos conteúdos para os estudantes, entretanto, faz-se importante
reconhecer que os princípios e conceitos defendidos nos temas trabalhados na EDH dizem respeito não
somente aos educandos e professores das disciplinas, mas de toda a comunidade escolar, sobretudo no
que tange aos dispositivos legais que estão estabelecidos pela Legislação vigente e devem ser de
conhecimento de todos os agentes envolvidos no processo educacional: professores, funcionários, corpo
gestor. 2. DIREITOS HUMANOS EM SÃO PAULO Quando analisamos conseguimos observar que houve um grande avanço no Brasil nos últimos anos
sobre esta temática, principalmente a partir da elaboração e publicação do Plano Nacional de Educação
em Direitos Humanos (PNEDH/2006), que define princípios norteadores para a educação em Direitos
Humanos, inclusive para a educação básica, objeto em questão. Neste caso, chamamos a atenção para
trechos do documento que afirma que “a prática escolar deve ser orientada para a educação em direitos
humanos, assegurando o seu caráter transversal e a relação dialógica entre os diversos atores sociais.” REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 5 SANTOS P. L. S. Entendemos dessa forma, que a Educação em Direitos Humanos deve permear os currículos da Educação
Básica, não sendo necessário o seu atendimento em formato disciplinar. Deve estar contida nos objetivos declarados junto ao projeto político pedagógico, de modo a
favorecer práticas educacionais democráticas no cotidiano das unidades escolares. O Plano Nacional de
Educação em Direitos Humanos (PNEDH) que preconiza que: [...] a educação em direitos humanos deve abarcar questões concernentes aos
campos da educação formal, à escola, aos procedimentos pedagógicos, às
agendas
e
instrumentos
que
possibilitem
uma
ação
pedagógica
conscientizadora e libertadora, voltada para o respeito e valorização da
diversidade, aos conceitos de sustentabilidade e de formação da cidadania ativa
(BRASIL, 2009, p. 23). [...] a educação em direitos humanos deve abarcar questões concernentes aos
campos da educação formal, à escola, aos procedimentos pedagógicos, às
agendas
e
instrumentos
que
possibilitem
uma
ação
pedagógica
conscientizadora e libertadora, voltada para o respeito e valorização da
diversidade, aos conceitos de sustentabilidade e de formação da cidadania ativa
(BRASIL, 2009, p. 23). Compreendemos assim, como versa o PNEDH/2006, que fazem parte da perspectiva da Educação
em Direitos Humanos, a inclusão no currículo de temáticas como Gênero, Identidade, Raça e Etnia,
Sexualidade, Diversidade, entre outros, bem como as inúmeras formas de discriminação e violações de
direitos na sociedade. Outro documento importante e que merece destaque são as Diretrizes Nacionais para a Educação
Básica: diversidade e inclusão, quando em seu capítulo 9, trata da Educação em Direitos, enfatizando a
importância da educação para mudança e transformação social pautada nos princípios de da dignidade
humana, igualdade de direitos, reconhecimentos e valorização das diversidades, laicidade do Estado,
democracia na educação, transversalidade, vivência, globalidade e sustentabilidade socioambiental. 3. CURRÍCULO ESTADUAL E DIREITOS HUMANOS No estado de São Paulo nos anos de 2017 e 2018, o Conselho Estadual de Defesa dos Direitos da
Pessoa Humana do Estado de São Paulo (CONDEPE) em parceria com órgãos públicos, universidades
públicas e movimentos sociais empreenderam esforços para a construção de um Plano de Educação em
Direitos Humanos. A iniciativa tinha como propósito central a elaboração de metas e objetivos para o
desenvolvimento de políticas públicas de educação em Direitos Humanos. Encontramos em nossa verificação que as DEEDH-SP são condizentes com o Currículo Paulista, para
as etapas do Ensino Fundamental Anos Finais e do Ensino Médio. O documento define um rol de
competências, habilidades e objetos de conhecimento que aborda temáticas diretamente relacionadas
aos Direitos Humanos, reiterando os pressupostos da Base Nacional Comum Curricular (BNCC) e da
legislação nacional referente à Educação em Direitos Humanos (Decreto nº 7.037/2009, Parecer CNE/CP
nº 8/2012 e Resolução CNE/CP nº 1/2012), dos quais destacamos duas Competências Gerais: 7. Argumentar com base em fatos, dados e informações confiáveis, para
formular, negociar e defender ideias, pontos de vista e decisões comuns que
respeitem e promovam os direitos humanos, a consciência socioambiental e o
consumo responsável em âmbito local, regional e global, com posicionamento
ético em relação ao cuidado de si mesmo, dos outros e do planeta. 9. Exercitar a empatia, o diálogo, a resolução de conflitos e a cooperação,
fazendo-se respeitar e promovendo o respeito ao outro e aos direitos humanos,
com acolhimento e valorização da diversidade de indivíduos e de grupos sociais,
seus saberes, identidades, culturas e potencialidades, sem preconceitos de
qualquer natureza. (SÃO PAULO, 2019, p.30.) 9. Exercitar a empatia, o diálogo, a resolução de conflitos e a cooperação,
fazendo-se respeitar e promovendo o respeito ao outro e aos direitos humanos,
com acolhimento e valorização da diversidade de indivíduos e de grupos sociais,
seus saberes, identidades, culturas e potencialidades, sem preconceitos de
qualquer natureza. (SÃO PAULO, 2019, p.30.) Educar os estudantes do século XXI, formar cidadãos globais que enfrentem o planeta em que
vivem, buscando um desenvolvimento humano completo são desafios que exigem ações de todos os
atores envolvidos no processo de educação. Há algum tempo são utilizados testes que buscam medir a
inteligência dos alunos testando suas habilidades cognitivas. presentes em nossa sociedade, além do fomento e promoção da cultura de paz. Em relação à formação dos professores da rede, a Secretaria de Estado da Educação, encontramos
em pesquisa que a formação e realizada através da Escola de Formação e Aperfeiçoamento de
Professores (EFAPE), com validação dos técnicos de Ciências Humanas da Coordenadoria de Gestão da
Educação Básica (CGEB), promove desde 2013, o curso Introdução aos Direitos Humanos e Estatuto da
Criança e Adolescente (ECA) para Educadores na modalidade EAD. Segunda Silva formação dos
professores sobre Direitos Humanos, deve refletir sua realidade para sala de aula: Frente a isso, o desafio dos educadores é descobrir, entre os muitos métodos
utilizados atualmente, qual deles é o que melhor se adapta à realidade social dos
alunos e quais os estimularão de forma efetiva. (SILVA, 2021, p.17) Segundo o regulamento do curso promovido pela Secretaria de Educação ele teve duração de 60
horas, sendo dividido em 10 módulos com duração de 6 horas cada. O público-alvo são professores,
professores mediadores escolares (PMEC), diretores, vice-diretores, professores coordenadores (PC) e
supervisores, conselheiros tutelares dos municípios do Estado de São Paulo, profissionais que atuam no
âmbito da SEDUC e demais servidor de apoio escolar. 2. DIREITOS HUMANOS EM SÃO PAULO Estes devem estar aptos quanto ao conhecimento do sistema de garantia de direitos, da rede de
proteção às crianças e aos adolescentes e da perspectiva da convivência humana democrática,
evidenciando no contexto escolar formas e estratégias (práticas) que visam o combate às desigualdades REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 6 questão de Direitos Humanos. Encontramos entre as competências específicas os aprendizados essenciais que devem ser
assegurados aos estudantes por um componente específico, ou por um conjunto de componentes
curriculares que integram determinada área. Uma competência implica na mobilização de conceitos,
procedimentos, habilidades, práticas cognitivas e socioemocionais, atitudes e valores para a resolução de
demandas da vida cotidiana. Na competência específica número 4 da área de Linguagens para os Anos
Finais do Ensino Fundamental, por exemplo, é salientado: 4. Utilizar diferentes linguagens para defender pontos de vista que respeitem o
outro e promovam os direitos humanos, a consciência socioambiental e o
consumo responsável em âmbito local, regional e global, atuando criticamente
frente a questões do mundo contemporâneo. (SÃO PAULO, 2019, p.96) [..] Essa competência trata da necessidade da promoção dos Direitos Humanos e
possui relação com a competência específica número 6 de Língua Portuguesa,
que trata da importância da análise crítica de conteúdos que violem os Direitos
Humanos: 6. Analisar informações, argumentos e opiniões manifestados em
interações sociais e nos meios de comunicação, posicionando-se ética e
criticamente em relação a conteúdos discriminatórios que ferem direitos
humanos e ambientais. (SÃO PAULO, 2019, p.30.) (SÃO PAULO, 2019, p.109) Por meio de exemplos como estes, é possível verificar que a educação em Direitos Humanos é uma
temática transversal que é abordada por diferentes áreas do Currículo Paulista dos Anos Finais do Ensino
Fundamental. Isso se reflete tanto nas competências maiores de diferentes áreas, quanto nas habilidades
de componentes curriculares específicos. Em relação ao Ensino Médio, indo ao encontro do que está
previsto na Resolução nº 03 de 21 de novembro de 2018, que atualizou as Diretrizes Curriculares Nacionais
para o Ensino Médio, dentre seus princípios específicos, o Currículo pressupõe que sejam assegurados: IV - respeito aos direitos humanos como direito universal; V - compreensão da diversidade e realidade dos sujeitos, das formas de produção
e de trabalho e das culturas. (SÃO PAULO, 2020, p.32) O que pode ser visto em uma série de habilidades de diferentes componentes e em competências
específicas da área de Ciências Humanas e Sociais Aplicadas e da área de Linguagens e suas Tecnologias. Como exemplo, duas competências específicas da área de Linguagens para o Ensino Médio que abordam
a temática dos Direitos Humanos: 2. 3. CURRÍCULO ESTADUAL E DIREITOS HUMANOS Assim o currículo das escolas pode ser o campo um diálogo pedagógico entre o que se aprende e
o que se ensina, sempre considerando a convivência e a formação de pessoas muito diferentes. (ARROYO,
2011). Desta forma as reflexões sobre Direitos Humanos presente neste documento formal têm grande
importância para formação dos estudantes. Ao analisar o Currículo Paulista para as etapas dos Anos Finais do Ensino Fundamental e Ensino
Médio, que está pautado em competências visa observar se ele apresenta esta visão e reflexão para a REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 7 SANTOS P. L. S. questão de Direitos Humanos. questão de Direitos Humanos. 90) Tal população é composta de uma diversidade étnica e cultural que inclui
comunidades indígenas, comunidades quilombolas, autodeclarados negros e
pardos, populações prisionais, adolescentes em cumprimento de medidas
socioeducativas,
comunidades
do
campo
(acampados,
assentados
e
comunidades tradicionais), estudantes migrantes internacionais, estudantes
itinerantes etc., é preciso criar possibilidades para que esses educandos
desenvolvam suas habilidades. Dessa forma, os conteúdos curriculares devem
ser adaptados, considerando as especificidades de cada estudante, respeitando,
assim, as potencialidades e dificuldades individuais – ritmos diferentes de
aprendizagem – por meio de estratégias diversificadas de ensino. (SÃO PAULO,
2020, p. 90) O segundo objetivo desta Diretriz Estaduais de Educação em Direitos Humanos do Estado de São
Paulo, evidencia que, para promoção de uma “educação com qualidade social, acesso e permanência” é
importante “desenvolver o respeito a diversas nacionalidades e suas culturas no currículo escolar”. Assim
sendo, é importante pontuar que uma das bases do Currículo Paulista é o princípio da equidade, que visa
assegurar a inclusão de todos os estudantes nas escolas e a garantia do direito a uma educação pública e
de qualidade, como prevê a Lei de Diretrizes e Bases da Educação. Já na apresentação do Currículo Paulista da etapa do Ensino Fundamental é enfatizado que para
garantia da equidade é necessário “respeitar a diversidade cultural, a socioeconômica, a étnico-racial, a
de gênero e os socioculturais presentes no território estadual”. (SÃO PAULO, 2019, p.27) Diferentes componentes curriculares possuem competências e habilidades que abordam temáticas
a este respeito e buscam inserir no repertório manifestações culturais de nacionalidades diferentes. Destacamos aqui a oitava competência específica de História, que vai ao encontro desta perspectiva: 8. Compreender a história e a cultura africana, afro-brasileira, imigrante e
indígena, bem como suas contribuições para o desenvolvimento social, cultural,
econômico, científico, tecnológico e político e tratar com equidade as diferentes
culturas. (SÃO PAULO, 2019, p. 461) Neste sentido, citamos também uma competência específica e uma habilidade de Ciências
Humanas e Sociais Aplicadas do Ensino Médio, que dentro da categoria Território e Fronteira vai trabalhar
a compreensão das relações de poder que envolvem os processos migratórios e suas implicações para os
direitos humanos: Competência Específica: 2. Analisar a formação de territórios e fronteiras em
diferentes tempos e espaços, mediante a compreensão das relações de poder
que determinam as territorialidades e o papel geopolítico dos Estados-nações. questão de Direitos Humanos. Compreender os processos identitários, conflitos e relações de poder que
permeiam as práticas sociais de linguagem, respeitando as diversidades e a
pluralidade de ideias e posições, e atuar socialmente com base em princípios e
valores assentados na democracia, na igualdade e nos Direitos Humanos,
exercitando o autoconhecimento, a empatia, o diálogo, a resolução de conflitos
e a cooperação, e combatendo preconceitos de qualquer natureza. (SÃO
PAULO, 2020, p. 84) 3. Utilizar diferentes linguagens (artísticas, corporais e verbais) para exercer,
com autonomia e colaboração, protagonismo e autoria na vida pessoal e
coletiva, de forma crítica, criativa, ética e solidária, defendendo pontos de vista
que respeitem o outro e promovam os Direitos Humanos, a consciência
socioambiental e o consumo responsável, em âmbito local, regional e global. (SÃO PAULO, 2020, p. 89) No que diz respeito às Diretrizes Estaduais de Educação em Direitos Humanos do Estado de São
Paulo, tem como eixo orientador Educação Básica, coloca em seu primeiro objetivo a necessidade de
acompanhamento dos educandos com dificuldades de aprendizagem, adolescentes em cumprimento de
medida socioeducativa e adolescentes ou crianças em medida protetiva. REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 8 Neste sentido, é importante situar que o Currículo Paulista da etapa do Ensino Médio prevê que,
para sua implementação nas redes de ensino do Estado de São Paulo deve-se pressupor um compromisso
com a garantia da Educação Básica para “segmentos da população paulista que, devido às distintas
características socioculturais e/ou às diversas situações de vulnerabilidades, requerem atendimento
educacional inclusivo para efetivar seu direito à educação”. (SÃO PAULO, 2020, p. 90) Ainda especifica as características de grupos que compõem esta população, colocando sobre a
necessidade da realização de adaptações curriculares considerando cada caso individualmente: Tal população é composta de uma diversidade étnica e cultural que inclui
comunidades indígenas, comunidades quilombolas, autodeclarados negros e
pardos, populações prisionais, adolescentes em cumprimento de medidas
socioeducativas,
comunidades
do
campo
(acampados,
assentados
e
comunidades tradicionais), estudantes migrantes internacionais, estudantes
itinerantes etc., é preciso criar possibilidades para que esses educandos
desenvolvam suas habilidades. Dessa forma, os conteúdos curriculares devem
ser adaptados, considerando as especificidades de cada estudante, respeitando,
assim, as potencialidades e dificuldades individuais – ritmos diferentes de
aprendizagem – por meio de estratégias diversificadas de ensino. (SÃO PAULO,
2020, p. para os direitos humanos. Os regimes políticos e a “produção” dá moral. Geografia: As correntes migratórias, a produção e circulação de mercadorias e
suas marcas na paisagem; conflitos socioespaciais e organização territorial. História: Processos migratórios, suas motivações e desdobramentos (questões
étnicas, xenofobia e conflitos territoriais). Geografia: As correntes migratórias, a produção e circulação de mercadorias e
suas marcas na paisagem; conflitos socioespaciais e organização territorial. História: Processos migratórios, suas motivações e desdobramentos (questões
étnicas, xenofobia e conflitos territoriais). Sociologia: Processos de gentrificação em territorialidades urbanas: xenofobia,
migrações, conflitos socioespaciais e territoriais. (SÃO PAULO, 2020, p. 182) Sociologia: Processos de gentrificação em territorialidades urbanas: xenofobia,
migrações, conflitos socioespaciais e territoriais. (SÃO PAULO, 2020, p. 182) O Currículo esclarece que a promoção da equidade pressupõe o uso de respostas adequadas “ao
público atendido nas modalidades da Educação Especial, Educação de Jovens e Adultos, Educação do
Campo, Educação Escolar Indígena e Educação Escolar Quilombola, segundo as necessidades locais”. (SÃO PAULO, 2020, p. 27) Encontramos na análise dos materiais produzidos pela Secretaria Educação para as escolas,
explícita sobre a repressão e a resistência à ditadura civil - militar, devem ter sua distribuição assegurada
aos estudantes. Neste sentido, destacamos que uma série de habilidades do Currículo Paulista trabalham
habilidades e objetos de conhecimentos relacionados à ditadura civil - militar. Os materiais, Currículo em Ação cadernos de atividades voltados para estudantes que são
distribuídos para toda a rede pública de ensino e que são produzidos pela própria Secretaria da Educação,
trabalham diretamente todas as habilidades do Currículo Paulista. No caso dos Anos Finais do Ensino
Fundamental, o contexto de repressão da ditadura civil – militar é trabalhado diretamente no caderno de
História do 9º ano no 3º e 4º bimestres, cujas habilidades implicam em: (EF09HI19) Identificar e compreender o processo que resultou na ditadura civil-
militar no Brasil e discutir a emergência de questões relacionadas à memória e à
justiça sobre os casos de violação dos direitos humanos. (EF09HI19) Identificar e compreender o processo que resultou na ditadura civil-
militar no Brasil e discutir a emergência de questões relacionadas à memória e à
justiça sobre os casos de violação dos direitos humanos. (EF09HI20) Discutir os processos de resistência e as propostas de reorganização
da sociedade brasileira durante a ditadura civil-militar. (EF09HI20) Discutir os processos de resistência e as propostas de reorganização
da sociedade brasileira durante a ditadura civil-militar. questão de Direitos Humanos. Habilidade: (EM13CHS201) Analisar e caracterizar as dinâmicas das populações,
das mercadorias e do capital nos diversos continentes, com destaque para a
mobilidade e a fixação de pessoas, grupos humanos e povos, em função de
eventos naturais, políticos, econômicos, sociais, religiosos e culturais, de modo
a compreender e posicionar-se criticamente em relação a esses processos e às
possíveis relações entre eles. Objetos de Conhecimento: Filosofia: A reflexão ética: as exigências morais do
homem moderno. As exigências morais da contemporaneidade e as implicações REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 9 SANTOS P. L. S. para os direitos humanos. Os regimes políticos e a “produção” dá moral. para os direitos humanos. Os regimes políticos e a “produção” dá moral. Objetos de conhecimento: Os anos 1960: revolução cultural; A ditadura civil-
militar e os processos de resistência; as questões indígena e negra e a ditadura. (SÃO PAULO, 2019, p. 483) No Currículo do Ensino Médio, a temática também é abordada pela área de Ciências Humanas e
Sociais Aplicadas, e dentre as competências específicas e habilidades que colaboram com seu
desenvolvimento destacamos a questão do combate às formas de injustiça por meio do respeito aos
Direitos Humanos: Competência Específica: 5. Identificar e combater as diversas formas de injustiça,
preconceito e violência, adotando princípios éticos, democráticos, inclusivos e
solidários, e respeitando os Direitos Humanos. Habilidade: (EM13CHS503) Identificar diversas formas de violência (física,
simbólica, psicológica etc.), suas principais vítimas, suas causas sociais,
psicológicas e afetivas, seus significados e usos políticos, sociais e culturais,
discutindo e avaliando mecanismos para combatê-las, com base em argumentos
éticos. Objetos de Conhecimento: Objetos de Conhecimento: Filosofia: Comportamentos opressores e modos de violência: pressupostos e
implicações da opressão, da violência e indiferença em relação aos fenômenos
sociais. O totalitarismo e o terrorismo como ameaça a democracia e aos Direitos
Humanos. Geografia: Representação cartográfica da violência. O discurso da violência nas
campanhas políticas, propagandas ideológicas, redes sociais e no uso político de REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 10 História: O uso institucional (político, social e cultural) da violência: regimes
ditatoriais e totalitários, golpes de Estado e terrorismo, Apartheid na África do
Sul e segregação étnico-racial no mundo. Sociologia: Formas e dimensões da violência: física, psicológica e simbólica. (SÃO
PAULO, 2020, p. 483) As temáticas que devem ser abordadas nos currículos e materiais didáticos de modo a assegurar
uma abordagem transversal na abordagem dos Direitos Humanos. Como já esclarecido, a Educação em
Direitos Humanos é uma temática transversal que é abordada por diferentes áreas do Currículo Paulista,
no entanto, a diretriz cita uma série de Leis Federais, que também teriam de ser tratadas pelo Currículo e
pelos materiais. Assim sendo, é importante situar que o que fora colocado no objetivo em questão está em
consonância com o que aparece na própria Base Nacional Comum Curricular (BNCC), que orientou o
Currículo Paulista. A BNCC prevê que os seguintes temas transversais estejam presentes nos currículos
do Ensino Fundamental e do Ensino Médio, sendo eles contemplados nos objetos de aprendizagem na
área de Ciências Humanas e Sociais Aplicadas, Ciências da Natureza e suas Tecnologias, e na área de
Linguagens e suas Tecnologias. Encontramos em nossa analise curricular das eletivas sugeridas pela Secretaria de Educação do
Estado de São Paulo no cardápio de sugestões aos professores da rede, uma eletiva como o nome de
direitos e Direitos e cidadania foi criada pelo Instituto Auschwitz a partir do projeto Cidadania e
democracia desde a escola no âmbito do programa Inova, para trabalhar com estudantes do 6º ano do
Ensino Fundamental até 3º ano do Ensino Médio nas escolas estaduais. Objetos de Conhecimento: Está eletiva se apresenta com objetivo segundo o material trabalhar com as atitudes e os
comportamentos habituais dos estudantes e desenvolve conteúdo para a compreensão dos valores e
procedimentos que sustentam a confiança cívica entre cidadãos numa democracia, tais como a
capacidade de se colocar no lugar do outro, a necessidade de aprender a dialogar no espaço público e a
importância de construir argumentos fundamentados para a defesa de suas posições com autonomia dos
estudantes que realizam estas aulas. Em sua justificativa para seu desenvolvimento pedagógico está baseada na presente Base Nacional
Comum Curricular, através das metodologias aplicadas em seu conteúdo com as dinâmicas, análise de
notícias e vídeos, rodas de conversa e pesquisa, envolvendo temas contemporâneos e pertinentes à
realidade dos estudantes. Com o material produzido pelo Instituto Auschwitz os professores da rede têm acesso à proposta
pedagógica com um material disponibilizado no site da instituição para realização das atividades
semestralmente, finalizando com uma atividade de encerramento chamada pela Secretaria de Educação
como culminância. As sugestões envolvem projetos artísticos (6º e 7º), de comunicação social (8º e 9º) e
de ações cidadãs (Ensino Médio) presentes nos materiais elaborados. Devemos sempre refletir sobre “a educação é uma prática social e política cujas ferramentas são
elementos simbólicos, produzidos e manuseados pela subjetividade e mediados pela cultura.”
(SEVERINO, 2002, p. 72), contarmos avanços, dificuldades e desafios sobre a Educação em Direitos
Humanos para a educação básica para a formação inicial dos estudantes de São Paulo. CONSIDERAÇÕES FINAIS Em relação ao Currículo dos Anos Finais do Ensino Fundamental e do Ensino Médio da educação
básica, essas diante do exposto, é possível concluir que as diretrizes relacionadas ao Currículo estão
alinhadas ao que é nele proposto em relação à Educação para os Direitos Humanos. Os seus objetivos
estão em consonância com os pressupostos do Currículo e com os seus Materiais produzidos para a
implementação do Currículo do Estado de São Paulo. Por fim, acreditamos que seja importante debater e refletir a questão da Educação em Direitos REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 11 SANTOS P. L. S. Humanos, pois este se constitui como um desafio para a sociedade como um todo, com destaque para o
papel da Universidade no que se refere a formação de quadros para atuação na Educação. Humanos, pois este se constitui como um desafio para a sociedade como um todo, com destaque para o
papel da Universidade no que se refere a formação de quadros para atuação na Educação. Essa discussão se faz pertinente para que possa ser incutida junto aos novos profissionais, uma
ética de trabalho em que sejam valorizados os vários aspectos importantes para a melhor a convivência
social, com respeito a vida e a dignidade dos seres humanos. Um pensar e agir capaz de permitir
vislumbrar caminhos possíveis para o pleno desenvolvimento humano de todos os educandos na
educação formal. ARROYO, Miguel. Currículo, território em disputa. Petrópolis: Vozes, 2011. BITTAR, Eduardo C. B. Democracia e políticas públicas de direitos humanos: a situação atual do Brasil. Revista USP, (119), 11-28, 2018. https://doi.org/10.11606/issn.2316-9036.v0i119. BITTAR, Eduardo C. B. Democracia e políticas públicas de direitos humanos: a situação atual do Brasil. Revista USP, (119), 11-28, 2018. https://doi.org/10.11606/issn.2316-9036.v0i119. BRASIL. Programa Nacional de Direitos Humanos (PNDH-1). Secretaria de Direitos Humanos da
Presidência da República. Brasília: SEDH/MJ, 1996. BRASIL. Programa Nacional de Direitos Humanos (PNDH-1). Secretaria de Direitos Humanos da
Presidência da República. Brasília: SEDH/MJ, 1996. BRASIL. Parâmetros Curriculares Nacionais. Ensino Médio. Parte IV – Ciências Humanas e suas
Tecnologias. Brasília, DF: Ministério da Educação, Secretaria de Educação Média e Tecnológica, 2000. BRASIL. Programa Nacional de Direitos Humanos (PNDH-2). Secretaria de Direitos Humanos da
Presidência da República. Brasília: SEDH/PR, 2002. Disponível em:
http://www.dhnet.org.br/dados/pp/edh/pndh_2_integral.pdf. Acesso em: 24 jul. 2021. BRASIL. Programa Nacional de Direitos Humanos (PNDH-3). Secretaria de Direitos Humanos da
Presidência da República. Brasília: SEDH/PR, 2010. Disponível em:
http://www.dhnet.org.br/dados/pp/a_pdf/pndh3_programa_nacional_direitos_humanos_3.pdf. Acesso
em: 24 jul. 2021. CANDAU, Vera M. Direitos Humanos, educação e interculturalidade: as tensões entre igualdade e
diferença. Revista Brasileira de Educação, v. 13, n. 37, p. 45-56, jan./abr. 2008. CANDAU, Vera Maria. Direito à educação, diversidade e educação em direitos humanos. Educação e
Sociedade, Campinas, v.33, n. 120, p. 715-726, 2012. HADDAD, Sérgio. O direito à educação no Brasil. In: Relatoria Nacional para o Direito Humano à
Educação. Curitiba: DhESC Brasil, 2004. SANTOS, Boaventura de Sousa. Direitos humanos, o desafio da interculturalidade. Revista Direitos
Humanos, n. 2, p. 10-18, 2009. SILVA, Itamar Nunes. Tensão entre universalismo e interculturalismo na trajetória das lutas por direitos
humanos no Brasil. Revista Interdisciplinar de Direitos Humanos. Bauru, v. 4, n. 1, p.11-32, jan/jun. 2016. PIOVESAN, Flávia. Concepção contemporânea de Direitos Humanos. In: HADDAD, S.; GERACIANO, M. (Orgs.). A educação entre os Direitos Humanos. São Paulo: Cortez e Associados/Ação Educativa, 2006. SÃO PAULO. Secretaria da Educação do Estado de São Paulo. União dos Dirigentes Municipais de
Educação do Estado de São Paulo. Currículo Paulista. São Paulo: SEESP/UNDIME-SP, 2019. Disponível
em: https://efape.educacao.sp.gov.br/curriculopaulista/wpcontent/uploads/sites/7/2019/09/curriculo-
paulista-26-07.pdf. Acesso em 19 dez. 2020. SÃO PAULO. Currículo Paulista: Etapa Ensino Médio. 2020. Disponível em:
https://efape.educacao.sp.gov.br/curriculopaulista/ensino-medio/. Acesso dia 08 de nov. de 2021. SÃO PAULO. Currículo Paulista: Etapa Ensino Médio. 2020. Disponível em:
https://efape.educacao.sp.gov.br/curriculopaulista/ensino-medio/. Acesso dia 08 de nov. de 2021. UNESCO. Educação para a cidadania global: preparando alunos para os desafios do século XXI. Brasília
UNESCO, 2015. UNESCO. ARROYO, Miguel. Currículo, território em disputa. Petrópolis: Vozes, 2011. Educação para a cidadania global: preparando alunos para os desafios do século XXI. Brasília:
UNESCO, 2015. RAMOS, Aura Helena. Educação em Direitos Humanos: local da diferença. Revista Brasileira de
Educação, v. 16, n. 46, p. 191-272, jan/abr, 2011. RAMOS, Aura Helena. Educação em Direitos Humanos: local da diferença. Revista Brasileira de
Educação, v. 16, n. 46, p. 191-272, jan/abr, 2011. ROSENDO, Daniela; LAPA, Fernanda Brandão. EDUCAÇÃO E(M) DIREITOS HUMANOS E BNCC: OSENDO, Daniela; LAPA, Fernanda Brandão. EDUCAÇÃO E(M) DIREITOS HUMANOS E BNCC: REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 12 competências socioemocionais e ética ambiental. Revista Espaço do Currículo, [S. l.], v. 3, n. 11, 2018. DOI: 10.22478/ufpb.1983-1579.2018v3n11.40385. Disponível em:
https://periodicos.ufpb.br/index.php/rec/article/view/ufpb.1983-1579.2018v3n11.40385. Acesso em: 14
set. 2022. SEVERINO, Antonio Joaquim. Educação, sujeito e história. 1. ed. São Paulo: Olho D’Água. 2002. SILVA, Phamella Rodrigues da. Direitos humanos e educação com ênfase na formação de professores. 2021. 25 f. Trabalho de Conclusão de Curso (Graduação em Pedagogia) – Universidade Federal de
Uberlândia, Uberlândia, 2021. competências socioemocionais e ética ambiental. Revista Espaço do Currículo, [S. l.], v. 3, n. 11, 2018. DOI: 10.22478/ufpb.1983-1579.2018v3n11.40385. Disponível em:
https://periodicos.ufpb.br/index.php/rec/article/view/ufpb.1983-1579.2018v3n11.40385. Acesso em: 14
set. 2022. SEVERINO A t
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Olh D’Á competências socioemocionais e ética ambiental. Revista Espaço do Currículo, [S. l.], v. 3, n. 11, 2018. DOI: 10.22478/ufpb.1983-1579.2018v3n11.40385. Disponível em:
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set. 2022. competências socioemocionais e ética ambiental. Revista Espaço do Currículo, [S. l.], v. 3, n. 11, 2018. DOI: 10.22478/ufpb.1983-1579.2018v3n11.40385. Disponível em:
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set. 2022. SEVERINO, Antonio Joaquim. Educação, sujeito e história. 1. ed. São Paulo: Olho D’Água. 2002. EVERINO, Antonio Joaquim. Educação, sujeito e história. 1. ed. São Paulo: Olho D’Água. 2002 SILVA, Phamella Rodrigues da. Direitos humanos e educação com ênfase na formação de professores. 2021. 25 f. Trabalho de Conclusão de Curso (Graduação em Pedagogia) – Universidade Federal de
Uberlândia, Uberlândia, 2021. SILVA, Phamella Rodrigues da. Direitos humanos e educação com ênfase na formação de professores. 2021. 25 f. Trabalho de Conclusão de Curso (Graduação em Pedagogia) – Universidade Federal de
Uberlândia, Uberlândia, 2021. Esta obra está licenciada com uma Licença Creative Commons Atribuição-NãoComercial 4.0 Internacional. Esta obra está licenciada com uma Licença Creative Commons Atribuição-NãoComercial 4.0 Internacional. REV. ESPAÇO DO CURRÍCULO (ONLINE), JOÃO PESSOA, V.16 N.1, P. 1-13, JAN/ABR. 2023. 13
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https://zenodo.org/records/1273479/files/article.pdf
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A low-cost test approach for embedded RF passive circuits
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|
https://openalex.org/W2551832670
|
https://boris.unibe.ch/99946/1/art%253A10.1007%252FJHEP11%25282016%2529112.pdf
|
English
| null |
Search for the Standard Model Higgs boson produced by vector-boson fusion and decaying to bottom quarks in s = 8 $$ \sqrt{s}=8 $$ TeV pp collisions with the ATLAS detector
|
The Journal of high energy physics/The journal of high energy physics
| 2,016
|
cc-by
| 23,310
|
Published for SISSA by
Springer Received: June 8, 2016
Revised: September 28, 2016
Accepted: November 10, 2016
Published: November 21, 2016 Received: June 8, 2016
Revised: September 28, 2016
Accepted: November 10, 2016
Published: November 21, 2016 Received: June 8, 2016
Revised: September 28, 2016
Accepted: November 10, 2016
Published: November 21, 2016 Search for the Standard Model Higgs boson produced
by vector-boson fusion and decaying to bottom quarks
in √s = 8 TeV pp collisions with the ATLAS detector JHEP11(2016)112 The ATLAS collaboration E-mail: atlas.publications@cern.ch E-mail: atlas.publications@cern.ch Abstract: A search with the ATLAS detector is presented for the Standard Model Higgs
boson produced by vector-boson fusion and decaying to a pair of bottom quarks, using
20.2 fb−1 of LHC proton-proton collision data at √s = 8 TeV. The signal is searched for
as a resonance in the invariant mass distribution of a pair of jets containing b-hadrons in
vector-boson-fusion candidate events. The yield is measured to be −0.8 ± 2.3 times the
Standard Model cross-section for a Higgs boson mass of 125 GeV. The upper limit on the
cross-section times the branching ratio is found to be 4.4 times the Standard Model cross-
section at the 95% confidence level, consistent with the expected limit value of 5.4 (5.7) in
the background-only (Standard Model production) hypothesis. Keywords: Hadron-Hadron scattering (experiments), Higgs physics, proton-proton scat-
tering ArXiv ePrint: 1606.02181 doi:10.1007/JHEP11(2016)112 doi:10.1007/JHEP11(2016)112 Contents
1
Introduction
1
2
The ATLAS detector
3
3
Data and simulation samples
3
4
Object reconstruction
4
5
Event pre-selection
5
6
Multivariate analysis
5
7
Invariant mass spectrum of the two b-jets
6
8
Sources of systematic uncertainty
9
8.1
Experimental uncertainties
10
8.2
Modelling uncertainties on the mbb shape of the non-resonant background
10
8.3
Theoretical uncertainties
10
9
Statistical procedure and results
11
10 Cut-based analysis
13
11 Summary
15
The ATLAS collaboration
20 Contents
1
Introduction
1
2
The ATLAS detector
3
3
Data and simulation samples
3
4
Object reconstruction
4
5
Event pre-selection
5
6
Multivariate analysis
5
7
Invariant mass spectrum of the two b-jets
6
8
Sources of systematic uncertainty
9
8.1
Experimental uncertainties
10
8.2
Modelling uncertainties on the mbb shape of the non-resonant background
10
8.3
Theoretical uncertainties
10
9
Statistical procedure and results
11
10 Cut-based analysis
13
11 Summary
15
The ATLAS collaboration
20 Contents
1
Introduction
1
2
The ATLAS detector
3
3
Data and simulation samples
3
4
Object reconstruction
4
5
Event pre-selection
5
6
Multivariate analysis
5
7
Invariant mass spectrum of the two b-jets
6
8
Sources of systematic uncertainty
9
8.1
Experimental uncertainties
10
8.2
Modelling uncertainties on the mbb shape of the non-resonant background
10
8.3
Theoretical uncertainties
10
9
Statistical procedure and results
11
10 Cut-based analysis
13
11 Summary
15
The ATLAS collaboration
20 1
3
3
4
5
5
6
9
10
10
10
11
13 JHEP11(2016)112 The ATLAS collaboration 1
Introduction Since the ATLAS and CMS collaborations reported the observation [1, 2] of a new par-
ticle with a mass of about 125 GeV and with properties consistent with those expected
for the Higgs boson in the Standard Model (SM) [3–5], more precise measurements have
strengthened the hypothesis that the new particle is indeed the Higgs boson [6–9]. These
measurements were performed primarily in the bosonic decay modes of the new particle:
H →γγ, ZZ, W +W −. It is essential to study whether it also directly decays into fermions
as predicted by the SM. Recently CMS and ATLAS reported evidence for the H →τ +τ −
decay mode at a significance level of 3.4 and 4.5 standard deviations, respectively [10–12],
and the combination of these results qualifies as an observation [13]. However, the H →b¯b
decay mode has not yet been observed [14–19], and the only direct evidence of its existence
so far has been obtained by the CDF and D0 collaborations [14] at the Tevatron collider. – 1 – q
q
¯b
W/Z
q
H
b
W/Z
q
Figure 1. An example Feynman diagram illustrating vector-boson-fusion production of the Higgs
boson and its decay to a b¯b pair. JHEP11(2016)112 Figure 1. An example Feynman diagram illustrating vector-boson-fusion production of the Higgs
boson and its decay to a b¯b pair. The production processes of Higgs bosons at the LHC include gluon fusion (gg →H,
denoted ggF), vector-boson fusion (qq →qqH, denoted VBF), Higgs-strahlung (q¯q′ →
WH, ZH, denoted WH/ZH or jointly V H), and production in association with a top-
quark pair (gg →t¯tH, denoted t¯tH). While an inclusive observation of the SM Higgs
boson decaying to a b¯b pair is difficult in hadron collisions because of the overwhelming
background from multijet production, the V H, VBF, and t¯tH processes offer viable options
for the observation of the b¯b decay channel. As reported in refs. [15–19], the leptonic decays
of vector bosons, the kinematic properties of the production process, and the identification
of top quarks are used to reduce the background for V H, VBF, and t¯tH, respectively. This article presents a search for VBF production of the SM Higgs boson in the b¯b decay
mode (VBF signal or VBF Higgs hereafter) using data recorded with the ATLAS detector
in proton-proton collisions at a centre-of-mass energy √s = 8 TeV. 1ATLAS uses a right-handed coordinate system with its origin at the nominal interaction point (IP) in
the centre of the detector and the z-axis along the beam pipe. The x-axis points from the IP to the centre
of the LHC ring, and the y-axis points upwards. Cylindrical coordinates (r, φ) are used in the transverse
plane, φ being the azimuthal angle around the z-axis. The pseudorapidity is defined in terms of the polar
angle θ as η = −ln tan(θ/2). Angular distance is measured in units of ∆R ≡
p
(∆η)2 + (∆φ)2. 2
The ATLAS detector The ATLAS experiment uses a multi-purpose particle detector [20] with a forward-
backward symmetric cylindrical geometry and a near 4π coverage in solid angle.1 It consists
of an inner tracking detector (ID) surrounded by a thin superconducting solenoid providing
a 2 T magnetic field, electromagnetic and hadronic calorimeters, and a muon spectrome-
ter (MS). The ID consists of silicon pixel and microstrip tracking detectors covering the
pseudorapidity range |η| < 2.5, and a transition radiation detector in the region |η| < 2.0. Lead/liquid-argon (LAr) sampling calorimeters in the region |η| < 3.2 provide electro-
magnetic energy measurements with high granularity. A hadron (steel/scintillator-tile)
calorimeter covers the range |η| < 1.7. The end-cap and forward regions are instrumented
with LAr calorimeters for both the electromagnetic and hadronic energy measurements up
to |η| = 4.9. The MS surrounds the calorimeters and is based on three large air-core toroid
superconducting magnets with eight coils each. It includes a system of tracking chambers
covering |η| < 2.7 and fast detectors for triggering in the range |η| < 2.4. The ATLAS
trigger system [21] consists of three levels: the first (L1) is a hardware-based system, and
the second and third levels are software-based systems which are collectively referred to as
the high-level trigger (HLT). JHEP11(2016)112 1
Introduction The signal is searched
for as a resonance in the invariant mass distribution (mbb) of a pair of jets containing
b-hadrons (b-jets) in vector-boson-fusion candidates. Events are selected by requiring four
energetic jets generated from the qqH →qqb¯b process as illustrated in figure 1: two light-
quark jets (VBF jets) at a small angle with respect to the beam line and two b-jets from the
Higgs boson decay in more central regions. Higgs bosons are colour singlets with no colour
line to the bottom quarks; thus little QCD radiation and hadronic activity is expected
between the two VBF jets, creating a rapidity gap between them. This feature is used
to distinguish signal events from multijet events, which form the dominant background
with a non-resonant contribution to the mbb distribution. Another relevant background
source arises from the decay of a Z boson to b¯b in association with two jets (Z →b¯b or Z
hereafter). This results in a resonant contribution to the mbb distribution. To improve the sensitivity, a multivariate analysis (MVA) is used to exploit the topol-
ogy of the VBF Higgs final state. An alternative analysis is performed using kinematic cuts
and the mbb distribution. The selected sample contains a minor contribution from Higgs
boson events produced via the ggF process in association with two jets. These events ex-
hibit an mbb distribution similar to that of VBF Higgs events, and are treated as signal in
this analysis. The possible contribution of V H production to the signal was also studied
but found to be negligible compared to VBF and ggF Higgs production for this analysis. – 2 – 3
Data and simulation samples The data used in this analysis were collected by the ATLAS experiment at a centre-of-
mass energy of 8 TeV during 2012, and correspond to an integrated luminosity of 20.2 fb−1
recorded in stable beam conditions and with all relevant sub-detectors providing high-
quality data. Events are primarily selected by a trigger requiring four jets with transverse momentum
pT > 15 GeV at L1 and pT > 35 GeV in the HLT, two of which must be identified as b-jets
by a dedicated HLT b-tagging algorithm (HLT b-jets). This trigger was available during
the entire 2012 data-taking period. Two triggers designed to enhance the acceptance for
VBF H →b¯b events (VBF Higgs triggers) were added during the 2012 data-taking period. They require either three L1 jets with pT > 15 GeV where one jet is in the forward region
(|η| > 3.2), or two L1 jets in the forward region with pT > 15 GeV. These criteria are
completed by the requirement of at least one HLT b-jet with pT > 35 GeV. The VBF Higgs
triggers were used for a data sample corresponding to an integrated luminosity of 4.4 fb−1,
resulting in an approximately 25% increase of the signal acceptance. VBF and ggF Higgs boson signal events and Z boson background events are modelled
by Monte Carlo (MC) simulations. The signal samples with a Higgs boson mass of 125 GeV
are generated by Powheg [22–24], which calculates the VBF and ggF Higgs production
processes up to next-to-leading order (NLO) in αS. Samples of Z boson + jets events – 3 – are generated using MadGraph5 [25], where the associated jets are produced via strong
or electroweak (EW) processes including VBF, and the matrix elements are calculated
for up to and including three partons at leading order. For all simulated samples, the
NLO CT10 parton distribution functions (PDF) [26] are used. The parton shower and the
hadronisation are modelled by Pythia8 [27], with the AU2 set of tuned parameters [28, 29]
for the underlying event. The VBF Higgs predictions are normalised to a cross-section calculation that in-
cludes full NLO QCD and EW corrections and approximate next-to-next-to-leading-order
(NNLO) QCD corrections [30]. The NLO EW corrections also affect the pT shape of the
Higgs boson [31]. The pT shape is reweighted, based on the shape difference between Hawk
calculations without and with NLO EW corrections included [32, 33]. 3
Data and simulation samples JHEP11(2016)112 The overall normalisation of the ggF process is taken from a calculation at NNLO in
QCD that includes soft-gluon resummation up to next-to-next-to-leading logarithmic terms
(NNLL) [30]. Corrections to the shape of the generated pT distribution of Higgs bosons are
applied to match the distribution from the NNLO calculation with the NNLL corrections
provided by the Hres program [34, 35]. In this calculation, the effects of finite masses
of the top and bottom quarks are included and dynamic renormalisation and factorisation
scales are used. A reweighting is derived such that the inclusive Higgs pT spectrum matches
the Hres prediction, and the Higgs pT spectrum of events with at least two jets matches
the the Minlo hjj [36] prediction, the most recent calculation in this phase space. The ATLAS simulation [37] of the detector is used for all MC events based on the
Geant4 program [38] except for the response of the calorimeters, for which a parameterised
simulation [39] is used. All simulated events are generated with a range of minimum-bias
interactions overlaid on the hard-scattering interaction to account for multiple pp inter-
actions that occur in the same or neighbouring bunch crossings (pile-up). The simulated
events are processed with the same reconstruction algorithms as the data. Corrections are
applied to the simulated samples to account for differences between data and simulation
in the trigger and reconstruction efficiencies and in pile-up contributions. 5
Event pre-selection Events with exactly four jets, each with pT > 50 GeV and |η| < 4.5, are retained. The
four jets are ordered in η such that η1 < η2 < η3 < η4. The jets associated with η1 and
η4 are labelled as VBF jets (or J1 and J2). The other two jets associated with η2 and η3
(Higgs jets or b1 and b2) are required to be within the tracker acceptance (|η| < 2.5), and
to be identified as b-jets. The two Higgs jets must be matched to the HLT b-jets for events
satisfying the primary trigger; for events satisfying the VBF Higgs triggers, one of the two
Higgs jets is required to be matched to an HLT b-jet. The 50 GeV cut on jet pT shapes
the mbb distribution for non-resonant backgrounds, creating a peak near 130 GeV, which
makes the extraction of a signal difficult. This shaping is removed by requiring the pT of
the b¯b system to exceed 100 GeV. Table 1 summarises the acceptances of these pre-selection
criteria, for the VBF and ggF Higgs MC events [30, 47] and the Z MC events. For the pre-selected events, corrections are applied to improve the b-jet energy mea-
surements. If muons with pT > 4 GeV and |η| < 2.5 are found within a b-jet, the four-
momentum of the muon closest to the jet axis is added to that of the jet (after correcting
for the expected energy deposited by the muon in the calorimeter material). Such muons
are reconstructed by combining measurements from the ID and MS systems, and are re-
quired to satisfy tight muon identification quality criteria [48]. In addition, a pT-dependent
correction of up to 5% is applied to account for biases in the response due to resolution
effects. This correction is determined from simulated WH/ZH events following ref. [15]. 4
Object reconstruction Charged-particle tracks are reconstructed with a pT threshold of 400 MeV. Event vertices
are formed from these tracks and are required to have at least three tracks. The primary
vertex is chosen as the vertex with the largest Σ p2
T of the associated tracks. Jets are reconstructed from topological clusters of energy deposits, after noise sup-
pression, in the calorimeters [40] using the anti-kt algorithm [41] with a radius parameter
R = 0.4. Jet energies are corrected for the contribution of pile-up interactions using a
jet-area-based technique [42], and calibrated using pT- and η-dependent correction factors
determined from MC simulations and in-situ data measurements of Z+jet, γ+jet and mul-
tijet events [43, 44]. To suppress jets from pile-up interactions, which are mainly at low
pT, a jet vertex tagger [45], based on tracking and vertexing information, is applied to jets
with pT < 50 GeV and |η| < 2.4. – 4 – Process
Cross-section × BR [pb]
Acceptance
VBF H →b¯b
0.9
6.9 ×10−3
ggF H →b¯b
11.1
4.2 ×10−4
Z →b¯b + 1, 2, or 3 partons
5.9 ×102
3.1 ×10−4
Table 1. Cross-sections times branching ratios (BRs) used for the VBF and ggF H →b¯b and
Z →b¯b MC generation, and acceptances of the pre-selection criteria for simulated samples. Table 1. Cross-sections times branching ratios (BRs) used for the VBF and ggF H →b¯b and
Z →b¯b MC generation, and acceptances of the pre-selection criteria for simulated samples. Table 1. Cross-sections times branching ratios (BRs) used for the VBF and ggF H →b¯b and
Z →b¯b MC generation, and acceptances of the pre-selection criteria for simulated samples. The b-jets are identified (b-tagged) by exploiting the relatively long lifetime and large
mass of b-hadrons. The b-tagging methods are based on the presence of tracks with a large
impact parameter with respect to the primary vertex, and secondary decay vertices. This
information is combined into a single neural-network discriminant [46]. This analysis uses
a b-tagging criterion that, in simulated t¯t events, provides an average efficiency of 70% for
b-jets and a c-jet (light-jet) mis-tag rate less than 20% (1%). JHEP11(2016)112 6
Multivariate analysis A Boosted Decision Tree [49, 50] (BDT) method, as implemented in the Toolkit for Multi-
variate Data Analysis package [51], is used to exploit the characteristics of VBF production. The BDT is trained to discriminate between VBF Higgs signal events and non-resonant – 5 – background events modelled using the data in the sideband regions of the mbb distribution
(70 < mbb < 90 GeV and 150 < mbb < 190 GeV). The input variables of the BDT are chosen to exploit the difference in topologies
between signal events and background events while keeping them as uncorrelated as possible
with mbb, to ensure that the sideband regions provide a good description of the non-resonant
background in the signal region. In order of decreasing discrimination power, which is
determined by removing variables one by one from the analysis, the variables are: the jet
widths of VBF jets having |η| < 2.1 (the jet width is defined as the pT-weighted angular
distance of the jet constituents from the jet axis, and is set to zero if |η| > 2.1), which
differs on average for quark and gluon jets; the scalar sum of the pT of additional jets with
pT > 20 GeV in the region |η| < 2.5, Σpjets
T ; the invariant mass of the two VBF jets, mJJ;
the η separation between the two VBF jets, ∆ηJJ; the maximum |η| of the two VBF jets,
max(|ηJ1|, |ηJ2|); the separation between the |η| average of the VBF jets and that of the
Higgs jets, (|ηJ1| + |ηJ2|)/2 −(|ηb1| + |ηb2|)/2; and the cosine of the polar angle of the cross
product of the VBF jets momenta, cos θ, which is sensitive to the production mechanism. JHEP11(2016)112 Figures 2 and 3 show the distributions of the BDT input variables in the data and the
simulated samples for the VBF H →b¯b, ggF H →b¯b, and Z →b¯b events that satisfy the
pre-selection criteria. The BDT responses to the pre-selected data and simulated events
are compared in figure 4. As expected, the BDT response to the VBF Higgs signal sample
is significantly different from its response to the data, which are primarily multijet events,
and also from its response to the Z and ggF Higgs samples. 7
Invariant mass spectrum of the two b-jets The signal is estimated using a fit to the mbb distribution in the range 70 < mbb < 300 GeV. The contributions to the distribution include H →b¯b events, from either VBF or ggF
production; Z →b¯b events produced in association with jets; and non-resonant processes
such as multijet, t¯t, single top, and W+jets production. In order to better exploit the
MVA discrimination power, the fit is performed simultaneously in four categories based on
the BDT output. The boundaries of the four categories, shown in table 2, were optimised
by minimising the relative statistical uncertainties, pNsig + Nbg/Nsig, where Nsig and Nbg
are the expected numbers of signal and background events, respectively. Table 2 shows, for
each category, the total number of events observed in the data and the number of Higgs
events expected from the VBF and ggF production processes, along with the number of Z
events expected in the entire mass range. The categories in table 2 are listed in order of
increasing sensitivity. The shapes of the mbb distributions for Higgs and Z boson events are taken from simu-
lation. Their shapes in the four categories are found to be comparable; therefore the inclu-
sive shapes are used. The mbb shapes for VBF and ggF Higgs boson events are similar, as
expected. In order to minimise the effects of the limited MC sample size, the resulting mbb
histograms for Higgs and Z events are smoothed using the 353QH algorithm [52]. The mbb
distributions used in the fit are shown in figure 5. The Higgs yield is left free to vary. The Z
yield is constrained to the SM prediction within its theoretical uncertainty (see section 8.3). 7
Invariant mass spectrum of the two b-jets – 6 – w = Calorimeter width J1, J2
0
0.05
0.1
0.15
0.2
0.25
0.3
(1/N) dN/dw
0.1
0.2
0.3
0.4
ATLAS
-1
= 8 TeV, 20.2 fb
s
data
b
b
→
Z
b
b
→
ggF H
b
b
→
VBF H
(a)
[GeV]
jets
T
p
Σ
w =
0
20
40
60
80
100
120
140
(1/N) dN/dw
-2
10
-1
10
1
ATLAS
-1
= 8 TeV, 20.2 fb
s
data
b
b
→
Z
b
b
→
ggF H
b
b
→
VBF H
(b)
[GeV]
JJ
w = m
0
500
1000
1500
2000
2500
3000
3500
(1/N) dN/dw
0.05
0.1
0.15
0.2
ATLAS
-1
= 8 TeV, 20.2 fb
s
data
b
b
→
Z
b
b
→
ggF H
b
b
→
VBF H
(c)
Figure 2. Distributions of the BDT input variables from the data (points) and the simulated
samples for VBF H →b¯b events (shaded histograms), ggF H →b¯b events (open dashed histograms)
and Z →b¯b events (open solid histograms). The pre-selection criteria are applied to these samples. The variables are: (a) the jet widths for the VBF jets having |η| < 2.1 (the jet width is set at zero if
|η| > 2.1); (b) the scalar sum of the pT of additional jets with pT > 20 GeV in the region |η| < 2.5,
Σpjets
T
(the peak at zero represents events without additional jets); and (c) the invariant mass of
the two VBF jets, mJJ. w = Calorimeter width J1, J2
0
0.05
0.1
0.15
0.2
0.25
0.3
(1/N) dN/dw
0.1
0.2
0.3
0.4
ATLAS
-1
= 8 TeV, 20.2 fb
s
data
b
b
→
Z
b
b
→
ggF H
b
b
→
VBF H JHEP11(2016)112 (a) a)
[GeV]
JJ
w = m
0
500
1000
1500
2000
2500
3000
3500
(1/N) dN/dw
0.05
0.1
0.15
0.2
ATLAS
-1
= 8 TeV, 20.2 fb
s
data
b
b
→
Z
b
b
→
ggF H
b
b
→
VBF H
(c) [GeV]
jets
T
p
Σ
w =
0
20
40
60
80
100
120
140
(1/N) dN/dw
-2
10
-1
10
1
ATLAS
-1
= 8 TeV, 20.2 fb
s
data
b
b
→
Z
b
b
→
ggF H
b
b
→
VBF H
(b) (c) (b) gure 2. 7
Invariant mass spectrum of the two b-jets Distributions of the BDT input variables from the data (points) and the simulated
mples for VBF H →b¯b events (shaded histograms), ggF H →b¯b events (open dashed histograms) Figure 2. Distributions of the BDT input variables from the data (points) and the simulated
samples for VBF H →b¯b events (shaded histograms), ggF H →b¯b events (open dashed histograms)
and Z →b¯b events (open solid histograms). The pre-selection criteria are applied to these samples. The variables are: (a) the jet widths for the VBF jets having |η| < 2.1 (the jet width is set at zero if
|η| > 2.1); (b) the scalar sum of the pT of additional jets with pT > 20 GeV in the region |η| < 2.5,
Σpjets
T
(the peak at zero represents events without additional jets); and (c) the invariant mass of
the two VBF jets, mJJ. Process
Pre-selection
Category I
Category II
Category III
Category IV
(−0.08 to 0.01)
(0.01 to 0.06)
(0.06 to 0.09)
(> 0.09)
VBF H →b¯b
130
39
33
23
19
ggF H →b¯b
94
31
8.5
3.8
1.6
Z →b¯b
3700
1100
350
97
49
Data
554302
176073
46912
15015
6493
T bl
2
E
t d
b
f
t f
VBF
d
F H
b¯b
d Z
b¯b
d th Process
Pre-selection
Category I
Category II
Category III
Category IV
(−0.08 to 0.01)
(0.01 to 0.06)
(0.06 to 0.09)
(> 0.09)
VBF H →b¯b
130
39
33
23
19
ggF H →b¯b
94
31
8.5
3.8
1.6
Z →b¯b
3700
1100
350
97
49
Data
554302
176073
46912
15015
6493
Table 2. Expected numbers of events for VBF and ggF H →b¯b and Z →b¯b processes, and the
observed numbers of events in data with 70 < mbb < 300 GeV, after the pre-selection criteria are
applied, in the four categories of the BDT response. The categories are listed in order of increasing
sensitivity. The values in the parentheses represent the boundaries of each BDT category. Table 2. Expected numbers of events for VBF and ggF H →b¯b and Z →b¯b processes, and the
observed numbers of events in data with 70 < mbb < 300 GeV, after the pre-selection criteria are
applied, in the four categories of the BDT response. The categories are listed in order of increasing
sensitivity. The values in the parentheses represent the boundaries of each BDT category. 7
Invariant mass spectrum of the two b-jets JJ
η
∆
w =
0
1
2
3
4
5
6
7
8
9
(1/N) dN/dw
0.05
0.1
ATLAS
-1
= 8 TeV, 20.2 fb
s
data
b
b
→
Z
b
b
→
ggF H
b
b
→
VBF H
(a) |)
J2
η
|,|
J1
η
w = max(|
0
1
2
3
4
5
6
(1/N) dN/dw
0.05
0.1
ATLAS
-1
= 8 TeV, 20.2 fb
s
data
b
b
→
Z
b
b
→
ggF H
b
b
→
VBF H
(b) (1/N) dN/dw JHEP11(2016)112 (a) (b) θ
w = cos
-1
-0.8 -0.6 -0.4 -0.2
0
0.2
0.4
0.6
0.8
1
(1/N) dN/dw
0
0.05
0.1
0.15
0.2
ATLAS
-1
= 8 TeV, 20.2 fb
s
data
b
b
→
Z
b
b
→
ggF H
b
b
→
VBF H
(d) *
J
η
w =
-2
-1
0
1
2
3
4
(1/N) dN/dw
0.05
0.1
ATLAS
-1
= 8 TeV, 20.2 fb
s
data
b
b
→
Z
b
b
→
ggF H
b
b
→
VBF H
(c) (d) (c) Figure 3. Distributions of the BDT input variables from the data (points) and the simulated
samples for VBF H →b¯b events (shaded histograms), ggF H →b¯b events (open dashed histograms)
and Z →b¯b events (open solid histograms). The pre-selection criteria are applied to these samples. The variables are: (a) the η separation between the two VBF jets, ∆ηJJ; (b) the maximum |η| of
the two VBF jets, max(|ηJ1|, |ηJ2|); (c) the separation between the |η| average of the VBF jets and
that of the Higgs jets, η∗
J = (|ηJ1| + |ηJ2|)/2 −(|ηb1| + |ηb2|)/2; and (d) the cosine of the polar angle
of the cross product of the VBF jets momenta, cos θ. A data-driven method is used to model the mbb distribution of the non-resonant back-
ground. Data in the sidebands of the mbb distribution are fit simultaneously to a function
which is then interpolated to the signal region. The analytic forms considered are Bernstein
polynomials [53], combinations of exponential functions, and combinations of Bernstein
polynomials and exponential functions with various numbers of coefficients, and functions
with a χ2 probability greater than 0.05, that do not introduce a bias, are selected. For
each form, the minimum number of coefficients is determined by performing an F-test, and
the corresponding function is chosen as a candidate function. 7
Invariant mass spectrum of the two b-jets – 7 – JJ
η
∆
w =
0
1
2
3
4
5
6
7
8
9
(1/N) dN/dw
0.05
0.1
ATLAS
-1
= 8 TeV, 20.2 fb
s
data
b
b
→
Z
b
b
→
ggF H
b
b
→
VBF H
(a)
|)
J2
η
|,|
J1
η
w = max(|
0
1
2
3
4
5
6
(1/N) dN/dw
0.05
0.1
ATLAS
-1
= 8 TeV, 20.2 fb
s
data
b
b
→
Z
b
b
→
ggF H
b
b
→
VBF H
(b)
*
J
η
w =
-2
-1
0
1
2
3
4
(1/N) dN/dw
0.05
0.1
ATLAS
-1
= 8 TeV, 20.2 fb
s
data
b
b
→
Z
b
b
→
ggF H
b
b
→
VBF H
(c)
θ
w = cos
-1
-0.8 -0.6 -0.4 -0.2
0
0.2
0.4
0.6
0.8
1
(1/N) dN/dw
0
0.05
0.1
0.15
0.2
ATLAS
-1
= 8 TeV, 20.2 fb
s
data
b
b
→
Z
b
b
→
ggF H
b
b
→
VBF H
(d)
Figure 3. Distributions of the BDT input variables from the data (points) and the simulated
samples for VBF H →b¯b events (shaded histograms), ggF H →b¯b events (open dashed histograms)
and Z →b¯b events (open solid histograms). The pre-selection criteria are applied to these samples. The variables are: (a) the η separation between the two VBF jets, ∆ηJJ; (b) the maximum |η| of
the two VBF jets, max(|ηJ1|, |ηJ2|); (c) the separation between the |η| average of the VBF jets and
that of the Higgs jets, η∗
J = (|ηJ1| + |ηJ2|)/2 −(|ηb1| + |ηb2|)/2; and (d) the cosine of the polar angle
of the cross product of the VBF jets momenta, cos θ. 7
Invariant mass spectrum of the two b-jets The fitted signal strength is
measured for each candidate function using toy samples. The function giving the smallest
bias is used as the nominal distribution. The function giving the second smallest bias is – 8 – w = BDT response
-0.3
-0.2
-0.1
0
0.1
(1/N) dN/dw
0.05
0.1
0.15
-1
= 8 TeV, 20.2 fb
s
data
b
b
→
Z
b
b
→
ggF H
b
b
→
VBF H
ATLAS
Figure 4. Distributions of the BDT response to the data (points) and to the simulated samples for
VBF H →b¯b events (shaded histogram), ggF H →b¯b events (open dashed histogram) and Z →b¯b
events (open solid histogram). The pre-selection criteria are applied to these samples. w = BDT response
-0.3
-0.2
-0.1
0
0.1
(1/N) dN/dw
0.05
0.1
0.15
-1
= 8 TeV, 20.2 fb
s
data
b
b
→
Z
b
b
→
ggF H
b
b
→
VBF H
ATLAS JHEP11(2016)112 Figure 4. Distributions of the BDT response to the data (points) and to the simulated samples for
VBF H →b¯b events (shaded histogram), ggF H →b¯b events (open dashed histogram) and Z →b¯b
events (open solid histogram). The pre-selection criteria are applied to these samples. [GeV]
bb
m
40
60
80
100
120
140
160
180
Entries / 5 GeV
2
4
6
8
10
ATLAS
Simulation
Category IV
b
b
→
Z
b
b
→
ggF H
b
b
→
VBF H
Figure 5. Simulated invariant mass distributions of two b-jets from decays of Higgs bosons,
summed for VBF (shaded histogram) and ggF (open dashed histogram) production, as well as from
decays of Z bosons (open solid histogram), normalised to the expected contributions in category
IV, which gives the highest sensitivity. [GeV]
bb
m
40
60
80
100
120
140
160
180
Entries / 5 GeV
2
4
6
8
10
ATLAS
Simulation
Category IV
b
b
→
Z
b
b
→
ggF H
b
b
→
VBF H Figure 5. Simulated invariant mass distributions of two b-jets from decays of Higgs bosons,
summed for VBF (shaded histogram) and ggF (open dashed histogram) production, as well as from
decays of Z bosons (open solid histogram), normalised to the expected contributions in category
IV, which gives the highest sensitivity. taken as an alternative distribution, and is used to estimate the systematic uncertainty due
to the choice of analytic function. 7
Invariant mass spectrum of the two b-jets The shapes of the mbb distributions are observed to be
different in the four categories. Bernstein polynomials of different degrees, fourth-order in
category I and third-order in the higher-sensitivity categories, are found to best describe
the mbb shape of the non-resonant background. The nominal and alternative functions are
summarised in table 3. 8
Sources of systematic uncertainty This section discusses sources of systematic uncertainty: experimental uncertainties, un-
certainties on the modelling of the non-resonant background, and theoretical uncertainties – 9 – category I
category II
category III
category IV
Nominal
4th Pol. 3rd Pol. 3rd Pol. 3rd Pol. Alternative
2nd Pol. × exponential
3 exponentials
2 exponentials
exponential
Table 3. Nominal and alternative functions describing the non-resonant background in the four
BDT categories. The fourth-, third-, and second-order Bernstein polynomials are referred to as 4th
Pol., 3rd Pol., and 2nd Pol. on the Higgs and Z processes. The uncertainties can affect the normalisation and the
kinematic distributions individually or both together. JHEP11(2016)112 8.2
Modelling uncertainties on the mbb shape of the non-resonant backgroun The uncertainties on the shape of the mbb distribution for the non-resonant background
is the largest source of systematic uncertainty, contributing about 80% to the total uncer-
tainty on the Higgs yield. The dominant contributions to this source come from the limited
number of events in the mbb sidebands of the data used for the fit to the nominal function,
and from the choice of the function. For the latter, an alternative function is chosen for
each BDT region, as described in section 7 and listed in table 3. Pseudo-data are generated
using the nominal functions and are fit simultaneously in the four BDT categories with
nominal and alternative functions. The bin-by-bin differences in the background yield pre-
dicted by the two alternative descriptions are used to estimate, by means of an eigenvector
decomposition, the corresponding systematic uncertainties. 8.1
Experimental uncertainties The dominant experimental uncertainties on the Higgs signal yield arise from the statistical
uncertainty due to the finite size of the MC samples, the jet energy scale uncertainty, and
the b-jet triggering and tagging, contributing 15%, 10–20%, and 10% respectively, to the
total uncertainty on the Higgs yield. Limited MC sizes affect the normalisation via the
acceptance of the signal events and the shape of the signal mbb distribution. Several
sources contribute to the uncertainty on the jet energy scale [44]. They include the in
situ jet calibration, pile-up-dependent corrections and the flavour composition of jets in
different event classes. The shape of the mbb distribution for the Higgs signal and the Z
background is affected by the jet energy scale uncertainty. Moreover, the change in the jet
energy modifies the value of the BDT output and can cause migration of events between
BDT categories. The b-jet trigger and tagging efficiencies are another source of systematic
uncertainty, contributing 10% to the total uncertainty. They are calibrated using multijet
events containing a muon and t¯t events, respectively [54]. The uncertainty on the jet energy
resolution contributes about 4%. The uncertainty on the integrated luminosity, 1.9% [55],
is included, but is negligible compared to the other uncertainties mentioned above. 8.3
Theoretical uncertainties The uncertainties on the MC modelling of the Higgs signal events contribute about 10% to
the total uncertainty on the Higgs yield. The sources for these uncertainties are higher order – 10 – QCD corrections, the modelling of the underlying event and the parton shower, the PDFs,
and the H →b¯b branching ratio. An uncertainty on higher order QCD corrections for the
cross-sections and acceptances is estimated by varying the factorisation and renormalisation
scales, µF and µR, independently by a factor of two around the nominal values [31] with
the constraint 0.5 ≤µF/µR ≤2. Higher order corrections to the pT spectrum of the Higgs
boson (described in section 3) are an additional source of the modelling uncertainties. This uncertainty is estimated by comparing the results between LO and NLO calculations
for VBF production and by varying the factorisation and renormalisation scales for ggF
production. Uncertainties related to the simulation of the underlying event and the parton
shower are estimated by comparing distributions obtained using Powheg+Pythia8 and
Powheg+Herwig [56]. The uncertainties on the acceptance due to uncertainties in the
PDFs are estimated by studying the change in the acceptance when different PDF sets such
as MSTW2008NLO [57] and NNPDF2.3 [58] are used or the CT10 PDF set parameters
are varied within their uncertainties. The largest variation in acceptance is taken as a
systematic uncertainty. The uncertainty on the H →b¯b branching ratio, 3.2% [47], is also
accounted for. JHEP11(2016)112 The uncertainty on higher order QCD corrections to the Z →b¯b yield is estimated
by varying the factorisation and renormalisation scales around the nominal value in the
manner described above. It is found to be about 40-50%, depending on the BDT category,
out of which about 25% is correlated. These correlated and uncorrelated uncertainties are
used to constrain the Z yield in the fit. This process results in about 20-25% to the total
uncertainty on the Higgs yield. 9
Statistical procedure and results A statistical fitting procedure based on the RooStats framework [59, 60] is used to estimate
the Higgs signal strength, µ, from the data, where µ is the ratio of the measured signal
yield to the SM prediction. A binned likelihood function is constructed as the product
of Poisson-probability terms of the bins in the mbb distributions, and of the four different
BDT categories. The impact of systematic uncertainties on the signal and background expectations,
presented in section 8, is described by a vector of nuisance parameters (NPs), ⃗θ. The
expected numbers of signal and background events in each bin and category are func-
tions of ⃗θ. For each NP with an a priori constraint, the prior is taken into account as a
Gaussian constraint in the likelihood. The NPs associated with uncertainties in the shape
and normalisation of the non-resonant background events, which do not have priors, are
determined from the data. The test statistic qµ is constructed according to the profile-likelihood ratio: qµ = 2 ln(L(µ, ⃗θµ)/L(ˆµ,⃗ˆθ)),
(9.1) (9.1) where ˆµ and ⃗ˆθ are the parameters that maximise the likelihood, and ⃗θµ are the nuisance
parameter values that maximise the likelihood for a given µ. This test statistic is used where ˆµ and ⃗ˆθ are the parameters that maximise the likelihood, and ⃗θµ are the nuisance
parameter values that maximise the likelihood for a given µ. This test statistic is used – 11 – Source of uncertainty
Uncertainty on µ
MVA
Cut-based
Experimental uncertainties
Detector-related
+0.2/−0.3
+1.6/−1.2
MC statistics
±0.4
±0.1
Theoretical uncertainties
MC signal modelling
±0.1
±1.3
Z yield
+0.6/−0.5
±1.4
Non-resonant background modelling
Choice of function
±1.0
±1.0
Sideband statistics
±1.7
±3.7
Statistical uncertainties
±1.3
Total
±2.3
+4.6/−4.4 Source of uncertainty
Uncertainty on µ
MVA
Cut-based
Experimental uncertainties
Detector-related
+0.2/−0.3
+1.6/−1.2
MC statistics
±0.4
±0.1
Theoretical uncertainties
MC signal modelling
±0.1
±1.3
Z yield
+0.6/−0.5
±1.4
Non-resonant background modelling
Choice of function
±1.0
±1.0
Sideband statistics
±1.7
±3.7
Statistical uncertainties
±1.3
Total
±2.3
+4.6/−4.4
Table 4. Summary of uncertainties on the Higgs signal strength for the MVA analysis, and for
the cut-based analysis. They are estimated at the central values of the signal strength, µ = −0.8
and −5.2 for the MVA and cut-based analyses, respectively. The two systematic uncertainties
accounting for non-resonant background modelling are strongly correlated. Their combined value
for the MVA analysis is 1.8. JHEP11(2016)112 Table 4. 9
Statistical procedure and results Summary of uncertainties on the Higgs signal strength for the MVA analysis, and for
the cut-based analysis. They are estimated at the central values of the signal strength, µ = −0.8
and −5.2 for the MVA and cut-based analyses, respectively. The two systematic uncertainties
accounting for non-resonant background modelling are strongly correlated. Their combined value
for the MVA analysis is 1.8. both to measure the compatibility of the background-only model with the data, and to
determine exclusion intervals using the CLS method [61, 62]. both to measure the compatibility of the background-only model with the data, and to
determine exclusion intervals using the CLS method [61, 62]. The robustness of the fit is validated by generating pseudo-data and estimating the
number of signal events for various values of µ. The results of the fit in the four categories
are shown in figure 6. The Z yield is constrained to the SM prediction within its theoretical
uncertainty, using four independent constraints in the four BDT regions (uncorrelated
terms) and a common constraint (correlated term) as described in section 8.3. The ratios
of Z yields to the SM predictions (µZ) are found to be compatible in all of the four BDT
regions. Combined over the four categories, the fit further constrains µZ to 0.7 ± 0.2. The combined Higgs signal strength is −0.8±2.3, where the uncertainty includes both
the statistical (±1.3) and systematic (+1.8/−1.9) components. The breakdown of the
systematic uncertainty on the estimated signal strength is given in table 4. The correlation
coefficient between the combined µ and the combined µZ is found to be 0.22. In the absence
of a signal, the limit on the Higgs signal strength at 95% confidence level (CL) is expected
to be 5.4. When Standard Model production is assumed, the expected limit is found to be
5.7. The observed limit is 4.4. The compatibility between the measured Z yield and its SM prediction is alternatively
tested by removing its a priori constraint from the fit. In this case a value of µZ = 0.3±0.3
is extracted from the fit, to be compared to the theory prediction of 1.0±0.4. The absence
of the Z constraint modifies the combined Higgs signal strength slightly, to −0.5 ± 2.3. 9
Statistical procedure and results – 12 – Entries / 4 GeV
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Higgs contributions. In the lower panels, the data after subtraction of the non-resonant back-
ground (points) are compared with the fit to the Z (open histogram) and Higgs (shaded histogram)
contributions. 9
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Higgs contributions. In the lower panels, the data after subtraction of the non-resonant back-
ground (points) are compared with the fit to the Z (open histogram) and Higgs (shaded histogram)
contributions. 10
Cut-based analysis An alternative analysis is performed based on kinematic cuts. While the MVA performs
a simultaneous fit to the mbb distributions of the four samples categorised by the BDT
response, the cut-based analysis performs a fit to one mbb distribution of the entire sample
in the mass range between 70 GeV and 300 GeV. Events are required to satisfy kinematic
criteria featuring the VBF Higgs final state. Events must not have any additional jet with
pT > 25 GeV and |η| < 2.4, and must satisfy |∆ηJJ| > 3.0 and mJJ > 650 GeV. Figure 7
shows the mbb distribution of 32906 events in the data that satisfy the selection criteria. The number of signal events in the data is expected to be 68.8, with about 15% coming
from ggF production. This can be compared to 158.9 events in the MVA, as obtained by
summing the corresponding numbers in table 2 over the four categories, where about 28%
comes from ggF production. – 13 – Entries / 4 GeV
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the data, and the histograms represent the non-resonant background, Z, and Higgs contributions. In the lower panel, the data after subtraction of the non-resonant background (points) are compared
with the fit to the Z (open histogram) and Higgs (shaded histogram) contributions. The Higgs yield
extracted from the fit is consistent with zero. Entries / 4 GeV
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100 JHEP11(2016)112 Figure 7. Distribution of mbb for events selected in the cut-based analysis. The points represent
the data, and the histograms represent the non-resonant background, Z, and Higgs contributions. In the lower panel, the data after subtraction of the non-resonant background (points) are compared
with the fit to the Z (open histogram) and Higgs (shaded histogram) contributions. The Higgs yield
extracted from the fit is consistent with zero. The cut-based analysis uses an unbinned maximum likelihood fit. 11
Summary A search for the Standard Model Higgs boson produced by vector-boson fusion and de-
caying into a pair of bottom quarks is presented. The dataset analysed corresponds to
an integrated luminosity of 20.2 fb−1 from pp collisions at √s = 8 TeV, recorded by the
ATLAS experiment during Run 1 of the LHC. Events are selected using the distinct fi-
nal state of the VBF H →b¯b signal, which is the presence of four energetic jets: two
b-jets from the Higgs boson decay in the central region of the detector and two jets
in the forward/backward region. To improve the sensitivity, a multivariate analysis is
used, exploiting the topology of the VBF Higgs final state and the properties of jets. The signal yield is estimated by performing a fit to the invariant mass distribution of
the two b-jets in the range 70 < mbb < 300 GeV and assuming a Higgs boson mass of
125 GeV. The ratio of the Higgs signal yield to the SM prediction is measured to be
µ = −0.8 ± 1.3(stat.)+1.8
−1.9(syst.) = −0.8 ± 2.3. The upper limit on µ is observed to be
µ = 4.4 at the 95% CL, which should be compared to the expected limits of 5.4 in the
background-only hypothesis and 5.7 if Standard Model production is assumed. An alterna-
tive analysis is performed using kinematic selection criteria and provides consistent results:
µ = −5.2+4.6
−4.4 and a 95% CL upper limit of 5.4. JHEP11(2016)112 10
Cut-based analysis The resonance
shapes of the mbb distributions for the Higgs and Z events are determined by a fit to
a Bukin function [63] using MC events. The analytic functions describing the non-resonant
background are studied by using events that satisfy the pre-selection criteria described in
section 5. A fourth-order polynomial is chosen as the nominal function and a fifth-order
polynomial is chosen as the alternative function. The Higgs yield is left free to vary, but the Z yield is fixed to its SM prediction. The
robustness of the fit is validated by generating pseudo-data and constructing pulls of the
estimated number of Higgs events for various values of µ. The fit results are presented
in figure 7. The Higgs signal strength is measured to be µ = −5.2 ± 3.7(stat.)+2.7
−2.5(syst.),
where the statistical uncertainty includes the statistical uncertainty on the non-resonant
background modelling (see table 4). The sources of systematic uncertainty are the same as
those for the MVA analysis as described in section 8 and are summarised in table 4. The
uncertainties on µ are estimated as the changes in µ when the sources are varied within
their uncertainties. Higher-order corrections to the Z samples and to the signal samples,
the choice of function describing the non-resonant background, and the jet energy scale are
the dominant sources of systematic uncertainty, each contributing about 40–50% to the
total systematic uncertainty on the Higgs signal strength. The magnitudes of experimental
and theoretical uncertianties are scaled with the central value of µ, as illustrated in table 4
except for the case of the MC statistical uncertainty. This is due to the fact that the MVA
divides the MC samples into four categories, and uses the signal mbb distribution directly
in the fit as a template while the cut-based analysis uses an interpolated function. The
upper limit on the strength is found to be 5.4 at the 95% CL, which can be compared to the
expected limit values of 8.5 in the background-only hypothesis and 9.5 if Standard Model
production is assumed. These results are consistent with those of the MVA. As expected,
the cut-based analysis is less sensitive than the MVA. – 14 – Acknowledgments We thank CERN for the very successful operation of the LHC, as well as the support staff
from our institutions without whom ATLAS could not be operated efficiently. We acknowledge the support of ANPCyT, Argentina; YerPhI, Armenia; ARC, Aus-
tralia; BMWFW and FWF, Austria; ANAS, Azerbaijan; SSTC, Belarus; CNPq and
FAPESP, Brazil; NSERC, NRC and CFI, Canada; CERN; CONICYT, Chile; CAS, MOST
and NSFC, China; COLCIENCIAS, Colombia; MSMT CR, MPO CR and VSC CR,
Czech Republic; DNRF and DNSRC, Denmark; IN2P3-CNRS, CEA-DSM/IRFU, France;
GNSF, Georgia; BMBF, HGF, and MPG, Germany; GSRT, Greece; RGC, Hong Kong
SAR, China; ISF, I-CORE and Benoziyo Center, Israel; INFN, Italy; MEXT and JSPS,
Japan; CNRST, Morocco; FOM and NWO, Netherlands; RCN, Norway; MNiSW and NCN,
Poland; FCT, Portugal; MNE/IFA, Romania; MES of Russia and NRC KI, Russian Fed-
eration; JINR; MESTD, Serbia; MSSR, Slovakia; ARRS and MIZˇS, Slovenia; DST/NRF,
South Africa; MINECO, Spain; SRC and Wallenberg Foundation, Sweden; SERI, SNSF
and Cantons of Bern and Geneva, Switzerland; MOST, Taiwan; TAEK, Turkey; STFC,
United Kingdom; DOE and NSF, United States of America. In addition, individual groups
and members have received support from BCKDF, the Canada Council, CANARIE, CRC,
Compute Canada, FQRNT, and the Ontario Innovation Trust, Canada; EPLANET, ERC,
FP7, Horizon 2020 and Marie Sk lodowska-Curie Actions, European Union; Investissements
d’Avenir Labex and Idex, ANR, R´egion Auvergne and Fondation Partager le Savoir, France;
DFG and AvH Foundation, Germany; Herakleitos, Thales and Aristeia programmes co-
financed by EU-ESF and the Greek NSRF; BSF, GIF and Minerva, Israel; BRF, Norway; – 15 – Generalitat de Catalunya, Generalitat Valenciana, Spain; the Royal Society and Leve
hulme Trust, United Kingdom. Generalitat de Catalunya, Generalitat Valenciana, Spain; the Royal Society and Lever-
hulme Trust, United Kingdom. The crucial computing support from all WLCG partners is acknowledged gratefully,
in particular from CERN, the ATLAS Tier-1 facilities at TRIUMF (Canada), NDGF
(Denmark, Norway, Sweden), CC-IN2P3 (France), KIT/GridKA (Germany), INFN-CNAF
(Italy), NL-T1 (Netherlands), PIC (Spain), ASGC (Taiwan), RAL (U.K.) and BNL
(U.S.A.), the Tier-2 facilities worldwide and large non-WLCG resource providers. Ma-
jor contributors of computing resources are listed in ref. [64]. Open Access. This article is distributed under the terms of the Creative Commons
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R. Aben108, O.S. AbouZeid138, N.L. Abraham150, H. Abramowicz154, H. Abreu153, R. Abreu117 Y. Abulaiti147a,147b, B.S. Acharya164a,164b,a, L. Adamczyk40a, D.L. Adams27, J. Adelman10 S. Adomeit101, T. Adye132, A.A. Affolder76, T. Agatonovic-Jovin14, J. Agricola56, J.A. Aguilar-Saavedra127a,127f, S.P. Ahlen24, F. Ahmadov67,b, G. Aielli134a,134b, H. Akerstedt147a,147b, T.P.A. ˚Akesson83, A.V. Akimov97, G.L. Alberghi22a,22b, J. Albert169 S. Albrand57, M.J. Alconada Verzini73, M. Aleksa32, I.N. Aleksandrov67, C. Alexa28b, r154, T. Alexopoulos10, M. Alhroob114, M. Aliev75a,75b, G. Alimonti93a, J. Alison33, G. Alexander154, T. Alexopoulos10, M. Alhroob114, M. Aliev75a,75b, G. Alimonti93a, J. Aliso JHEP11(2016)112 S.P. Alkire37, B.M.M. Allbrooke150, B.W. Allen117, P.P. Allport19, A. Aloisio105a,105b, A. Alo F. Alonso73, C. Alpigiani139, M. Alstaty87, B. Alvarez Gonzalez32, D. ´Alvarez Piqueras167, Alonso73, C. Alpigiani139, M. Alstaty87, B. Alvarez Gonzalez32, D. ´Alvarez Piqueras167, M.G. Alviggi105a,105b, B.T. Amadio16, K. Amako68, Y. Amaral Coutinho26a, C. Amelung25, G. Alviggi105a,105b, B.T. Amadio16, K. Amako68, Y. Amaral Coutinho26a, C. Amelung25, Amidei91, S.P. Amor Dos Santos127a,127c, A. Amorim127a,127b, S. Amoroso32, G. Amundsen25, D. Amidei91, S.P. Amor Dos Santos127a,127c, A. Amorim127a,127b, S. Amoroso32, G. Amundsen25, C. Anastopoulos140, L.S. Ancu51, N. Andari109, T. Andeen11, C.F. Anders60b, G. Anders32, J.K. Anders76, K.J. Anderson33, A. Andreazza93a,93b, V. Andrei60a, S. Angelidakis9, I. Angelozzi108, P. Anger46, A. Angerami37, F. Anghinolfi32, A.V. Anisenkov110,c, N. Anjos13, g
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A. Annovi125a,125b, M. Antonelli49, A. Antonov99, F. Anulli133a, M. Aoki68, L. Aperio Bella19, S. Argyropoulos65, M. Arik20a, A.J. Armbruster144, L.J. Armitage78, O. Arnaez32, H. Arnold50, M. Arratia30, O. Arslan23, A. Artamonov98, G. Artoni121, S. Artz85, S. Asai156, N. Asbah44, A. Ashkenazi154, B. ˚Asman147a,147b, L. Asquith150, K. Assamagan27, R. Astalos145a, M. Atkinson166, N.B. Atlay142, K. Augsten129, G. Avolio32, B. Axen16, M.K. Ayoub118, G. Azuelos96,d, M.A. Baak32, A.E. Baas60a, M.J. Baca19, H. Bachacou137, K. Bachas75a,75b, M. Backes32, M. Backhaus32, P. Bagiacchi133a,133b, P. Bagnaia133a,133b, Y. Bai35a, J.T. Baine O.K. Baker176, E.M. Baldin110,c, P. Balek130, T. Balestri149, F. Balli137, W.K. Balunas123, nas41, Sw. Banerjee173,e, A.A.E. Bannoura175, L. Barak32, E.L. Barberio90, E. Banas41, Sw. Banerjee173,e, A.A.E. Bannoura175, L. Barak32, E.L. Barberio90, D. Barberis52a,52b, M. Barbero87, T. Barillari102, T. Barklow144, N. Barlow30, S.L. Barnes86, rberis52a,52b, M. Barbero87, T. Barillari102, T. Barklow144, N. Barlow30, S.L. Barnes86, B.M. Barnett132, R.M. Barnett16, Z. Barnovska5, A. Baroncelli135a, G. Barone25, A.J. Barr121, L. Barranco Navarro167, F. Barreiro84, J. Barreiro Guimar˜aes da Costa35a, R. Bartoldus144, A.E. Barton74, P. The ATLAS collaboration Bartos145a, A. Basalaev124, A. Bassalat118, R.L. Bates55, S.J. Batista159, M.D. Beattie74, T. Beau82, P.H. Beauchemin162, P. Bechtle23, H.P. Beck18,g, K. Becker121, M. Bedognetti108, C.P. Bee149, L.J. Beemster108, T.A. Beermann32, M. Begel27, J.K. Behr44, C. Belanger-Champagne89, A.S. Bell80, G. Bella154, L. Bellagamba22a, A. Bellerive31, M. Bellomo88, K. Belotskiy99, O. Beltramello32, N.L. Belyaev99, O. Benary154, Bellomo88, K. Belotskiy99, O. Beltramello32, N.L. Belyaev99, O. Benary154, D. Benchekroun136a, M. Bender101, K. Bendtz147a,147b, N. Benekos10, Y. Benha E. Benhar Noccioli176, J. Benitez65, D.P. Benjamin47, J.R. Bensinger25, S. Bentvelsen108, Benhar Noccioli176, J. Benitez65, D.P. Benjamin47, J.R. Bensinger25, S. Bentvelsen108, L. Beresford121, M. Beretta49, D. Berge108, E. Bergeaas Kuutmann165, N. Berger5, J. Ber Beresford121, M. Beretta49, D. Berge108, E. Bergeaas Kuutmann165, N. Berger5, J. Beringer16, S. Berlendis57, N.R. Bernard88, C. Bernius111, F.U. Bernlochner23, T. Berry79, P. Berta13 C. Bertella85, G. Bertoli147a,147b, F. Bertolucci125a,125b, I.A. Bertram74, C. Bertsche44, D. Bertsche114, G.J. Besjes38, O. Bessidskaia Bylund147a,147b, M. Bessner44, N. Besson137, j
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C. Betancourt50, S. Bethke102, A.J. Bevan78, W. Bhimji16, R.M. Bianchi126, L. Bianchini25, – 20 – W. Blum85,∗, U. Blumenschein56, S. Blunier34a, G.J. Bobbink108, V.S. Bobrovnikov110,c, S.S. Bocchetta83, A. Bocci47, C. Bock101, M. Boehler50, D. Boerner175, J.A. Bogaerts32, g
D. Bogavac14, A.G. Bogdanchikov110, C. Bohm147a, V. Boisvert79, P. Bokan14, T. Bold40a, A.S. Boldyrev164a,164c, M. Bomben82, M. Bona78, M. Boonekamp137, A. Borisov131, G. Boris J. Bortfeldt101, D. Bortoletto121, V. Bortolotto62a,62b,62c, K. Bos108, D. Boscherini22a, Bortfeldt101, D. Bortoletto121, V. Bortolotto62a,62b,62c, K. Bos108, D. Boscherini22a, M. Bosman13, J.D. Bossio Sola29, J. Boudreau126, J. Bouffard2, E.V. Bouhova-Thacker74, Bosman13, J.D. Bossio Sola29, J. Boudreau126, J. Bouffard2, E.V. Bouhova-Thacker74, D. Boumediene36, C. Bourdarios118, S.K. Boutle55, A. Boveia32, J. Boyd32, I.R. Boyko67, J. Bracinik19, A. Brandt8, G. Brandt56, O. Brandt60a, U. Bratzler157, B. Brau88, J.E. Brau117, J. Bracinik19, A. Brandt8, G. Brandt56, O. Brandt60a, U. Bratzler157, B. Brau88, J.E. Brau117, R. Brenner165, S. Bressler172, T.M. Bristow48, D. Britton55, D. Britzger44, F.M. Brochu30, R. Brenner165, S. Bressler172, T.M. Bristow48, D. Britton55, D. Britzger44, F.M. Brochu30, I. Brock23, R. Brock92, G. Brooijmans37, T. Brooks79, W.K. Brooks34b, J. Brosamer16, JHEP11(2016)112 E. Brost117, J.H Broughton19, P.A. Bruckman de Renstrom41, D. Bruncko145b, R. Brune A. Bruni22a, G. Bruni22a, BH Brunt30, M. Bruschi22a, N. Bruscino23, P. Bryant33, L. Bryngemark83, T. Buanes15, Q. Buat143, P. Buchholz142, A.G. Buckley55, I.A. Buda F. Buehrer50, M.K. Bugge120, O. Bulekov99, D. Bullock8, H. Burckhart32, S. Burdin76, C.D. Burgard50, B. Burghgrave109, K. Burka41, S. Burke132, I. Burmeister45, E. Busato36, D. B¨uscher50, V. B¨uscher85, P. Bussey55, J.M. The ATLAS collaboration Butler24, C.M. Buttar55, J.M. Butterworth80, P. Butti108, W. Buttinger27, A. Buzatu55, A.R. Buzykaev110,c, S. Cabrera Urb´an167, D. Caforio129, V.M. Cairo39a,39b, O. Cakir4a, N. Calace51, P. Calafiura16, A. Calandri87, G. Calderini82, P. Calfayan101, L.P. Caloba26a, D. Calvet36, S. Calvet36, T.P. Calvet87, R. Camacho Toro33, S. Camarda32, P. Camarri134a,134b, D. Cameron120, R. Caminal Armadans166, C. Camincher57, S. Campana32, M. Campanelli80, A. Camplani93 A. Campoverde149, V. Canale105a,105b, A. Canepa160a, M. Cano Bret35e, J. Cantero115, R. Cantrill127a, T. Cao42, M.D.M. Capeans Garrido32, I. Caprini28b, M. Caprini28b, M. Capua39a,39b, R. Caputo85, R.M. Carbone37, R. Cardarelli134a, F. Cardillo50, I. Car M. Capua39a,39b, R. Caputo85, R.M. Carbone37, R. Cardarelli134a, F. Cardillo50, I. Carli130, M. Capua39a,39b, R. Caputo85, R.M. Carbone37, R. Cardarelli134a, F. Cardillo50,
T. Carli32, G. Carlino105a, L. Carminati93a,93b, S. Caron107, E. Carquin34b, . Carli32, G. Carlino105a, L. Carminati93a,93b, S. G.D. Carrillo-Montoya32, J.R. Carter30, J. Carvalho127a,127c, D. Casadei19, M.P. Casado13,h M. Casolino13, D.W. Casper163, E. Castaneda-Miranda146a, R. Castelijn108, A. Castelli108, V. Castillo Gimenez167, N.F. Castro127a,i, A. Catinaccio32, J.R. Catmore120, A. Cattai32, J. Caudron85, V. Cavaliere166, E. Cavallaro13, D. Cavalli93a, M. Cavalli-Sforza13, V. Cavasinni125a,125b, F. Ceradini135a,135b, L. Cerda Alberich167, B.C. Cerio47, A.S. Cerqueira26 A. Cerri150, L. Cerrito78, F. Cerutti16, M. Cerv32, A. Cervelli18, S.A. Cetin20d, A. Chafaq136a, D. Chakraborty109, S.K. Chan59, Y.L. Chan62a, P. Chang166, J.D. Chapman30, D.G. Charlton19 A. Chatterjee51, C.C. Chau159, C.A. Chavez Barajas150, S. Che112, S. Cheatham74, A. Chatterjee
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A. Chegwidden92, S. Chekanov6, S.V. Chekulaev160a, G.A. Chelkov67,j, M.A. Chelstowska91,
C. Chen66, H. Chen27, K. Chen149, S. Chen35c, S. Chen156, X. Chen35f, Y. Chen69, H.C. Cheng91,
H.J Cheng35a, Y. Cheng33, A. Cheplakov67, E. Cheremushkina131, R. Cherkaoui El Moursli136e,
V. Chernyatin27,∗, E. Cheu7, L. Chevalier137, V. Chiarella49, G. Chiarelli125a,125b, G. Chiodini75a,
A.S. Chisholm19, A. Chitan28b, M.V. Chizhov67, K. Choi63, A.R. Chomont36, S. Chouridou9,
B.K.B. Chow101, V. Christodoulou80, D. Chromek-Burckhart32, J. Chudoba128, A.J. Chuinard89,
J.J. Chwastowski41, L. Chytka116, G. Ciapetti133a,133b, A.K. Ciftci4a, D. Cinca55, V. Cindro77,
I.A. Cioara23, A. Ciocio16, F. Cirotto105a,105b, Z.H. Citron172, M. Citterio93a, M. Ciubancan28b,
A. Clark51, B.L. Clark59, M.R. Clark37, P.J. Clark48, R.N. Clarke16, C. Clement147a,147b,
Y. Coadou87, M. Cobal164a,164c, A. Coccaro51, J. Cochran66, L. Coffey25, L. Colasurdo107,
B. Cole37, A.P. Colijn108, J. Collot57, T. Colombo32, G. Compostella102, P. Conde Mui˜no127a,127b,
E. Coniavitis50, S.H. Connell146b, I.A. Connelly79, V. Consorti50, S. Constantinescu28b,
G. Conti32, F. Conventi105a,k, M. Cooke16, B.D. Cooper80, A.M. Cooper-Sarkar121,
K.J.R. Cormier159, T. Cornelissen175, M. Corradi133a,133b, F. Corriveau89,l, A. The ATLAS collaboration Dann86, M. Danninger168, M. Dano Hoffmann137, V. Dao50, G. Darbo52a, M. Davies154, P. Davison80, E. Dawe90, I. Dawson140, R.K. Daya-Ishmukhametova88, K. De8, ,
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R. de Asmundis105a, A. De Benedetti114, S. De Castro22a,22b, S. De Cecco82, N. De Groot107, JHEP11(2016)112 P. de Jong108, H. De la Torre84, F. De Lorenzi66, A. De Maria56, D. De Pedis133a, A. De Salvo133a, U. De Sanctis150, A. De Santo150, J.B. De Vivie De Regie118, W.J. Dearnaley74, R. Debbe27, C. Debenedetti138, D.V. Dedovich67, N. Dehghanian3, I. Deigaard108, M. Del Gaudio39a,39b, J. Del Peso84, T. Del Prete125a,125b, D. Delgove118, F. Deliot137, C.M. Delitzsch51, M. Deliyergiyev77, A. Dell’Acqua32, L. Dell’Asta24, M. Dell’Orso125a,125b, M. Della Pietra105a,k, D. della Volpe51, M. Delmastro5, P.A. Delsart57, C. Deluca108, D.A. DeMarco159, S. Demers176, M. Demichev67, A. Demilly82, S.P. Denisov131, D. Denysiuk137, D. Derendarz41, J.E. Derkaoui136 F. Derue82, P. Dervan76, K. Desch23, C. Deterre44, K. Dette45, P.O. Deviveiros32, A. Dewhurst13 S. Dhaliwal25, A. Di Ciaccio134a,134b, L. Di Ciaccio5, W.K. Di Clemente123, C. Di Donato133a,13 A. Di Girolamo32, B. Di Girolamo32, B. Di Micco135a,135b, R. Di Nardo32, A. Di Simone50, R. Di Sipio159, D. Di Valentino31, C. Diaconu87, M. Diamond159, F.A. Dias48, M.A. Diaz34a, E.B. Diehl91, J. Dietrich17, S. Diglio87, A. Dimitrievska14, J. Dingfelder23, P. Dita28b, S. Dita28b, F. Dittus32, F. Djama87, T. Djobava53b, J.I. Djuvsland60a, M.A.B. do Vale26c, D. Dobos32, M. Dobre28b, C. Doglioni83, T. Dohmae156, J. Dolejsi130, Z. Dolezal130, B.A. Dolgoshein99,∗, M. Dobre28b, C. Doglioni83, T. Dohmae156, J. Dolejsi130, Z. Dolezal130, B.A. Dolgoshein99,∗,
M. Donadelli26d, S. Donati125a,125b, P. Dondero122a,122b, J. Donini36, J. Dopke132, A. Doria105 M.T. Dova73, A.T. Doyle55, E. Drechsler56, M. Dris10, Y. Du35d, J. Duarte-Campderros154, E. Duchovni172, G. Duckeck101, O.A. Ducu96,m, D. Duda108, A. Dudarev32, E.M. Duffield16, L. Duflot118, L. Duguid79, M. D¨uhrssen32, M. Dumancic172, M. Dunford60a, H. Duran Yildiz4a, M. D¨uren54, A. Durglishvili53b, D. Duschinger46, B. Dutta44, M. Dyndal44, C. Eckardt44, K.M. Ecker102, R.C. Edgar91, N.C. Edwards48, T. Eifert32, G. Eigen15, K. Einsweiler16, T. Ekelof165, M. El Kacimi136c, V. Ellajosyula87, M. Ellert165, S. Elles5, F. Ellinghaus175, ot169, N. Ellis32, J. Elmsheuser27, M. Elsing32, D. Emeliyanov132, Y. Enari156, A.A. Elliot169, N. Ellis32, J. Elmsheuser27, M. Elsing32, D. Emeliyanov132, Y. Enari156, O.C. Endner85, M. Endo119, J.S. Ennis170, J. Erdmann45, A. Ereditato18, G. Ernis175, J. Ernst2 dner85, M. Endo119, J.S. Ennis170, J. Erdmann45, A. Ereditato18, G. Ernis175, J. Ernst2, M. Ernst27, S. Errede166, E. Ertel85, M. Escalier118, H. Esch45, C. Escobar126, B. Esposito49, A.I. Etienvre137, E. The ATLAS collaboration Corso-Radu163, A. Chegwidden92, S. Chekanov6, S.V. Chekulaev160a, G.A. Chelkov67,j, M.A. Chelstowska91, C. Chen66, H. Chen27, K. Chen149, S. Chen35c, S. Chen156, X. Chen35f, Y. Chen69, H.C. Cheng9 H.J Cheng35a, Y. Cheng33, A. Cheplakov67, E. Cheremushkina131, R. Cherkaoui El Moursli136e, V. Chernyatin27,∗, E. Cheu7, L. Chevalier137, V. Chiarella49, G. Chiarelli125a,125b, G. Chiodini75a, A.S. Chisholm19, A. Chitan28b, M.V. Chizhov67, K. Choi63, A.R. Chomont36, S. Chouridou9, A.S. Chisholm
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B.K.B. Chow101, V. Christodoulou80, D. Chromek-Burckhart32, J. Chudoba128, A.J. Chuinard89,
J.J. Chwastowski41, L. Chytka116, G. Ciapetti133a,133b, A.K. Ciftci4a, D. Cinca55, V. Cindro77,
I.A. Cioara23, A. Ciocio16, F. Cirotto105a,105b, Z.H. Citron172, M. Citterio93a, M. Ciubancan28b,
A. Clark51, B.L. Clark59, M.R. Clark37, P.J. Clark48, R.N. Clarke16, C. Clement147a,147b,
Y. Coadou87, M. Cobal164a,164c, A. Coccaro51, J. Cochran66, L. Coffey25, L. Colasurdo107,
B. Cole37, A.P. Colijn108, J. Collot57, T. Colombo32, G. Compostella102, P. Conde Mui˜no127a,127b,
E. Coniavitis50, S.H. Connell146b, I.A. Connelly79, V. Consorti50, S. Constantinescu28b,
G. Conti32, F. Conventi105a,k, M. Cooke16, B.D. Cooper80, A.M. Cooper-Sarkar121,
K.J.R. Cormier159, T. Cornelissen175, M. Corradi133a,133b, F. Corriveau89,l, A. Corso-Radu163, B.K.B. Chow101, V. Christodoulou80, D. Chromek-Burckhart32, J. Chudoba128, A.J. Chuinard89, y
I.A. Cioara23, A. Ciocio16, F. Cirotto105a,105b, Z.H. Citron172, M. Citterio93a, M. Ciubancan28b, A. Clark51, B.L. Clark59, M.R. Clark37, P.J. Clark48, R.N. Clarke16, C. Clement147a,147b, ,
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Y. Coadou87, M. Cobal164a,164c, A. Coccaro51, J. Cochran66, L. Coffey25, L. Colasurdo107, B. Cole37, A.P. Colijn108, J. Collot57, T. Colombo32, G. Compostella102, P. Conde Mui˜no127a,12 B. Cole
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F. Crescioli82, W.A. Cribbs147a,147b, M. Crispin Ortuzar121, M. Cristinziani23, V. Croft107, G. Crosetti39a,39b, T. Cuhadar Donszelmann140, J. Cummings176, M. Curatolo49, J. C´uth85, C. Cuthbert151, H. Czirr142, P. Czodrowski3, G. D’amen22a,22b, S. D’Auria55, M. D’Onofrio76, M.J. Da Cunha Sargedas De Sousa127a,127b, C. Da Via86, W. Dabrowski40a, T. Dado145a, T. Dai91, O. Dale15, F. Dallaire96, C. Dallapiccola88, M. Dam38, J.R. Dandoy33, N.P. Dang50, A.C. Daniells19, N.S. The ATLAS collaboration Etzion154, H. Evans63, A. Ezhilov124, F. Fabbri22a,22b, L. Fabbri22a,22b, G. Facini33, R.M. Fakhrutdinov131, S. Falciano133a, R.J. Falla80, J. Faltova130, Y. Fang35a, cini33, R.M. Fakhrutdinov131, S. Falciano133a, R.J. Falla80, J. Faltova130, Y. Fang35a, nti93a,93b, A. Farbin8, A. Farilla135a, C. Farina126, T. Farooque13, S. Farrell16, M. Fanti93a,93b, A. Farbin8, A. Farilla135a, C. Farina126, T. Farooque13, S. Farrell16, S.M. Farrington170, P. Farthouat32, F. Fassi136e, P. Fassnacht32, D. Fassouliotis9, M. Faucci Giannelli79, A. Favareto52a,52b, W.J. Fawcett121, L. Fayard118, O.L. Fedin124,n, M. Faucci Giannelli79, A. Favareto52a,52b, W.J. Fawcett121, L. Fayard118, O.L. Fedin124,n, L. Feremenga8, P. Fernandez Martinez167, S. Fernandez Perez13, J. Ferrando55, A. Ferrari165, L. Feremenga8, P. Fernandez Martinez167, S. Fernandez Perez13, J. Ferrando55, A. Ferrari165, P. Ferrari108, R. Ferrari122a, D.E. Ferreira de Lima60b, A. Ferrer167, D. Ferrere51, C. Ferretti A. Ferretto Parodi52a,52b, F. Fiedler85, A. Filipˇciˇc77, M. Filipuzzi44, F. Filthaut107, M. Fincke-Keeler169, K.D. Finelli151, M.C.N. Fiolhais127a,127c, L. Fiorini167, A. Firan4 A. Fischer2, C. Fischer13, J. Fischer175, W.C. Fisher92, N. Flaschel44, I. Fleck142, P. Fleischmann91, G.T. Fletcher140, R.R.M. Fletcher123, T. Flick175, A. Floderus83, L.R. Flores Castillo62a, M.J. Flowerdew102, G.T. Forcolin86, A. Formica137, A. Forti86, – 22 – A.G. Foster19, D. Fournier118, H. Fox74, S. Fracchia13, P. Francavilla82, M. Franchini22a,22b, D. Francis32, L. Franconi120, M. Franklin59, M. Frate163, M. Fraternali122a,122b, D. Freeborn8 S.M. Fressard-Batraneanu32, F. Friedrich46, D. Froidevaux32, J.A. Frost121, C. Fukunaga157, E. Fullana Torregrosa85, T. Fusayasu103, J. Fuster167, C. Gabaldon57, O. Gabizon175, Gabrielli22a,22b, A. Gabrielli16, G.P. Gach40a, S. Gadatsch32, S. Gadomski51, A. Gabrielli22a,22b, A. Gabrielli16, G.P. Gach40a, S. Gadatsch32, S. Gadomski51, G. Gagliardi52a,52b, L.G. Gagnon96, P. Gagnon63, C. Galea107, B. Galhardo127a,127c, Gagliardi52a,52b, L.G. Gagnon96, P. Gagnon63, C. Galea107, B. Galhardo127a,127c, E.J. Gallas121, B.J. Gallop132, P. Gallus129, G. Galster38, K.K. Gan112, J. Gao35b,87, Y. Gao J. Gallas121, B.J. Gallop132, P. Gallus129, G. Galster38, K.K. Gan112, J. Gao35b,87, Y. Gao48, Y.S. Gao144,f, F.M. Garay Walls48, C. Garc´ıa167, J.E. Garc´ıa Navarro167, M. Garcia-Sciveres16, Y.S. Gao144,f, F.M. Garay Walls48, C. Garc´ıa167, J.E. Garc´ıa Navarro167, M. Garcia-Sciveres16, A. Gaudiello52a,52b, G. Gaudio122a, B. Gaur142, L. Gauthier96, I.L. Gavrilenko97, C. Gay168, A. Gaudiello52a,52b, G. Gaudio122a, B. Gaur142, L. Gauthier96, I.L. Gavrilenko97, C. Gay168, Gaycken23, E.N. Gazis10, Z. Gecse168, C.N.P. Gee132, Ch. Geich-Gimbel23, M. Geisen85, G. Gaycken23, E.N. Gazis10, Z. Gecse168, C.N.P. Gee132, Ch. Geich-Gimbel23, M. Geisen85, JHEP11(2016)112 M.P. Geisler60a, C. Gemme52a, M.H. Genest57, C. Geng35b,o, S. Gentile133a,133b, S. George79, M.P. Geisler60a, C. Gemme52a, M.H. Genest57, C. Geng35b,o, S. Gentile133a,133b, S. George79, D. Gerbaudo13, A. Gershon154, S. Ghasemi142, H. Ghazlane136b, M. Ghneimat23, B. The ATLAS collaboration Giacobbe22 S. Giagu133a,133b, P. Giannetti125a,125b, B. Gibbard27, S.M. Gibson79, M. Gignac168, M. Gilchriese16, T.P.S. Gillam30, D. Gillberg31, G. Gilles175, D.M. Gingrich3,d, N. Giokaris9, M.P. Giordani164a,164c, F.M. Giorgi22a, F.M. Giorgi17, P.F. Giraud137, P. Giromini59, D. Giugni93a, F. Giuli121, C. Giuliani102, M. Giulini60b, B.K. Gjelsten120, S. Gkaitatzis155, Gkialas155, E.L. Gkougkousis118, L.K. Gladilin100, C. Glasman84, J. Glatzer32, I. Gkialas155, E.L. Gkougkousis118, L.K. Gladilin100, C. Glasman84, J. Glatzer32, P.C.F. Glaysher48, A. Glazov44, M. Goblirsch-Kolb102, J. Godlewski41, S. Goldfarb91, C.F. Glaysher48, A. Glazov44, M. Goblirsch-Kolb102, J. Godlewski41, S. Goldfarb91, T. Golling51, D. Golubkov131, A. Gomes127a,127b,127d, R. Gon¸calo127a, J. Goncalves Pinto Firmino Da Costa137, L. Gonella19, A. Gongadze67, S. Gonz´alez de la Hoz167 G. Gonzalez Parra13, S. Gonzalez-Sevilla51, L. Goossens32, P.A. Gorbounov98, H.A. Gordon2 I. Gorelov106, B. Gorini32, E. Gorini75a,75b, A. Goriˇsek77, E. Gornicki41, A.T. Goshaw47, C. G¨ossling45, M.I. Gostkin67, C.R. Goudet118, D. Goujdami136c, A.G. Goussiou139, N. Govender146b,p, E. Gozani153, L. Graber56, I. Grabowska-Bold40a, P.O.J. Gradin57, N. Govender146b,p, E. Gozani153, L. Graber56, I. Grabowska-Bold40a, P.O.J. Gradin57, P.M. Gravila28e, H.M. Gray32, E. Graziani135a, Z.D. Greenwood81,q, C. Grefe23, K. Gregers P.M. Gravila28e, H.M. Gray32, E. Graziani135a, Z.D. Greenwood81,q, C. Grefe23, K. Gregersen8 I.M. Gregor44, P. Grenier144, K. Grevtsov5, J. Griffiths8, A.A. Grillo138, K. Grimm74, S. Grinstein13,r, Ph. Gris36, J.-F. Grivaz118, S. Groh85, J.P. Grohs46, E. Gross172, J. Grosse-Knetter56, G.C. Grossi81, Z.J. Grout150, L. Guan91, W. Guan173, J. Guenther129, J. Grosse-Knetter56, G.C. Grossi81, Z.J. Grout150, L. Guan91, W. Guan173, J. Guenther129,
F. Guescini51, D. Guest163, O. Gueta154, E. Guido52a,52b, T. Guillemin5, S. Guindon2, U. Gul55,
C Gumpert32 J Guo35e Y Guo35b,o S Gupta121 G Gustavino133a,133b P Gutierrez114 F. Guescini51, D. Guest163, O. Gueta154, E. Guido52a,52b, T. Guillemin5, S. Guindon2, U. Gul55, C. Gumpert32, J. Guo35e, Y. Guo35b,o, S. Gupta121, G. Gustavino133a,133b, P. Gutierrez114, N.G. Gutierrez Ortiz80, C. Gutschow46, C. Guyot137, C. Gwenlan121, C.B. Gwilliam76, Gutierrez Ortiz80, C. Gutschow46, C. Guyot137, C. Gwenlan121, C.B. Gwilliam76, A. Haas111, C. Haber16, H.K. Hadavand8, N. Haddad136e, A. Hadef87, P. Haefner23, geb¨ock23, Z. Hajduk41, H. Hakobyan177,∗, M. Haleem44, J. Haley115, G. Halladjian92, S. Hageb¨ock23, Z. Hajduk41, H. Hakobyan177,∗, M. Haleem44, J. Haley115, G. Halladjian92, G.D. Hallewell87, K. Hamacher175, P. Hamal116, K. Hamano169, A. Hamilton146a, G.N. Ha D. Hallewell87, K. Hamacher175, P. Hamal116, K. Hamano169, A. Hamilton146a, G.N. Hamity140, P.G. Hamnett44, L. Han35b, K. Hanagaki68,s, K. Hanawa156, M. Hance138, B. Haney123, P.G. Hamnett44, L. Han35b, K. Hanagaki68,s, K. Hanawa156, M. Hance138, B. Haney123, P. Hanke60a, R. Hanna137, J.B. Hansen38, J.D. Hansen38, M.C. Hansen23, P.H. The ATLAS collaboration Hansen38, P. Hanke60a, R. Hanna137, J.B. Hansen38, J.D. Hansen38, M.C. Hansen23, P.H. Hansen38, K. Hara161, A.S. Hard173, T. Harenberg175, F. Hariri118, S. Harkusha94, R.D. Harrington48, P.F. Harrison170, F. Hartjes108, N.M. Hartmann101, M. Hasegawa69, Y. Hasegawa141, A. Hasib1 P.F. Harrison170, F. Hartjes108, N.M. Hartmann101, M. Hasegawa69, Y. Hasegawa141, A. Hasib114, R.J. Hawkings32, D. Hayden92, C.P. Hays121, J.M. Hays78, H.S. Hayward76, S.J. Haywood132, R.J. Hawkings32, D. Hayden92, C.P. Hays121, J.M. Hays78, H.S. Hayward76, S.J. Haywood132, S.J. Head19, T. Heck85, V. Hedberg83, L. Heelan8, S. Heim123, T. Heim16, B. Heinemann16, J.J. Heinrich101, L. Heinrich111, C. Heinz54, J. Hejbal128, L. Helary24, S. Hellman147a,147b, C. Helsens32, J. Henderson121, R.C.W. Henderson74, Y. Heng173, S. Henkelmann168, A.M. Henriques Correia32, S. Henrot-Versille118, G.H. Herbert17, Y. Hern´andez Jim´enez167, G. Herten50, R. Hertenberger101, L. Hervas32, G.G. Hesketh80, N.P. Hessey108, J.W. Hetherl Herten50, R. Hertenberger101, L. Hervas32, G.G. Hesketh80, N.P. Hessey108, J.W. Hetherly42, R. Hickling78, E. Hig´on-Rodriguez167, E. Hill169, J.C. Hill30, K.H. Hiller44, S.J. Hillier19, – 23 – I. Hinchliffe16, E. Hines123, R.R. Hinman16, M. Hirose158, D. Hirschbuehl175, J. Hobbs149, N. Hod160a, M.C. Hodgkinson140, P. Hodgson140, A. Hoecker32, M.R. Hoeferkamp106, F. Hoenig101, D. Hohn23, T.R. Holmes16, M. Homann45, T.M. Hong126, B.H. Hooberman166, W.H. Hopkins117, Y. Horii104, A.J. Horton143, J-Y. Hostachy57, S. Hou152, A. Hoummada136 J. Howarth44, M. Hrabovsky116, I. Hristova17, J. Hrivnac118, T. Hryn’ova5, A. Hrynevich95, C. Hsu146c, P.J. Hsu152,t, S.-C. Hsu139, D. Hu37, Q. Hu35b, Y. Huang44, Z. Hubacek129, F. Hubaut87, F. Huegging23, T.B. Huffman121, E.W. Hughes37, G. Hughes74, M. Huhtin T.A. H¨ulsing85, P. Huo149, N. Huseynov67,b, J. Huston92, J. Huth59, G. Iacobucci O. Igonkina108,u, T. Iizawa171, Y. Ikegami68, M. Ikeno68, Y. Ilchenko11,v, D. Iliadis155, N. Ilic14 T. Ince102, G. Introzzi122a,122b, P. Ioannou9,∗, M. Iodice135a, K. Iordanidou37, V. Ippolito59 JHEP11(2016)112 M. Ishino70, M. Ishitsuka158, R. Ishmukhametov112, C. Issever121, S. Istin20a, F. Ito161, J.M. Iturbe Ponce86, R. Iuppa134a,134b, W. Iwanski41, H. Iwasaki68, J.M. Izen43, V. Izzo105a, S. Jabbar3, B. Jackson123, M. Jackson76, P. Jackson1, V. Jain2, K.B. Jakobi85, K. Jakobs50, S. Jakobsen32, T. Jakoubek128, D.O. Jamin115, D.K. Jana81, E. Jansen80, R. Jansky64, Janssen23, M. Janus56, G. Jarlskog83, N. Javadov67,b, T. Jav˚urek50, F. Jeanneau137, J. Janssen23, M. Janus56, G. Jarlskog83, N. Javadov67,b, T. Jav˚urek50, F. Jeanneau137, anty16, J. Jejelava53a,w, G.-Y. Jeng151, D. Jennens90, P. Jenni50,x, J. Jentzsch45, C. Jeske170, L. Jeanty16, J. Jejelava53a,w, G.-Y. Jeng151, D. Jennens90, P. Jenni50,x, J. Jentzsch45, C. J´ez´equel5, H. Ji173, J. Jia149, H. Jiang66, Y. Jiang35b, S. Jiggins80, J. Jimenez Pena167, S. J´ez´equel5, H. Ji173, J. The ATLAS collaboration Jia149, H. Jiang66, Y. Jiang35b, S. Jiggins80, J. Jimenez Pena167, Jin35a, A. Jinaru28b, O. Jinnouchi158, P. Johansson140, K.A. Johns7, W.J. Johnson139, S. Jin35a, A. Jinaru28b, O. Jinnouchi158, P. Johansson140, K.A. Johns7, W.J. Johnson139, Jon-And147a,147b, G. Jones170, R.W.L. Jones74, S. Jones7, T.J. Jones76, J. Jongmanns60a, K. Jon-And147a,147b, G. Jones170, R.W.L. Jones74, S. Jones7, T.J. Jones76, J. Jongmann P.M. Jorge127a,127b, J. Jovicevic160a, X. Ju173, A. Juste Rozas13,r, M.K. K¨ohler172, M. Jorge127a,127b, J. Jovicevic160a, X. Ju173, A. Juste Rozas13,r, M.K. K¨ohler172, aczmarska41, M. Kado118, H. Kagan112, M. Kagan144, S.J. Kahn87, E. Kajomovitz47, A. Kaczmarska41, M. Kado118, H. Kagan112, M. Kagan144, S.J. Kahn87, E. Kajomovitz47, C.W. Kalderon121, A. Kaluza85, S. Kama42, A. Kamenshchikov131, N. Kanaya156, S. Kanet C.W. Kalderon121, A. Kaluza85, S. Kama42, A. Kamenshchikov131, N. Kanaya156, S. Kaneti30, L. Kanjir77, V.A. Kantserov99, J. Kanzaki68, B. Kaplan111, L.S. Kaplan173, A. Kapliy33, 77, V.A. Kantserov99, J. Kanzaki68, B. Kaplan111, L.S. Kaplan173, A. Kapliy33, D. Kar146c, K. Karakostas10, A. Karamaoun3, N. Karastathis10, M.J. Kareem56, E. Karentzos1 D. Kar146c, K. Karakostas10, A. Karamaoun3, N. Karastathis10, M.J. Kareem56, E. Karentzos10 A.N. Karyukhin131, K. Kasahara161, L. Kashif173, R.D. Kass112, A. Kastanas15, Y. Kataoka156, A.N. Karyukhin131, K. Kasahara161, L. Kashif173, R.D. Kass112, A. Kastanas15, Y. Kataoka156, C. Kato156, A. Katre51, J. Katzy44, K. Kawagoe72, T. Kawamoto156, G. Kawamura56, S. Kazama156, V.F. Kazanin110,c, R. Keeler169, R. Kehoe42, J.S. Keller44, J.J. Kempster S. Kazama156, V.F. Kazanin110,c, R. Keeler169, R. Kehoe42, J.S. Keller44, J.J. Kempster79, K Kentaro104, H. Keoshkerian159, O. Kepka128, B.P. Kerˇsevan77, S. Kersten175, R.A. Keye F. Khalil-zada12, A. Khanov115, A.G. Kharlamov110,c, T.J. Khoo51, V. Khovanskiy98, mov67, J. Khubua53b,y, S. Kido69, H.Y. Kim8, S.H. Kim161, Y.K. Kim33, N. Kimura155, E. Khramov67, J. Khubua53b,y, S. Kido69, H.Y. Kim8, S.H. Kim161, Y.K. Kim33, N. Kimur O.M. Kind17, B.T. King76, M. King167, S.B. King168, J. Kirk132, A.E. Kiryunin102, T. Kishimoto69, D. Kisielewska40a, F. Kiss50, K. Kiuchi161, O. Kivernyk137, E. Kladiva145b, M.H. Klein37, M. Klein76, U. Klein76, K. Kleinknecht85, P. Klimek147a,147b, A. Klimentov27, R. Klingenberg45, J.A. Klinger140, T. Klioutchnikova32, E.-E. Kluge60a, P. Kluit108, S. Kluth102 J. Knapik41, E. Kneringer64, E.B.F.G. Knoops87, A. Knue55, A. Kobayashi156, D. Kobayashi158 T. Kobayashi156, M. Kobel46, M. Kocian144, P. Kodys130, T. Koffas31, E. Koffeman108, T. Koi144, H. Kolanoski17, M. Kolb60b, I. Koletsou5, A.A. Komar97,∗, Y. Komori156, T. Kondo68, N. Kondrashova44, K. K¨oneke50, A.C. K¨onig107, T. Kono68,z, R. Konoplich111,aa, N. Konstantinidis80, R. Kopeliansky63, S. Koperny40a, L. K¨opke85, A.K. Kopp50, K. Korcyl41,
K. Kordas155, A. Korn80, A.A. Korol110,c, I. Korolkov13, E.V. Korolkova140, O. The ATLAS collaboration Kortner102, N. Konstantinidis80, R. Kopeliansky63, S. Koperny40a, L. Kopke85, A.K. Kopp50, K. Korcyl41,
K. Kordas155, A. Korn80, A.A. Korol110,c, I. Korolkov13, E.V. Korolkova140, O. Kortner102,
S. Kortner102, T. Kosek130, V.V. Kostyukhin23, A. Kotwal47, A. Kourkoumeli-Charalampidi155,
C. Kourkoumelis9, V. Kouskoura27, A.B. Kowalewska41, R. Kowalewski169, T.Z. Kowalski40a,
C. Kozakai156, W. Kozanecki137, A.S. Kozhin131, V.A. Kramarenko100, G. Kramberger77,
D. Krasnopevtsev99, M.W. Krasny82, A. Krasznahorkay32, J.K. Kraus23, A. Kravchenko27,
M. Kretz60c, J. Kretzschmar76, K. Kreutzfeldt54, P. Krieger159, K. Krizka33, K. Kroeninger45,
H. Kroha102, J. Kroll123, J. Kroseberg23, J. Krstic14, U. Kruchonak67, H. Kr¨uger23, K. Kordas155, A. Korn80, A.A. Korol110,c, I. Korolkov13, E.V. Korolkova140, O. Kortner102, – 24 – N. Krumnack66, A. Kruse173, M.C. Kruse47, M. Kruskal24, T. Kubota90, H. Kucuk80, S. Kuday4 ,
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J.T. Kuechler175, S. Kuehn50, A. Kugel60c, F. Kuger174, A. Kuhl138, T. Kuhl44, V. Kukhtin67, ,
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R. Kukla137, Y. Kulchitsky94, S. Kuleshov34b, M. Kuna133a,133b, T. Kunigo70, A. Kupco128, H. Kurashige69, Y.A. Kurochkin94, V. Kus128, E.S. Kuwertz169, M. Kuze158, J. Kvita116, T. Kwan169, D. Kyriazopoulos140, A. La Rosa102, J.L. La Rosa Navarro26d, L. La Rotonda39 C. Lacasta167, F. Lacava133a,133b, J. Lacey31, H. Lacker17, D. Lacour82, V.R. La E. Ladygin67, R. Lafaye5, B. Laforge82, T. Lagouri176, S. Lai56, S. Lammers63, W. Lampl7, E. Lan¸con137, U. Landgraf50, M.P.J. Landon78, V.S. Lang60a, J.C. Lange13, A.J. L F. Lasagni Manghi22a,22b, M. Lassnig32, P. Laurelli49, W. Lavrijsen16, A.T. Law138, P. Laycock76, g
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T. Lazovich59, M. Lazzaroni93a,93b, B. Le90, O. Le Dortz82, E. Le Guirriec87, E.P. Le Quilleuc13 JHEP11(2016)112 M. LeBlanc169, T. LeCompte6, F. Ledroit-Guillon57, C.A. Lee27, S.C. Lee152, L. Lee1, G. Lefebvre82, M. Lefebvre169, F. Legger101, C. Leggett16, A. Lehan76, G. Lehmann Miotto32, X. Lei7, W.A. Leight31, A. Leisos155,ab, A.G. Leister176, M.A.L. Leite26d, R. Leitner130, D. Lellouch172, B. Lemmer56, K.J.C. Leney80, T. Lenz23, B. Lenzi32, R. Leone7, S. Leone125a,125 C. Leonidopoulos48, S. Leontsinis10, G. Lerner150, C. Leroy96, A.A.J. Lesage137, C.G. Lester30, M. Levchenko124, J. Levˆeque5, D. Levin91, L.J. Levinson172, M. Levy19, D. Lewis78, A.M. Leyko2 M. Leyton43, B. Li35b,o, H. Li149, H.L. Li33, L. Li47, L. Li35e, Q. Li35a, S. Li47, X. Li86, Y. Li142 Z. Liang35a, B. Liberti134a, A. Liblong159, P. Lichard32, K. Lie166, J. Liebal23, W. Liebig15, A. Limosani151, S.C. Lin152,ac, T.H. Lin85, B.E. Lindquist149, A.E. Lionti51, E. Lipeles123, A. Lipniacka15, M. Lisovyi60b, T.M. Liss166, A. Lister168, A.M. Litke138, B. Liu152,ad, D. Liu152 H. The ATLAS collaboration Liu91, H. Liu27, J. Liu87, J.B. Liu35b, K. Liu87, L. Liu166, M. Liu47, M. Liu35b, Y.L. Liu35b, Y. Liu35b, M. Livan122a,122b, A. Lleres57, J. Llorente Merino35a, S.L. Lloyd78, F. Lo Sterzo152, E. Lobodzinska44, P. Loch7, W.S. Lockman138, F.K. Loebinger86, A.E. Loevschall-Jensen38, K.M. Loew25, A. Loginov176, T. Lohse17, K. Lohwasser44, M. Lokajicek128, B.A. Long24, B. Lopez Paredes140, I. Lopez Paz13, A. Lopez Solis82, J. Lorenz101, N. Lorenzo Martinez63, M. Losada21, P.J. L¨osel101, X. Lou35a, A. Lounis118, J. Love6, P.A. Love74, H. Lu62a, N. Lu91, H.J. Lubatti139, C. Luci133a,133b, A. Lucotte57, C. Luedtke50, F. Luehring63, W. Lukas64, L. Luminari133a, O. Lundberg147a,147b, B. Lund-Jensen148, P.M. Luzi82, D. Lynn27, R. Lysa E. Lytken83, V. Lyubushkin67, H. Ma27, L.L. Ma35d, Y. Ma35d, G. Maccarrone49, A. Macchiolo102, C.M. Macdonald140, B. Maˇcek77, J. Machado Miguens123,127b, D. R. Madar36, H.J. Maddocks165, W.F. Mader46, A. Madsen44, J. Maeda69, S. Maeland15, Madar36, H.J. Maddocks165, W.F. Mader46, A. Madsen44, J. Maeda69, S. Maeland15, Maeno27, A. Maevskiy100, E. Magradze56, J. Mahlstedt108, C. Maiani118, C. Maidantchik26a, T. Maeno27, A. Maevskiy100, E. Magradze56, J. Mahlstedt108, C. Maiani118, C. Maidantchik A. Maier102, T. Maier101, A. Maio127a,127b,127d, S. Majewski117, Y. Makida68, N. Makovec118, A.A. Maier102, T. Maier101, A. Maio127a,127b,127d, S. Majewski117, Y. Makida68, N. Makovec B. Malaescu82, Pa. Malecki41, V.P. Maleev124, F. Malek57, U. Mallik65, D. Malon6, C. Malone144, I. Mandi´c77, J. Maneira127a,127b, L. Manhaes de Andrade Filho26b, J. Manjarres Ramos160b, S. Manzoni93a,93b, L. Mapelli32, G. Marceca29, L. March51, G. Marchiori82, M. Marcis S. Marti-Garcia167, B. Martin92, T.A. Martin170, V.J. Martin48, B. Martin dit Lat M. Martinez13,r, S. Martin-Haugh132, V.S. Martoiu28b, A.C. Martyniuk80, M. Marx139, – 25 – J.A. Mcfayden80, G. Mchedlidze56, S.J. McMahon132, R.A. McPherson169,l, M. Medinnis44, y
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D. Melini167, B.R. Mellado Garcia146c, M. Melo145a, F. Meloni18, A. Mengarelli22a,22b, S. Menke102, E. Meoni162, S. Mergelmeyer17, P. Mermod51, L. Merola105a,105b, C. Meroni93a F.S. Merritt33, A. Messina133a,133b, J. Metcalfe6, A.S. Mete163, C. Meyer85, C. Meyer123, J-P. Meyer137, J. Meyer108, H. Meyer Zu Theenhausen60a, F. Miano150, R.P. Middleton132, S. Miglioranzi52a,52b, L. Mijovi´c23, G. Mikenberg172, M. Mikestikova128, M. Mikuˇz77, M. Milesi9 A. Milic64, D.W. Miller33, C. Mills48, A. Milov172, D.A. Milstead147a,147b, A.A. Minaenko13 ,
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L.M. Mir13, K.P. Mistry123, T. Mitani171, J. Mitrevski101, V.A. Mitsou167, A. Miucci51, P.S. Miyagawa140, J.U. Mj¨ornmark83, T. Moa147a,147b, K. Mochizuki96, S. Mohapatra37, JHEP11(2016)112 S. Molander147a,147b, R. The ATLAS collaboration Moles-Valls23, R. Monden70, M.C. Mondragon92, K. M¨onig44, J. Monk38, E. Monnier87, A. Montalbano149, J. Montejo Berlingen32, F. Monticelli73, S. Monzani93a,93b, R.W. Moore3, N. Morange118, D. Moreno21, M. Moreno Ll´acer56, P. Morettini52a, D. Mori143 T. Mori156, M. Morii59, M. Morinaga156, V. Morisbak120, S. Moritz85, A.K. Morley151, Mornacchi32, J.D. Morris78, S.S. Mortensen38, L. Morvaj149, M. Mosidze53b, J. Moss144, G. Mornacchi32, J.D. Morris78, S.S. Mortensen38, L. Morvaj149, M. Mosidze53b, J. Moss144, K. Motohashi158, R. Mount144, E. Mountricha27, S.V. Mouraviev97,∗, E.J.W. Moyse88, Motohashi158, R. Mount144, E. Mountricha27, S.V. Mouraviev97,∗, E.J.W. Moyse88, S. Muanza87, R.D. Mudd19, F. Mueller102, J. Mueller126, R.S.P. Mueller101, T. Mueller30, Muanza87, R.D. Mudd19, F. Mueller102, J. Mueller126, R.S.P. Mueller101, T. Mueller30, D. Muenstermann74, P. Mullen55, G.A. Mullier18, F.J. Munoz Sanchez86, J.A. Murillo Quija stermann74, P. Mullen55, G.A. Mullier18, F.J. Munoz Sanchez86, J.A. Murillo Quijada19, W.J. Murray170,132, H. Musheghyan56, M. Muˇskinja77, A.G. Myagkov131,ae, M. Myska129, rray170,132, H. Musheghyan56, M. Muˇskinja77, A.G. Myagkov131,ae, M. Myska129, B.P. Nachman144, O. Nackenhorst51, K. Nagai121, R. Nagai68,z, K. Nagano68, Y. Nagasaka6 B.P. Nachman144, O. Nackenhorst51, K. Nagai121, R. Nagai68,z, K. Nagano68, Y. Nagasaka61, K. Nagata161, M. Nagel50, E. Nagy87, A.M. Nairz32, Y. Nakahama32, K. Nakamura68, T. Nakamura156, I. Nakano113, H. Namasivayam43, R.F. Naranjo Garcia44, R. Narayan11, T. Nakamura156, I. Nakano113, H. Namasivayam43, R.F. Naranjo Garcia44, R. Narayan11, D.I. Narrias Villar60a, I. Naryshkin124, T. Naumann44, G. Navarro21, R. Nayyar7, H.A. Neal91,
P.Yu. Nechaeva97, T.J. Neep86, P.D. Nef144, A. Negri122a,122b, M. Negrini22a, S. Nektarijevic107, P.Yu. Nechaeva97, T.J. Neep86, P.D. Nef144, A. Negri122a,122b, M. Negrini22a, S. Nektarijevic107, M.S. Neubauer166, M. Neumann175, R.M. Neves111, P. Nevski27, P.R. Newman19, D.H. Nguyen6 T. Nguyen Manh96, R.B. Nickerson121, R. Nicolaidou137, J. Nielsen138, A. Nikiforov17, V. Nikolaenko131,ae, I. Nikolic-Audit82, K. Nikolopoulos19, J.K. Nilsen120, P. Nilsson27, Y. Ninomiya156, A. Nisati133a, R. Nisius102, T. Nobe156, L. Nodulman6, M. Nomachi119 miya156, A. Nisati133a, R. Nisius102, T. Nobe156, L. Nodulman6, M. Nomachi119, I. Nomidis31, T. Nooney78, S. Norberg114, M. Nordberg32, N. Norjoharuddeen121, is31, T. Nooney78, S. Norberg114, M. Nordberg32, N. Norjoharuddeen121, orodova46, S. Nowak102, M. Nozaki68, L. Nozka116, K. Ntekas10, E. Nurse80, F. Nuti90, O. Novgorodova46, S. Nowak102, M. Nozaki68, L. Nozka116, K. Ntekas10, E. Nurse80 O’grady7, D.C. O’Neil143, A.A. O’Rourke44, V. O’Shea55, F.G. Oakham31,d, H. Oberlack102, F. O’grady7, D.C. O’Neil143, A.A. O’Rourke44, V. O’Shea55, F.G. Oakham31,d, H. Oberlack1 T. Obermann23, J. Ocariz82, A. Ochi69, I. Ochoa37, J.P. Ochoa-Ricoux34a, S. Oda72, S. Odak Obermann23, J. Ocariz82, A. Ochi69, I. Ochoa37, J.P. Ochoa-Ricoux34a, S. Oda72, S. Odaka68, H. Ogren63, A. The ATLAS collaboration Oh86, S.H. Oh47, C.C. Ohm16, H. Ohman165, H. Oide32, H. Okawa161, Ogren63, A. Oh86, S.H. Oh47, C.C. Ohm16, H. Ohman165, H. Oide32, H. Okawa161, Okumura33, T. Okuyama68, A. Olariu28b, L.F. Oleiro Seabra127a, S.A. Olivares Pino48, Y. Okumura33, T. Okuyama68, A. Olariu28b, L.F. Oleiro Seabra127a, S.A. Olivares Pino48 D. Oliveira Damazio27, A. Olszewski41, J. Olszowska41, A. Onofre127a,127e, K. Onogi104, D. Oliveira Damazio27, A. Olszewski41, J. Olszowska41, A. Onofre127a,127e, K. Onogi104, U.E. Onyisi11,v, M.J. Oreglia33, Y. Oren154, D. Orestano135a,135b, N. Orlando62b, R.S. Orr159, B. Osculati52a,52b, R. Ospanov86, G. Otero y Garzon29, H. Otono72, M. Ouchrif136d, Osculati52a,52b, R. Ospanov86, G. Otero y Garzon29, H. Otono72, M. Ouchrif136d, F. Ould-Saada120, A. Ouraou137, K.P. Oussoren108, Q. Ouyang35a, M. Owen55, R.E. Owen F. Ould-Saada120, A. Ouraou137, K.P. Oussoren108, Q. Ouyang35a, M. Owen55, R.E. Ow M. Pag´aˇcov´a50, S. Pagan Griso16, F. Paige27, P. Pais88, K. Pajchel120, G. Palacino160b, M. Pag´aˇcov´a50, S. Pagan Griso16, F. Paige27, P. Pais88, K. Pajchel120, G. Palacino160b g
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E. Pandini82, J.G. Panduro Vazquez79, P. Pani147a,147b, S. Panitkin27, D. Pantea28b, C.E. Pandini82, J.G. Panduro Vazquez79, P. Pani147a,147b, S. Panitkin27, D. Pantea28b Paolozzi51, Th.D. Papadopoulou10, K. Papageorgiou155, A. Paramonov6, L. Paolozzi51, Th.D. Papadopoulou10, K. Papageorgiou155, A. Paramonov6, Paredes Hernandez176, A.J. Parker74, M.A. Parker30, K.A. Parker140, F. Parodi52a,52b, D. Paredes Hernandez176, A.J. Parker74, M.A. Parker30, K.A. Parker140, F. Parodi52a Paredes Hernandez176, A.J. Parker74, M.A. Parker30, K.A. Parker140, F. Parodi52a,52b,
A. Parsons37, U. Parzefall50, V.R. Pascuzzi159, E. Pasqualucci133a, S. Passaggio52a, D. Paredes Hernandez176, A.J. Parker74, M.A. Parker30, K.A. Parker140, F. Parodi52a,52b
J.A. Parsons37, U. Parzefall50, V.R. Pascuzzi159, E. Pasqualucci133a, S. Passaggio52a, J.A. Parsons37, U. Parzefall50, V.R. Pascuzzi159, E. Pasqualucci133a, S. Passaggio52a, Fr. Pastore79, G. P´asztor31,ag, S. Pataraia175, J.R. Pater86, T. Pauly32, J. Pearce169, – 26 – B. Pearson114, L.E. Pedersen38, M. Pedersen120, S. Pedraza Lopez167, R. Pedro127a,127b, ,
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B.S. Peralva26b, M.M. Perego137, D.V. Perepelitsa27, E. Perez Codina160a, L. Perini93a,93b, H. Pernegger32, S. Perrella105a,105b, R. Peschke44, V.D. Peshekhonov67, K. Peters44, R.F.Y. Peters86, B.A. Petersen32, T.C. Petersen38, E. Petit57, A. Petridis1, C. Petridou155, P. Petroff118, E. Petrolo133a, M. Petrov121, F. Petrucci135a,135b, N.E. The ATLAS collaboration Pettersson88, A. Peyau R. Pezoa34b, P.W. Phillips132, G. Piacquadio144, E. Pianori170, A. Picazio88, E. Piccaro78, M. Piccinini22a,22b, M.A. Pickering121, R. Piegaia29, J.E. Pilcher33, A.D. Pilk M. Pitt172, L. Plazak145a, M.-A. Pleier27, V. Pleskot85, E. Plotnikova67, P. Plucinski92, D. Pluth M. Pitt172, L. Plazak145a, M.-A. Pleier27, V. Plesk M. Pitt 7 , L. Plazak
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R. Poettgen147a,147b, L. Poggioli118, D. Pohl23, G. Polesello122a, A. Poley44, A. Policicchio39a,39b JHEP11(2016)112 R. Polifka159, A. Polini22a, C.S. Pollard55, V. Polychronakos27, K. Pomm`es32, L. Pontecorvo133a, B.G. Pope92, G.A. Popeneciu28c, D.S. Popovic14, A. Poppleton32, S. Pospisil129, K. Potamianos I.N. Potrap67, C.J. Potter30, C.T. Potter117, G. Poulard32, J. Poveda32, V. Pozdnyakov67, M.E. Pozo Astigarraga32, P. Pralavorio87, A. Pranko16, S. Prell66, D. Price86, L.E. Price6, M. Primavera75a, S. Prince89, M. Proissl48, K. Prokofiev62c, F. Prokoshin34b, S. Protopopescu27 J. Proudfoot6, M. Przybycien40a, D. Puddu135a,135b, D. Puldon149, M. Purohit27,ai, P. Puzo118, J. Qian91, G. Qin55, Y. Qin86, A. Quadt56, W.B. Quayle164a,164b, M. Queitsch-Maitland86, D. Quilty55, S. Raddum120, V. Radeka27, V. Radescu60b, S.K. Radhakrishnan149, P. Radloff117, P. Rados90, F. Ragusa93a,93b, G. Rahal178, J.A. Raine86, S. Rajagopalan27, M. Rammensee32 dos90, F. Ragusa93a,93b, G. Rahal178, J.A. Raine86, S. Rajagopalan27, M. Rammensee32, C. Rangel-Smith165, M.G. Ratti93a,93b, F. Rauscher101, S. Rave85, T. Ravenscroft55, ngel-Smith165, M.G. Ratti93a,93b, F. Rauscher101, S. Rave85, T. Ravenscroft55, I. Ravinovich172, M. Raymond32, A.L. Read120, N.P. Readioff76, M. Reale75a,75b, D.M. Rebuzzi122a,122b, A. Redelbach174, G. Redlinger27, R. Reece138, K. Reeves43, L. Rehnisch17, D.M. Rebuzzi122a,122b, A. Redelbach174, G. Redlinger27, R. Reece138, K. Reeves43, L. Rehn J. Reichert123, H. Reisin29, C. Rembser32, H. Ren35a, M. Rescigno133a, S. Resconi93a, O.L. Rezanova110,c, P. Reznicek130, R. Rezvani96, R. Richter102, S. Richter80, E. Richter-Was40b, O.L. Rezanova110,c, P. Reznicek130, R. Rezvani96, R. Richter102, S. Richter80, E. Richter-Was40b O.L. Rezanova110,c, P. Reznicek130, R. Rezvani96, R. Richter102, S. Richter80, E. Richter
O. Ricken23, M. Ridel82, P. Rieck17, C.J. Riegel175, J. Rieger56, O. Rifki114, M. Rijssenb A. Rimoldi122a,122b, M. Rimoldi18, L. Rinaldi22a, B. Risti´c51, E. Ritsch32, I. Riu13, A. Rimoldi122a,122b, M. Rimoldi18, L. Rinaldi22a, B. Risti´c51, E. Ritsch32, I. Riu13, F. Rizatdinova115, E. Rizvi78, C. Rizzi13, S.H. Robertson89,l, A. Robichaud-Veronneau89 Robinson30, J.E.M. Robinson44, A. Robson55, C. Roda125a,125b, Y. Rodina87, D. Robinson30, J.E.M. Robinson44, A. Robson55, C. Roda125a,125b, Y. Rodina87, driguez Perez13, D. Rodriguez Rodriguez167, S. Roe32, C.S. Rogan59, O. Røhne120, A. Rodriguez Perez13, D. Rodriguez Rodriguez167, S. Roe32, C.S. Rogan59, O. Røhne120, A. Romaniouk99, M. Romano22a,22b, S.M. Romano Saez36, E. The ATLAS collaboration Simard5, Lj. Simic14, jacki14, J. Silva127a,127d, S.B. Silverstein147a, V. Simak129, O. Simard5, Lj. Simic14, S. Simion118, E. Simioni85, B. Simmons80, D. Simon36, M. Simon85, P. Sinervo159, N.B. Sinev117,
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F. Socher46, A. Soffer154, D.A. Soh152, G. Sokhrannyi77, C.A. Solans Sanchez32, M. Solar129 E.Yu. Soldatov99, U. Soldevila167, A.A. Solodkov131, A. Soloshenko67, O.V. Solovyanov131, E.Yu. Soldatov99, U. Soldevila167, A.A. Solodkov131, A. Soloshenko67, O.V. Solovyanov131,
124
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12 D. South44, B.C. Sowden79, S. Spagnolo75a,75b, M. Spalla125a,125b, M. Spangenberg170, an`o79, D. Sperlich17, F. Spettel102, R. Spighi22a, G. Spigo32, L.A. Spiller90, M. Spousta130, F. Span`o79, D. Sperlich17, F. Spettel102, R. Spighi22a, G. Spigo32, L.A. Spiller90, M. Sp R.D. St. Denis55,∗, A. Stabile93a, R. Stamen60a, S. Stamm17, E. Stanecka41, R.W. Stanek6, St. Denis55,∗, A. Stabile93a, R. Stamen60a, S. Stamm17, E. Stanecka41, R.W. Stanek6, C. Stanescu135a, M. Stanescu-Bellu44, M.M. Stanitzki44, S. Stapnes120, E.A. Starchenko131, Stanescu135a, M. Stanescu-Bellu44, M.M. Stanitzki44, S. Stapnes120, E.A. Starchenko131, H. Stark33, J. Stark57, P. Staroba128, P. Starovoitov60a, S. St¨arz32, R. Staszewski41, G.H. Stark33, J. Stark57, P. Staroba128, P. Starovoitov60a, S. St¨arz32, R. Staszewski41, Steinberg27, B. Stelzer143, H.J. Stelzer32, O. Stelzer-Chilton160a, H. Stenzel54, G.A. Stewart55, P. Steinberg27, B. Stelzer143, H.J. Stelzer32, O. Stelzer-Chilton160a, H. Stenzel54, G.A. Stewa J.A. Stillings23, M.C. Stockton89, M. Stoebe89, G. Stoicea28b, P. Stolte56, S. Stonjek102, A. Stillings23, M.C. Stockton89, M. Stoebe89, G. Stoicea28b, P. Stolte56, S. Stonjek102, A.R. Stradling8, A. Straessner46, M.E. Stramaglia18, J. Strandberg148, S. Strandberg147a, R. Stradling8, A. Straessner46, M.E. Stramaglia18, J. Strandberg148, S. Strandberg147a,147b, A. Strandlie120, M. Strauss114, P. Strizenec145b, R. Str¨ohmer174, D.M. Strom117, A. Strandlie120, M. Strauss114, P. Strizenec145b, R. Str¨ohmer174, D.M. Strom117, y
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R. Subramaniam81, S. Suchek60a, Y. Sugaya119, M. Suk129, V.V. Sulin97, S. Sultansoy4c, R. The ATLAS collaboration Schaarschmidt172, P. Schacht102, B.M. Schachtner101, D. Schaefer32, R. Schaefer44, ,
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J. The ATLAS collaboration Tsirintanis9, S. Tsiskaridze13, V. Tsiskaridze50, E.G. Tskhadadze53a, K.M. Tsui62a, I.I. Tsukerman98, V. Tsulaia16, S. Tsuno68, D. Tsybychev149, A. Tudorache28b, V. Tud . Tsukerman98, V. Tsulaia16, S. Tsuno68, D. Tsybychev149, A. Tudorache28b, V. Tudorache28b, N. Tuna59, S.A. Tupputi22a,22b, S. Turchikhin100,al, D. Turecek129, D. Turgeman172, A.N. Tuna59, S.A. Tupputi22a,22b, S. Turchikhin100,al, D. Turecek129, D. Turgeman172, R. Turra93a,93b, A.J. Turvey42, P.M. Tuts37, M. Tyndel132, G. Ucchielli22a,22b, I. Ueda156, R. Turra93a,93b, A.J. Turvey42, P.M. Tuts37, M. Tyndel132, G. Ucchielli22a,22b, I. Ueda156 R. Ueno31, M. Ughetto147a,147b, F. Ukegawa161, G. Unal32, A. Undrus27, G. Unel163, R. Ueno31, M. Ughetto147a,147b, F. Ukegawa161, G. Unal32, A. Undrus27, G. Unel163, F.C. Ungaro90, Y. Unno68, C. Unverdorben101, J. Urban145b, P. Urquijo90, P. Urrejola85, F.C. Ungaro90, Y. Unno68, C. Unverdorben101, J. Urban145b, P. Urquijo90, P. Urrejola85, G. Usai8, A. Usanova64, L. Vacavant87, V. Vacek129, B. Vachon89, C. Valderanis101, G. Usai8, A. Usanova64, L. Vacavant87, V. Vacek129, B. Vachon89, C. Valderanis101, E. Valdes Santurio147a,147b, N. Valencic108, S. Valentinetti22a,22b, A. Valero167, L. Valery13, E. Valdes Santurio147a,147b, N. Valencic108, S. Valentinetti22a,22b, A. Valero167, L. Valery1 S. Valkar130, S. Vallecorsa51, J.A. Valls Ferrer167, W. Van Den Wollenberg108, S. Valkar130, S. Vallecorsa51, J.A. Valls Ferrer167, W. Van Den Wollenberg108, P.C. Van Der Deijl108, R. van der Geer108, H. van der Graaf108, N. van Eldik153, P.C. Van Der Deijl108, R. van der Geer108, H. van der Graaf108, N. van Eldik153, P. van Gemmeren6, J. Van Nieuwkoop143, I. van Vulpen108, M.C. van Woerden32, van Gemmeren6, J. Van Nieuwkoop143, I. van Vulpen108, M.C. van Woerden32, M. Vanadia133a,133b, W. Vandelli32, R. Vanguri123, A. Vaniachine6, P. Vankov108, M. Vanadia133a,133b, W. Vandelli32, R. Vanguri123, A. Vaniachine6, P. Vankov108, A. Vartapetian8, K.E. Varvell151, J.G. Vasquez176, F. Vazeille36, T. Vazquez Schroeder A. Vartapetian8, K.E. Varvell151, J.G. Vasquez176, F. Vazeille36, T. Vazquez Schroeder J. Veatch56, L.M. Veloce159, F. Veloso127a,127c, S. Veneziano133a, A. Ventura75a,75b, M Veatch56, L.M. Veloce159, F. Veloso127a,127c, S. Veneziano133a, A. Ventura75a,75b, M. Venturi169 N. Venturi159, A. Venturini25, V. Vercesi122a, M. Verducci133a,133b, W. Verkerke108, N. Venturi159, A. Venturini25, V. Vercesi122a, M. Verducci133a,133b, W. Verkerke108, C. Vermeulen108, A. Vest46,aq, M.C. Vetterli143,d, O. Viazlo83, I. Vichou166, T. Vickey140, J.C. Vermeulen108, A. Vest46,aq, M.C. Vetterli143,d, O. Viazlo83, I. Vichou166, T. Vic O.E. Vickey Boeriu140, G.H.A. Viehhauser121, S. Viel16, L. Vigani121, R. Vigne64, M. Villa22a,22b,
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H. Takai27, R. Takashima71, T. Takeshita141, Y. Takubo68, M. Talby87, A.A. Talyshev110,c,
K.G. Tan90, J. Tanaka156, R. Tanaka118, S. Tanaka68, B.B. Tannenwald112, S. Tapia Araya34b,
S. Tapprogge85, S. Tarem153, G.F. Tartarelli93a, P. Tas130, M. Tasevsky128, T. Tashiro70, H. Takai27, R. Takashima71, T. Takeshita141, Y. Takubo68, M. Talby87, A.A. Talyshev110,c, S. Tapprogge85, S. Tarem153, G.F. Tartarelli93a, P. Tas130, M. Tasevsky128, T. Tashiro70, – 28 – E. Tassi39a,39b, A. Tavares Delgado127a,127b, Y. Tayalati136d, A.C. Taylor106, G.N. Taylor90, P.T.E. Taylor90, W. Taylor160b, F.A. Teischinger32, P. Teixeira-Dias79, K.K. Temming50, y
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M.W. Wolter41, H. Wolters127a,127c, B.K. Wosiek41, J. Wotschack32, M.J. Woudstra86, W. Wolter41, H. Wolters127a,127c, B.K. Wosiek41, J. Wotschack32, M.J. Woudstra86, ,
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K.W. Wozniak41, M. Wu57, M. Wu33, S.L. Wu173, X. Wu51, Y. Wu91, T.R. Wyatt86, ,
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W. Wozniak41, M. Wu57, M. Wu33, S.L. Wu173, X. Wu51, Y. Wu91, T.R. Wyatt86, B.M. Wynne48, S. Xella38, D. Xu35a, L. Xu27, B. Yabsley151, S. Yacoob146a, R. Yakabe69, B.M. Wynne48, S. Xella38, D. Xu35a, L. Xu27, B. Yabsley151, S. Yacoob146a, R. Yakabe69 D. Yamaguchi158, Y. Yamaguchi119, A. Yamamoto68, S. Yamamoto156, T. Yamanaka156, D. Yamaguchi158, Y. Yamaguchi119, A. Yamamoto68, S. Yamamoto156, T. Yamanaka156, K. Yamauchi104, Y. Yamazaki69, Z. Yan24, H. Yang35e, H. Yang173, Y. Yang152, Z. Yang15, K. Yamauchi104, Y. Yamazaki69, Z. Yan24, H. Yang35e, H. Yang173, Y. Yang152, Z. Yang1 W-M. Yao16, Y.C. Yap82, Y. Yasu68, E. Yatsenko5, K.H. Yau Wong23, J. Ye42, S. The ATLAS collaboration Ye27, I. Yeletskikh67, A.L. Yen59, E. Yildirim85, K. Yorita171, R. Yoshida6, K. Yosh S.P.Y. Yuen23, I. Yusuff30,ar, B. Zabinski41, R. Zaidan35d, A.M. Zaitsev131,ae, N JHEP11(2016)112 M. Zeman129, A. Zemla40a, J.C. Zeng166, Q. Zeng144, K. Zengel25, O. Zenin131, T. ˇZeniˇs145a, D. Zerwas118, D. Zhang91, F. Zhang173, G. Zhang35b,am, H. Zhang35c, J. Zhang6, L. Zhang50, R. Zhang23, R. Zhang35b,as, X. Zhang35d, Z. Zhang118, X. Zhao42, Y. Zhao35d, Z. Zhao35b, R. Zhang23, R. Zhang35b,as, X. Zhang35d, Z. Zhang118, X. Zhao42, Y. Zhao35d, Z. Zhao35b,
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149 A. Zhemchugov67, J. Zhong121, B. Zhou91, C. Zhou47, L. Zhou37, L. Zhou42, M. Zhou149, N. Zhou35f, C.G. Zhu35d, H. Zhu35a, J. Zhu91, Y. Zhu35b, X. Zhuang35a, K. Zhukov97, A. Zibell174, D. Zieminska63, N.I. Zimine67, C. Zimmermann85, S. Zimmermann50, Z. Zinonos56
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M. Zinser85, M. Ziolkowski142, L. ˇZivkovi´c14, G. Zobernig173, A. Zoccoli22a,22b, M. zur Nedden17,
G. Zurzolo105a,105b and L. Zwalinski32 Zinser85, M. Ziolkowski142, L. ˇZivkovi´c14, G. Zobernig173, A. Zoccoli22a,22b, M. zur Nedden17,
Zurzolo105a,105b and L. Zwalinski32 G. Zurzolo105a,105b and L. The ATLAS collaboration Zwalinski32 1 Department of Physics, University of Adelaide, Adelaide, Australia 2 Physics Department, SUNY Albany, Albany NY, United States of America 3 Department of Physics, University of Alberta, Edmonton AB, Canada Division of Physics, TOBB University of Economics and Technology, Ankara, Turkey Division of Physics, TOBB University of Economics and Technology, Ankara, Turkey 5 LAPP, CNRS/IN2P3 and Universit´e Savoie Mont Blanc, Annecy-le-Vieux, France 5 LAPP, CNRS/IN2P3 and Universit´e Savoie Mont Blanc, Annecy-le-Vieux, France 6 High Energy Physics Division, Argonne National Laboratory, Argonne IL, United Sta 6 High Energy Physics Division, Argonne National Laboratory, Argonne IL, United States of Amer 7 Department of Physics, University of Arizona, Tucson AZ, United States of Ameri
8 7 Department of Physics, University of Arizona, Tucson AZ, United States of America 8 Department of Physics, The University of Texas at Arlington, Arlington TX, United States of
America sics Department, University of Athens, Athens, Gree 10 Physics Department, National Technical University of Athens, Zografou, Greece 10 Physics Department, National Technical University of Athens, Zografou, Greece 11 Department of Physics, The University of Texas at Austin, Austin TX, United States of America 11 Department of Physics, The University of Texas at Austin, Austin TX, United 12 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 12 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 13 Institut de F´ısica d’Altes Energies (IFAE), The Barcelona Institute of Science and Technology,
Barcelona, Spain, Spain 14 Institute of Physics, University of Belgrade, Belgrade, Serbia 14 Institute of Physics, University of Belgrade, Belgrade, Serbia 16 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley
CA, United States of America 16 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley
CA, United States of America 17 Department of Physics, Humboldt University, Berlin, Germany 17 Department of Physics, Humboldt University, Berlin, Germany 18 Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics,
University of Bern, Bern, Switzerland 18 Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, stein Center for Fundamental Physics and Laboratory 20
(a) Department of Physics, Bogazici University, Istanbul; (b) Department of Physics Engineering,
Gaziantep University, Gaziantep; (d) Istanbul Bilgi University, Faculty of Engineering and Natural
Sciences, Istanbul,Turkey; (e) Bahcesehir University, Faculty of Engineering and Natural Sciences,
Istanbul, Turkey, Turkey 21 Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colombia 22
(a) INFN Sezione di Bologna; (b) Dipartimento di Fisica e Astronomia, Universit`a di Bologna,
Bologna, Italy – 30 – 23 Physikalisches Institut, University of Bonn, Bonn, Germany 24 Department of Physics, Boston University, Boston MA, United States of America 25 Department of Physics, Brandeis University, Waltham MA, United States of America Department of Physics, Brandeis University, Waltham MA, United States of America
26
(a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b) Electrical Circuits
Department, Federal University of Juiz de Fora (UFJF), Juiz de Fora; (c) Federal University of Sao
Joao del Rei (UFSJ), Sao Joao del Rei; (d) Instituto de Fisica, Universidade de Sao Paulo, Sao
Paulo, Brazil 27 Physics Department, Brookhaven National Laboratory, Upton NY, United States of America
( )
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(a) Transilvania University of Brasov, Brasov, Romania; (b) National Institute of Physics and
Nuclear Engineering, Bucharest; (c) National Institute for Research and Development of Isotopic
and Molecular Technologies, Physics Department, Cluj Napoca; (d) University Politehnica
Bucharest, Bucharest; (e) West University in Timisoara, Timisoara, Romania JHEP11(2016)112 29 Departamento de F´ısica, Universidad de Buenos Aires, Buenos Aires, Argentina 30 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 31 Department of Physics, Carleton University, Ottawa ON, Canada 32 CERN, Geneva, Switzerland 32 CERN, Geneva, Switzerland 33 Enrico Fermi Institute, University of Chicago, Chicago IL, United States of America 34
(a) Departamento de F´ısica, Pontificia Universidad Cat´olica de Chile, Santiago; (b) Departamento
de F´ısica, Universidad T´ecnica Federico Santa Mar´ıa, Valpara´ıso, Chile 35
(a) Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b) Department of
Modern Physics, University of Science and Technology of China, Anhui; (c) Department of Physics,
Nanjing University, Jiangsu; (d) School of Physics, Shandong University, Shandong; (e) Department
of Physics and Astronomy, Shanghai Key Laboratory for Particle Physics and Cosmology, Shanghai
Jiao Tong University, Shanghai; (also affiliated with PKU-CHEP); (f) Physics Department,
Tsinghua University, Beijing 100084, China Tsinghua University, Beijing 100084, China 36 Laboratoire de Physique Corpusculaire, Clermont Universit´e and Universit´e Blaise Pascal and
CNRS/IN2P3, Clermont-Ferrand, France 37 Nevis Laboratory, Columbia University, Irvington NY, United States of America 37 Nevis Laboratory, Columbia University, Irv 38 Niels Bohr Institute, University of Copenhagen, Kobenhavn, Denmark
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(a) INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati; (b)
Fisica, Universit`a della Calabria, Rende, Italy 39
(a) INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati; (b) Dipartimento di 39
(a) INFN Gruppo Collegato di Cosenza, Laborat
Fisica, Universit`a della Calabria, Rende, Italy 39
(a) INFN Gruppo Collegato di Cosenza, Laborat Fisica, Universit`a della Calabria, Rende, Italy 40
(a) AGH University of Science and Technology, Faculty of Physics and Applied Computer Science
Krakow; (b) Marian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland 40
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43 Physics Department, University of Texas at Dallas, Richardson TX, United States of America
44 DESY, Hamburg and Zeuthen, Germany 43 Physics Department, University of Texas at Dallas, Richardson TX, United States of America
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49 INFN Laboratori Nazionali di Frascati, Frascati, Italy UPA - School of Physics and Astronomy, University 49 INFN Laboratori Nazionali di Frascati, Frascati, Italy 49 INFN Laboratori Nazionali di Frascati, Frascati, Italy 50 Fakult¨at f¨ur Mathematik und Physik, Albert-Ludwigs-Universit¨at, Freiburg, Germany 51 Section de Physique, Universit´e de Gen`eve, Geneva, Switzerland 52
(a) INFN Sezione di Genova; (b) Dipartimento di Fisica, Universit`a di Genova, Genova, Italy 53
(a) E. The ATLAS collaboration Andronikashvili Institute of Physics, Iv. Javakhishvili Tbilisi State University, Tbilisi; (b
High Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia 53
(a) E. Andronikashvili Institute of Physics, Iv. The ATLAS collaboration Javakhishvili Tbilisi State University, Tbilisi; (b) High Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia 54 II Physikalisches Institut, Justus-Liebig-Universit¨at Giessen, Giessen, Germany hes Institut, Justus-Liebig-Universit¨at Giessen, Giess 55 SUPA - School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom 56 II Physikalisches Institut, Georg-August-Universit¨at, G¨ottingen, Germany 57 Laboratoire de Physique Subatomique et de Cosmologie, Universit´e Grenoble-Alpes, CNRS/IN2P3,
Grenoble, France – 31 – 58 Department of Physics, Hampton University, Hampton VA, United States of America 59 Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge MA, United States
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(a) Kirchhoff-Institut f¨ur Physik, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg; (b)
Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg; (c) ZITI Insti 60
(a) Kirchhoff-Institut f¨ur Physik, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg; (b)
Physikalisches Institut, Ruprecht-Karls-Universit¨at Heidelberg, Heidelberg; (c) ZITI Institut f¨ur
technische Informatik, Ruprecht-Karls-Universit¨at Heidelberg, Mannheim, Germany technische Informatik, Ruprecht-Karls-Universit¨at Heidelberg, Mannheim, Germany 61 Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Japan 62
(a) Department of Physics, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong; (b)
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(a) INFN Sezione di Lecce; (b) Dipartimento di Matematica e Fisica, Universit`a del Salento, Lecc
Italy 76 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 78 School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom nd Astronomy, Queen Mary University of London, L 79 Department of Physics, Royal Holloway University of London, Surrey, United Kingdom p
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81 80 Department of Physics and Astronomy, University College London, London, United Kingdom 81 Louisiana Tech University, Ruston LA, United States of America 82 Laboratoire de Physique Nucl´eaire et de Hautes Energies, UPMC and Universit´e Paris-Diderot and
CNRS/IN2P3, Paris, France Fysiska institutionen, Lunds universitet, Lund, Swed 84 Departamento de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spa Departamento de Fisica Teorica C-15, Universidad A 85 Institut f¨ur Physik, Universit¨at Mainz, Mainz, Germany 86 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 86 School of Physics and Astronomy, University of Manchester, Ma 87 CPPM, Aix-Marseille Universit´e and CNRS/IN2P3, Marseille, France CPPM, Aix-Marseille Universit´e and CNRS/IN2P 88 Department of Physics, University of Massachusetts, Amherst MA, United States of America 89 Department of Physics, McGill University, Montreal QC, Canada of Physics, University of Melbourne, Victoria, Austr 90 School of Physics, University of Melbourne, Victoria, Australia 91 Department of Physics, The University of Michigan, Ann Arbor MI, United States of America 91 Department of Physics, The University of Michigan, Ann Arbor MI, United States of America
92 Department of Physics and Astronomy, Michigan State University, East Lansing MI, United States
of America 91 Department of Physics, The University of Michigan, Ann Arbor MI, United States of America
92 Department of Physics and Astronomy, Michigan State University, East Lansing MI, United States 92 Department of Physics and Astronomy, Michigan State University, East Lansing MI, United States
of America 93
(a) INFN Sezione di Milano; (b) Dipartimento di Fisica, Universit`a di Milano, Milano, Italy 93
(a) INFN Sezione di Milano; (b) Dipartimento di Fisica, Universit`a di Milano, Milano, Italy
94 B.I. The ATLAS collaboration Dodge Department of Physics and Astronomy, University of Oklahoma, Norman OK,
United States of America 115 Department of Physics, Oklahoma State University, Stillwater OK, United States of America Department of Physics, Oklahoma State University, Stillwater OK, United States of America
Palack´y University, RCPTM, Olomouc, Czech Republic 116 Palack´y University, RCPTM, Olomouc, Czech Republic 116 Palack´y University, RCPTM, Olomouc, Czech Republic alacky University, RC
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(a) INFN Sezione di Pavia; (b) Dipartimento di Fisica, Universit`a di Pavia, Pavia, Italy 123 Department of Physics, University of Pennsylvania, Philadelphia PA, United States of America 123 Department of Physics, University of Pennsylvania, Philadelphia PA, United States of America
124 National Research Centre “Kurchatov Institute” B.P.Konstantinov Petersburg Nuclear Physics 124 National Research Centre “Kurchatov Institute” B.P.Konstantinov Petersburg Nuclear Physics
Institute, St. Petersburg, Russia 125
(a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. The ATLAS collaboration Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Republic of
Belarus 94 B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Republic of
Belarus 95 National Scientific and Educational Centre for Particle and High Energy Physics, Minsk, Republic
of Belarus 96 Group of Particle Physics, University of Montreal, Montreal QC, Canada 97 P.N. Lebedev Physical Institute of the Russian Academy of Sciences, Moscow, Russ 98 Institute for Theoretical and Experimental Physics (ITEP), Moscow, Russia 99 National Research Nuclear University MEPhI, Moscow, Russia – 32 – 100 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow,
Russia 101 Fakult¨at f¨ur Physik, Ludwig-Maximilians-Universit¨at M¨unchen, M¨unchen, Germany 102 Max-Planck-Institut f¨ur Physik (Werner-Heisenberg-Institut), M¨unchen, German agasaki Institute of Applied Science, Nagasaki, Japan 104 Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan
( )
( ) 104 Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan
105
(a) INFN Sezione di Napoli; (b) Dipartimento di Fisica Universit`a di Napoli Napoli Italy Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan
105
(a) INFN Sezione di Napoli; (b) Dipartimento di Fisica, Universit`a di Napoli, Napoli, Italy 105
(a) INFN Sezione di Napoli; (b) Dipartimento di Fisica, Universit`a di Napoli 106 Department of Physics and Astronomy, University of New Mexico, Albuquerque NM, United States
of America 107 Institute for Mathematics, Astrophysics and Particle Physics, Radboud University
Nijmegen/Nikhef, Nijmegen, Netherlands 108 Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam,
Netherlands JHEP11(2016)112 109 Department of Physics, Northern Illinois University, DeKalb IL, United States of America 110 Budker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia 111 Department of Physics, New York University, New York NY, United States of America ent of Physics, New York University, New York NY, 112 Ohio State University, Columbus OH, United States of America 113 Faculty of Science, Okayama University, Okayama, Japan 114 Homer L. The ATLAS collaboration Fermi, Universit`a di Pisa, Pisa, Italy 126 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh PA, United States of
America 127
(a) Laborat´orio de Instrumenta¸c˜ao e F´ısica Experimental de Part´ıculas - LIP, Lisboa; (b) Faculdade
de Ciˆencias, Universidade de Lisboa, Lisboa; (c) Department of Physics, University of Coimbra,
Coimbra; (d) Centro de F´ısica Nuclear da Universidade de Lisboa, Lisboa; (e) Departamento de
Fisica, Universidade do Minho, Braga; (f) Departamento de Fisica Teorica y del Cosmos and
CAFPE, Universidad de Granada, Granada (Spain); (g) Dep Fisica and CEFITEC of Faculdade de
Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal gia, Universidade Nova de Lisboa, Caparica, Portuga 128 Institute of Physics, Academy of Sciences of the Czech Republic, Praha, Czech Re 129 Czech Technical University in Prague, Praha, Czech Republic 130 Faculty of Mathematics and Physics, Charles University in Prague, Praha, Czech Republic 130 Faculty of Mathematics and Physics, Charles University in Pra Research Center Institute for High Energy Physics (P 131 State Research Center Institute for High Energy Physics (Protvino), NRC KI, Russia 132 Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 133
(a) INFN Sezione di Roma; (b) Dipartimento di Fisica, Sapienza Universit`a di Roma, Roma, Italy 134
(a) INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Universit`a di Roma Tor
Vergata, Roma, Italy 135
(a) INFN Sezione di Roma Tre; (b) Dipartimento di Matematica e Fisica, Universit`a Roma Tre,
Roma, Italy 136
(a) Facult´e des Sciences Ain Chock, R´eseau Universitaire de Physique des Hautes Energies -
Universit´e Hassan II, Casablanca; (b) Centre National de l’Energie des Sciences Techniques – 33 – Nucleaires, Rabat; (c) Facult´e des Sciences Semlalia, Universit´e Cadi Ayyad, LPHEA-Marrakech; Nucleaires, Rabat; (c) Facult´e des Sciences Semlalia, Universit´e Cadi Ayyad, LPHEA-Marrakech;
(d) Facult´e des Sciences, Universit´e Mohamed Premier and LPTPM, Oujda; (e) Facult´e des
sciences Universit´e Mohammed V Rabat Morocco Nucleaires, Rabat;
Faculte des Sciences Semlalia, Universite Cadi Ayyad, LPHEA-Marrakech;
(d) Facult´e des Sciences, Universit´e Mohamed Premier and LPTPM, Oujda; (e) Facult´e des
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(Commissariat `a l’Energie Atomique et aux Energies Alternatives), Gif-sur-Yvette, France 138 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz CA,
United States of America 139 Department of Physics, University of Washington, Seattle WA, United States of America 140 Department of Physics and Astronomy, University of Sheffield, S 140 Department of Physics and Astronomy, University of Sheffield, Sheffield, United Kingdom 141 Department of Physics, Shinshu University, Nagano, Japan 142 Fachbereich Physik, Universit¨at Siegen, Siegen, Germany epartment of Physics, Simon Fraser University, Burn JHEP11(2016)112 144 SLAC National Accelerator Laboratory, Stanford CA, United States of America 145
(a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b)
Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of
Sciences, Kosice, Slovak Republic 145
(a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b)
Department of Subnuclear Physics Institute of Experimental Physics of the Slovak Acade 145
(a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b)
Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of
Sciences, Kosice, Slovak Republic Sciences, Kosice, Slovak Republic Sciences, Kosice, Slovak Republic 146
(a) Department of Physics, University of Cape Town, Cape Town; (b) Department of Physics,
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(a) Department of Physics, Stockholm University; (b) The Oskar Klein Centre, Stockholm, Sweden 148 Physics Department, Royal Institute of Technology, Stockholm, Sweden 149 Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook NY,
United States of America 150 Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 151 School of Physics, University of Sydney, Sydney, Australia 152 Institute of Physics, Academia Sinica, Taipei, Taiwan 152 Institute of Physics, Academia Sinica, Taipei, Taiwan
153 152 Institute of Physics, Academia Sinica, Taipei, Taiwan
153 Department of Physics Technion: Israel Institute of Technology Haifa Israel 153 Department of Physics, Technion: Israel Institute of Technology, Haifa, Israel
154 154 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv,
Israel 155 Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 156 International Center for Elementary Particle Physics and Department of Physics, The Universit
of Tokyo, Tokyo, Japan 157 Graduate School of Science and Technology, Tokyo Metropolitan Univer 158 Department of Physics, Tokyo Institute of Technology, Tokyo, Japan nt of Physics, Tokyo Institute of Technology, Tokyo, J 159 Department of Physics, University of Toronto, Toronto ON, Canada 160
(a) TRIUMF, Vancouver BC; (b) Department of Physics and Astronomy, York University, Toronto
ON, Canada 161 Faculty of Pure and Applied Sciences, and Center for Integrated Research in Fundamental Science
and Engineering, University of Tsukuba, Tsukuba, Japan 162 Department of Physics and Astronomy, Tufts University, Medford MA, United States of America
163 partment of Physics and Astronomy, Tufts University 163 Department of Physics and Astronomy, University of California Irvine, Irvine CA, United States of
America 164
(a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP, Trieste; (c) 164
(a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP, Trieste; (c)
Dipartimento di Chimica Fisica e Ambiente Universit`a di Udine Udine Italy ( ) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; ( ) ICTP, Trieste
Dipartimento di Chimica, Fisica e Ambiente, Universit`a di Udine, Udine, Italy Dipartimento di Chimica, Fisica e Ambiente, Universit`a di Udine, Udine, Italy 165 Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden
166 166 Department of Physics, University of Illinois, Urbana IL, United States of America 167 Instituto de Fisica Corpuscular (IFIC) and Departamento de Fisica Atomica, Molecular y Nuclear
and Departamento de Ingenier´ıa Electr´onica and Instituto de Microelectr´onica de Barcelona
(IMB-CNM) University of Valencia and CSIC Valencia Spain Instituto de Fisica Corpuscular (IFIC) and Departamento de Fisica Atomica, Molecular y Nuclear
and Departamento de Ingenier´ıa Electr´onica and Instituto de Microelectr´onica de Barcelona and Departamento de Ingenier´ıa Electr´onica and Instituto de Microelectr´onica de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain 168 Department of Physics, University of British Columbia, Vancouver BC, Canada 169 Department of Physics and Astronomy, University of Victoria, Victoria BC, Canada 170 Department of Physics, University of Warwick, Coventry, United Kingdom – 34 – 171 Waseda University, Tokyo, Japan 172 Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel 173 Department of Physics, University of Wisconsin, Madison WI, United States of America 174 Fakult¨at f¨ur Physik und Astronomie, Julius-Maximilians-Universit¨at, W¨urzburg, Germany 175 Fakult¨at f¨ur Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universit¨at
Wuppertal, Wuppertal, Germany Wuppertal, Wuppertal, Germany 176 Department of Physics, Yale University, New Haven CT, United States of America 177 Yerevan Physics Institute, Yerevan, Armenia
178 177 Yerevan Physics Institute, Yerevan, Armenia 178 Centre de Calcul de l’Institut National de Physique Nucl´eaire et de Physique des Particules
(IN2P3), Villeurbanne, France JHEP11(2016)112 b Also at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan b Also at Institute of Physics, Azerbaijan Academy of Sciences c Also at Novosibirsk State University, Novosibirsk, Russia d Also at TRIUMF, Vancouver BC, Canada e Also at Department of Physics & Astronomy, University of Louisville, Louisville, KY, United
States of America States of America f Also at Department of Physics, California State University, Fresno CA, United States of America partment of Physics, University of Fribourg, Fribourg h Also at Departament de Fisica de la Universitat Autonoma de Barcelona, Barcelona, Spain i Also at Departamento de Fisica e Astronomia, Faculdade de Ciencias, Universidade do Porto,
Portugal j Also at Tomsk State University, Tomsk, Russia k Also at Universita di Napoli Parthenope, Napoli, Italy l Also at Institute of Particle Physics (IPP), Canada m Also at National Institute of Physics and Nuclear Engineering, Bucharest, Romania n Also at Department of Physics, St. Petersburg State Polytechnical University, St. States of America Petersburg,
Russia o Also at Department of Physics, The University of Michigan, Ann Arbor MI, United States of
America p Also at Centre for High Performance Computing, CSIR Campus, Rosebank, Cape Town, South
Africa r Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barc s Also at Graduate School of Science, Osaka University, Osaka, Japan s Also at Graduate School of Science, Osaka University, Osaka, Japan t Also at Department of Physics, National Tsing Hua University, Taiwan t Also at Department of Physics, National Tsing Hua University, Taiwan u Also at Institute for Mathematics, Astrophysics and Particle Physics, Radbou
Nijmegen/Nikhef, Nijmegen, Netherlands u Also at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University u Also at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands v Also at Department of Physics, The University of Texas at Austin, Austin TX, United States of
America v Also at Department of Physics, The University of Texas at Austin, Austin TX, United States of
America w Also at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia w Also at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia lso at Institute of Theoretical Physics, Ilia State Univ x Also at CERN, Geneva, Switzerland y Also at Georgian Technical University (GTU),Tbilisi, Georgia y Also at Georgian Technical University (GTU),Tbilisi, Georgia y Also at Georgian Technical University z Also at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan z Also at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan aa Also at Manhattan College, New York NY, United States of America aa Also at Manhattan College, New York NY, United States of America Also at Manhattan College, New York NY, United S ab Also at Hellenic Open University, Patras, Greece ac Also at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan ac Also at Academia Sinica Grid Computing, Institute of Physics, Acad ad Also at School of Physics, Shandong University, Shandong, China o at School of Physics, Shandong University, Shando ae Also at Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia ae Also at Moscow Institute of Physics and Technology State University, Dolgoprudny, R af Also at Section de Physique, Universit´e de Gen`eve, Geneva, Switzerland af Also at Section de Physique, Universit´e de Gen`eve, Geneva, Switzerland ag Also at Eotvos Lorand University, Budapest, Hungary ag Also at Eotvos Lorand University, Budapest, Hungary ah Also at International School for Advanced Studies (SISSA), Trieste, Italy ah Also at International School for Advanced Studies (SISSA), Trieste, Italy – 35 – ai Also at Department of Physics and Astronomy, University of South Carolina, Columbia SC, United
States of America aj Also at School of Physics and Engineering, Sun Yat-sen University, Guangzhou, China ak Also at Institute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Academy of
Sciences, Sofia, Bulgaria al Also at Faculty of Physics, M.V.Lomonosov Moscow State University, Moscow, Russia am Also at Institute of Physics, Academia Sinica, Taipei, Taiwan an Also at National Research Nuclear University MEPhI, Moscow, Russia ao Also at Department of Physics, Stanford University, Stanford CA, United States of America ap Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapes
Hungary JHEP11(2016)112 ar Also at University of Malaya, Department of Physics, Kuala Lumpur, Mala as Also at CPPM, Aix-Marseille Universit´e and CNRS/IN2P3, Marseille, France as Also at CPPM, Aix-Marseille Universit´e and CNRS/IN2P3, Marseille, France
∗Deceased ,
/
,
,
∗Deceased – 36 –
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https://openalex.org/W2100959617
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https://ro-journal.biomedcentral.com/counter/pdf/10.1186/1748-717X-6-7
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English
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Enhancement of radiosensitivity in human glioblastoma cells by the DNA N-mustard alkylating agent BO-1051 through augmented and sustained DNA damage response
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Radiation oncology
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cc-by
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* Correspondence: uf004693@mail2000.com.tw
† Contributed equally
1Graduate Institutes of Life Sciences, National Defense Medical Center &
Department of Neurological Surgery, Tri-Service General Hospital, Taipei,
Taiwan
Full list of author information is available at the end of the article RESEARCH Open Access Open Access © 2011 Chu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. Abstract Background: 1-{4-[Bis(2-chloroethyl)amino]phenyl}-3-[2-methyl-5-(4-methylacridin-9-ylamino)phenyl]urea (BO-1051)
is an N-mustard DNA alkylating agent reported to exhibit antitumor activity. Here we further investigate the effects
of this compound on radiation responses of human gliomas, which are notorious for the high resistance to
radiotherapy. Methods: The clonogenic assay was used to determine the IC50 and radiosensitivity of human glioma cell lines
(U87MG, U251MG and GBM-3) following BO-1051. DNA histogram and propidium iodide-Annexin V staining were
used to determine the cell cycle distribution and the apoptosis, respectively. DNA damage and repair state were
determined by g-H2AX foci, and mitotic catastrophe was measure using nuclear fragmentation. Xenograft tumors
were measured with a caliper, and the survival rate was determined using Kaplan-Meier method. Results: BO-1051 inhibited growth of human gliomas in a dose- and time-dependent manner. Using the dosage
at IC50, BO-1051 significantly enhanced radiosensitivity to different extents [The sensitizer enhancement ratio was
between 1.24 and 1.50 at 10% of survival fraction]. The radiosensitive G2/M population was raised by BO-1051,
whereas apoptosis and mitotic catastrophe were not affected. g-H2AX foci was greatly increased and sustained by
combined BO-1051 and g-rays, suggested that DNA damage or repair capacity was impaired during treatment. In vivo studies further demonstrated that BO-1051 enhanced the radiotherapeutic effects on GBM-3-beared
xenograft tumors, by which the sensitizer enhancement ratio was 1.97. The survival rate of treated mice was also
increased accordingly. Conclusions: These results indicate that BO-1051 can effectively enhance glioma cell radiosensitivity in vitro and
in vivo. It suggests that BO-1051 is a potent radiosensitizer for treating human glioma cells. Enhancement of radiosensitivity in human
glioblastoma cells by the DNA N-mustard
alkylating agent BO-1051 through augmented
and sustained DNA damage response Pei-Ming Chu1, Shih-Hwa Chiou2,3,4†, Tsann-Long Su5†, Yi-Jang Lee6†, Li-Hsin Chen3, Yi-Wei Chen4,7,
Sang-Hue Yen7, Ming-Teh Chen8, Ming-Hsiung Chen8, Yang-Hsin Shih8, Pang-Hsien Tu5, Hsin-I Ma1* Chu et al. Radiation Oncology 2011, 6:7
http://www.ro-journal.com/content/6/1/7 Materials and Methods
Cell lines and treatment This research followed the tenets of the Declaration of
Helsinki. All samples were obtained after patients pro-
vided informed consent. The study was approved by the
Institutional Ethics Committee/Institutional Review
Board of Tri-Service General Hospital. The commercial
available U87MG, and U251MG glioma cell lines as well
as primary GBM cell line (GBM-3), which was isolated
from tumor sample obtained from patient undergoing
surgery for a GBM (World Health Organizing Grade 4
astrocytoma), were grown as attached monolayers in
75-cm2 flasks in DMEM media (Invitrogen) supplemen-
ted with glutamate (5 mmol/L) and 10% fetal bovine
serum. Cells were incubated at the exponential growth
phase in humidified 5% CO2/95% air atmosphere at
37℃. The GBM-3 cells used for the experiments had
already undergone > 100 passages. 1-{4-[bis(2-chlor-
oethyl)amino]phenyl}-3-[2-methyl-5- (4-methylacridin-9-
ylamino)phenyl]urea (BO-0151, Figure 1A) was dissolved
in DMSO to a stock concentration of 5 mM and stored
at -20℃. Gamma radiation (ionizing irradiation) was
delivered with a T-1000 Theratronic cobalt unit (Thera-
tronic International, Inc., Ottawa, Canada) at a dose rate
of 1.1 Gy/min (SSD = 57.5 cm). BO-0742 was found to have a potent therapeutic effi-
cacy against human leukemia and solid tumor cell growth
in vitro. Also, it has a good therapeutic index with leuke-
mia being 10-40 times more sensitive than hematopoietic
progenitors. Administration of BO-0742 at an optimal
dose schedule, based on its pharmacokinetics, signifi-
cantly suppressed the growth of xenograft tumors in
mice bearing human breast and ovarian cancers. How-
ever, BO-0742’s bioavailability is low because it has a nar-
row therapeutic window and is chemically unstable in
mice (half-life < 25 min) [12]. To improve the poor phar-
macokinetics of BO-0742, we have recently synthesized a
series of phenyl N-mustard-9-anilinoacridine conjugates
via a urea linker [13,14]. Of these agents, BO-1051 was
found to be more chemically stable than BO-0742 in rat
plasma (54.2 vs. 0.4 h). BO-1051, an agent capable of
inducing marked dose-dependent levels of DNA inter-
strand cross-linking (ICLs), revealed a broad spectrum of
anti-cancer activities in vitro without cross-resistance to
taxol or vinblastine. Due to BO-1051’s hydrophobic abil-
ity, it can penetrate through the blood-brain barrier to
brain cortex. BO-1051 has been shown to possess thera-
peutic efficacy in nude mice bearing human breast MX-1
tumors and human glioma in vivo [14]. Interestingly, we
found that obvious tumor suppression was observed in
mice and sustained over 70 days without relapse [14]. Background growth and recurrence rate [1]. A multicenter study has
shown that addition of concurrent temozolomide (TMZ)
to radical radiation therapy improves the survival in
patients who suffered from GBM [2,3]. These studies
have demonstrated an improvement for patients who
received TMZ, compared to those who did not, in the
median survival time and in the 2-year survival rate (14.6
vs. 12 months, 27% vs. 10%, respectively). Unfortunately,
the survival rate remains low using TMZ, and it prompts
investigators to seek new and more effective chemothera-
peutic agents for the treatment of malignant gliomas. Malignant gliomas account for approximately 30% of all
intracranial tumors, and of them, glioblastoma multi-
forme (GBM) is considered as the most frequent and
aggressive type. Removal of GBM by surgical resection is
usually not feasible due to the highly diffuse infiltrative Chu et al. Radiation Oncology 2011, 6:7
http://www.ro-journal.com/content/6/1/7 Page 2 of 13 Chu et al. Radiation Oncology 2011, 6:7
http://www.ro-journal.com/content/6/1/7 Chu et al. Radiation Oncology 2011, 6:7
http://www.ro-journal.com/content/6/1/7 Chu et al. Radiation Oncology 2011, 6:7
http://www.ro-journal.com/content/6/1/7 DNA alkylating agents are used widely for treatment of
a variety of pediatric and adult cancers because the cyto-
toxic effects of these agents can directly modify DNA and
cause DNA lesions [4]. However, the development of new
alkylating N-mustard agents is slow due to their low
tumor specificity, high chemical reactivity and an induc-
tion of bone marrow toxicity [5,6]. To overcome these
drawbacks, one strategy has been to design DNA-
directed alkylating agents by linking the alkylating
pharmacophore to the DNA-affinity molecules (e.g.,
DNA intercalating agents, DNA minor groove binder)
[7,8]. In most cases, the DNA-directed alkylating agents
have more selective, cytotoxic and potential than the cor-
responding untargeted derivatives [8-10]. Among these
agents, the compound BO-0742 exhibited significant
cytotoxicity (107-fold higher) on human lymphoblastic
leukemic cells than its parent analogue 3-(9-acridinyla-
mino)-5-hydroxymethylaniline [9,11]. treatment with BO-1051 at nanomolar concentrations
sensitizes the glioma cells to radiation-induced cellular
lethality. These data indicate that BO-1051 enhances
tumor radiosensitivity in vitro and in vivo. Moreover,
this sensitization correlates with its enhancement arrest
in the radiosensitive cell cycle phase and the delayed
dispersion of phosphorylated histone H2AX (g-H2AX)
foci, which suggests an inhibition of the repair to the
DNA double-strand breaks (DBSs). SF
PE
PE
xnM,thr
0nM,thr
xnM thr
,
= Materials and Methods
Cell lines and treatment The results indicated that BO-1051 was more potent
than cyclophosphamide with low toxicity to the host
(15% body-weight drop) suggesting that this agent is a
promising candidate for preclinical studies. SF
PE
PE
xnM,thr
0nM,thr
xnM thr
,
= Assay of BO-1051 cytotoxicity
KͲϭϬϱϭ 0.1
1
6 hours
12 hours
24 hours
Surviving fraction
Dose of BO-1051 (nM) – U87MG
400
50
100
200 Surviving fraction 0.1
1
6 hours
12 hours
24 hours
Surviving fraction
Dose of BO-1051 (nM) – U251MG
400
50
100
200 0.1
1
6 hours
12 hours
24 hours
0.1
1.0
6 hours
12 hours
24 hours
24 hours
Surviving fraction
Surviving fraction
Dose of BO-1051 (nM
Dose of BO-1051 (nM) – U251MG
50
100
400
50
100
200 Surviving fraction Surviving fraction Figure 1 Clonogenic survival of human glioma cells treated with BO-1051. (A) Chemical structure of 1-{4-[bis(2-chloroethyl)amino]phenyl}-3-
[2-methyl-5-(4- methylacridin-9-ylamino)phenyl]urea (BO-1051). (B) U87MG, (C) U251MG and (D) GBM-3 cells were exposed to escalating doses
(50-400 nM) of BO-1051 or vehicle (DMSO). At 6, 12 and 24 h after the addition of BO-1051, the BO-1051- containing medium was removed,
rinsed, and then fed with fresh growth media. Colony- forming efficiency was determined 10-14 days later, and the survival fractions of BO-1051-
treated cells were calculated after normalizing for the plating efficiencies of untreated cells. Points: mean for at least 3 independent experiments;
bars, SD. generated after normalizing for the amount of
BO-1051-induced cell death. The radiation SF of cells
pretreated with × nM BO-1051 was calculated as [15]: This protocol was used in an attempt to eliminate any
effects of trypsinization on post-treatment or post-irradia-
tion signaling/recovery processes [16-20]. Moreover, this
protocol allows for the irradiation of single cells but not
microcolonies, which eliminates the confounding para-
meter of multiplicity and its effects on the radiosensitivity. SF
PE
PE
xnM,DGy
xnM,DGy
xnM,0Gy
= Assay of BO-1051 cytotoxicity y
y
y
For these studies, a specified number of single cells were
seeded into a 25-T flask, and after 6 h, to allow for cell
attachment (but no division), the cells were treated with
0, 50, 100, 200 or 400 nM BO-1051. At 0, 6, 12 and
24 h after the BO-1051 addition, the BO-1051-contain-
ing medium was removed; the cells were washed with
sterile PBS, and fresh media was added. After 10 to 14
days of incubation, colonies were fixed with methanol
and stained with Giemsa. The number of colonies con-
taining at least 50 cells was determined, and the plating
efficiency (PE) and surviving fractions (SF) were calcu-
lated. The SF of cells exposed to × nM BO-1051 for t h
was calculated as [15]: Given that radiotherapy is considered to be the most
effective adjuvant treatment with surgery, we tested if
the therapeutic ability of BO-1051 could be translated
into antitumor activity. In this study, we investigated the
effects of BO-1051 on the radiosensitivity of a panel of
three human glioma cell lines, and we found that SF
PE
PE
xnM,thr
0nM,thr
xnM thr
,
= SF
PE
PE
xnM,thr
0nM,thr
xnM thr
,
= Chu et al. Radiation Oncology 2011, 6:7
http://www.ro-journal.com/content/6/1/7 Page 3 of 13 0.1
1
6 hours
12 hours
24 hours
0.1
1.0
6 hours
12 hours
24 hours
0.1
1
6 hours
12 hours
24 hours
Surviving fraction
KͲϭϬϱϭ
Surviving fraction
Surviving fraction
Dose of BO-1051 (nM) – GBM-3
Dose of BO-1051 (nM) – U87MG
Dose of BO-1051 (nM) – U251MG
400
50
100
200
400
50
100
200
400
50
100
200
Figure 1 Clonogenic survival of human glioma cells treated with BO-1051. (A) Chemical structure of 1-{4-[bis(2-chloroethyl)amino]phenyl}-3-
[2-methyl-5-(4- methylacridin-9-ylamino)phenyl]urea (BO-1051). (B) U87MG, (C) U251MG and (D) GBM-3 cells were exposed to escalating doses
(50-400 nM) of BO-1051 or vehicle (DMSO). At 6, 12 and 24 h after the addition of BO-1051, the BO-1051- containing medium was removed,
rinsed, and then fed with fresh growth media. Colony- forming efficiency was determined 10-14 days later, and the survival fractions of BO-1051-
treated cells were calculated after normalizing for the plating efficiencies of untreated cells. Points: mean for at least 3 independent experiments;
bars, SD. Annexin V-PI apoptosis assay g
y
y
The tumor re-growth delay assay measures the time
required for a tumor to reach a given size post-treatment. When tumors grew to a mean volume of ~150 mm3, mice
were randomly assigned to one of four treatment groups:
vehicle control (14 animals), BO-1051 (12 animals), 4 Gy
irradiation (9 animals), or combined BO-1051 and radia-
tion (8 animals). BO-1051 treatment was performed,
which consisted of an intraperitoneal (i.p.) injection proto-
col of 50 mg/kg administered at 3-day intervals over a
6-day period (3 injections on days 0, 3, 6; Q3D × 3). For
irradiation, unanesthetized animals were immobilized in a
lead jig that allowed for the localized irradiation of the
implanted tumors. Gamma radiation was delivered by a
T-1000 Theratronic cobalt unit (Theratronic International,
Inc.; Ottawa, Canada) at a dose rate of 1.1 Gy/min (SSD =
57.5 cm). For the BO-1051-plus-radiation group, BO-1051
(50 mg/kg) was delivered via i.p. injection on days 0, 3,
and 6, with day 0 being the day of randomization. Radia-
tion (4 Gy) was delivered to animals restrained in a cus-
tom lead jig 24 h after the first injection of BO-1051 (day
1 after randomization). Tumor volume is a critical para-
meter in determining radiation-induced growth delay with
smaller tumors appearing more radiosensitive. To ensure
BO-1051-induced growth delay did not bias the results of
the combination treatment (BO-1051 plus 4 Gy), it was
important that the two irradiated groups (4 Gy and BO-
1051 plus 4 Gy) received radiation when the tumors were
approximately the same size. To obtain tumor growth
curves, perpendicular diameter measurements of each
tumor were made every day with digital calipers, and the
volumes were calculated using the formula for volume of
an ellipsoid: 4Π/3 × L/2 × W/2 × H/2, where L = length,
W = width, and H = height. The time for the tumor
to grow again to ten times the initial volume (about
1500 mm3) was calculated for each animal. Absolute To evaluate apoptosis as a mechanism of cell death,
approximately 2 × 106 cells were plated in 100-mm
petri dishes. Cells were exposed to 200 nM or higher
concentration (1.2 μM) of BO-1051 prior to irradiation
and were stained at 24 and 48 h postirradiation (2 Gy). Both adherent and detached cells were collected, centri-
fuged, and double stained with Annexin V-FITC and
propidium iodide (PI). Combination of BO-1051 and irradiation The combined therapeutic effects based on drug and
ionizing irradiation was obtained by the survival frac-
tions measured by separate treatment as reported pre-
viously [21]. The expected effect by two separate
treatments was determined by the formula SF(Drug) × SF
(Rad), which was compared to the observed survival
fraction. After allowing the cells time to attach, the culture
medium was then replaced with fresh medium that
contained 200 nM BO-1051, and the flasks were irra-
diated 24 h later. Immediately after irradiation, the
growth media was aspirated, and fresh media was
added. Colonies were stained with Giemsa 10 to
14 days after seeding. Survival curves were then Chu et al. Radiation Oncology 2011, 6:7
http://www.ro-journal.com/content/6/1/7 Chu et al. Radiation Oncology 2011, 6:7
http://www.ro-journal.com/content/6/1/7 Page 4 of 13 Chu et al. Radiation Oncology 2011, 6:7
http://www.ro-journal.com/content/6/1/7 In vivo tumor model Six-week-old female nude mice were used in these stu-
dies. Mice were caged in groups of five or less, and all
animals were fed a diet of animal chow and water ad
libitum. All procedures involving animals were per-
formed in accordance with the institutional animal wel-
fare guidelines of the Taipei Veterans General Hospital. Tumors were generated by injecting 5 × 106 GBM-3
cells subcutaneous (s.c.) into the right hind leg. Irradia-
tion was performed using a T-1000 Theratronic cobalt
unit (Theratronic International, Inc.; Ottawa, Canada)
irradiator with animals restrained in a custom jig. Annexin V-PI apoptosis assay Apoptotic cells were quantified
with flow cytometry using a FACSCalibur cytometer
(BD FACS Caliber, Mountain View, CA). Cell-cycle analysis analysis software package (Scanalytics, Inc.; Fairfax, VA)
running on a Macintosh G3 computer. For each treat-
ment condition, g-H2AX foci were determined in at
least 150 cells. After treatment, cells were prepared for fluorescence-
activated cell sorting (FACS) to assess the relative distri-
bution in the respective phases of the cell cycle. Cells
were harvested 24 h after of treatment with BO-1051,
pelleted by centrifugation, re-suspended in PBS, fixed in
70% ethanol and stored at -20℃. Immediately before
flow cytometry, the cells were washed in cold PBS (4℃),
incubated in Ribonuclease A (Sigma) for 20 min at
room temperature, labeled by adding an equal volume
of propidium iodide solution (100 μg/ml) and incubated
in the dark for 20 min at 4℃. These samples were mea-
sured (20,000 events collected from each) in a FACSCa-
libur cytometer (BD FACS Caliber; Mountain View,
CA). The data shown are for one experiment, but the
results were reproduced and confirmed in at least three
identical experiments. Immunofluorescent staining for g-H2AX g
g
Cells were treated with or without BO-1051 for 24 h
prior to irradiation (2 Gy) and fed with BO-1051-free
medium, and the average number of foci per cell was
measured beginning at 1 h after irradiation and followed
thereafter for 24 h. At specified times, the media were
aspirated and cells were fixed in 1% paraformaldehyde
for 10 min at room temperature. Paraformaldehyde was
aspirated, and the cells were treated with a 0.2% NP40/
PBS solution for 15 min. Cells were then washed in PBS
twice, and the anti-gH2AX antibody was added at a
dilution of 1:500 in 1% BSA and incubated overnight at
4℃. Again, the cells were washed twice in PBS before
incubating in the dark for 1 h with a FITC-labeled sec-
ondary antibody at a dilution of 1:100 in 1% BSA. The
secondary antibody solution was then aspirated, and the
cells were washed twice in PBS. The cells were then
incubated in the dark with PI (1 μg/ml) in PBS for
30 min, washed twice, and coverslips were mounted
with an anti-fade solution (Dako Corp.; Carpinteria,
CA). Slides were examined with a confocal fluorescent
microscope (Wetzlar, Germany). Images were captured
by a Photometrics Sensys CCD camera (Roper Scientific;
Tucson, AZ) and imported into the IP Labs image Chu et al. Radiation Oncology 2011, 6:7
http://www.ro-journal.com/content/6/1/7 Page 5 of 13 tumor growth delay was calculated as the number of days
for the treated tumors to reach ten times the initial tumor
volume minus the number of days for the control group
to reach the same size. Enhancement of radiosensitivity in glioma cells by BO-1051
To investigate if BO-1051 enhances the cellular sensitiv-
ity to ionizing radiation, the glioma cells were exposed
to BO-1051 for 24 h before irradiation and subjected to
the clonogenic assay. The results showed that the SFs at
different radiation dosages were apparently reduced in
U87MG, U251MG and GBM-3 cells after they were
exposed to BO-1051 (Figure. 2A-C). SFs after 2 Gy of
BO-1051-pretreated cells were significantly lower than
those of untreated cells (Figure 2D). Besides, the SERs
were 1.50 for U87MG, 1.24 for U251MG and 1.31 for
GBM-3 at a 10% cell survival (0.1). At 50% cell survival
(0.5), the SERs were 1.87 for U87MG, 1.83 for U251MG
and 1.68 for GBM-3 (Figure 2A-C, and 2E). Determination of the cytotoxicity of BO-1051 on different
human glioma cell lines Determination of the cytotoxicity of BO-1051 on different
human glioma cell lines To determine the effects of BO-1051 on glioma cell cyto-
toxicity by clonogenic survival, MTT assay was per-
formed in a panel of 3 human malignant glioma cell lines
(U87MG, U251MG and GBM-3). The IC50 (concentra-
tion resulting in cell viability of 50% of control) values of
BO-1051 for U87MG, U251MG and GBM-3 cells were
2.7, 2.5 and 1.5 μM, respectively. However, the
clonogenic survival analysis showed little or no colony
formation for 24 h post-exposure to the concentrations
of BO-1051 > 400 nM. We found that the appropriate
dosage range of BO-1051 for colony formation in these
glioma cell lines was between 50 and 400 nM. The cyto-
toxicity of U87MG, U251MG and GBM-3 cells were sig-
nificantly influenced by BO-1051 in a time-dependent
and dose-dependent manner. The 24-h treatment of
200 nM BO-1051 resulted in SFs of 0.470 ± 0.091, 0.485
± 0.041 and 0.510 ± 0.042 for U87MG, U251MG, and
GBM-3, respectively (Figure 1). Because approximately
50% of survival fractions were reached using 200 nM
BO-1051 treatments on each glioma cells at 24 h, we
chose this dose for the following experiments. Induction of a G2/M phase arrest in glioma cells exposed
to BO-1051 Given that radiosensitivity is distinct in different phases
of the cell cycle, we tested the cell cycle distribution in
BO-1051 treated glioma cells [22,23]. Cells were treated
with BO-1051 for 24 h and then subjected to flow cyto-
metric analysis. As illustrated in the DNA histograms,
BO-1051 treatment significantly disturbed the cell cycle
progression and showed a dramatic increase in G2/M
phase populations in U87MG cells compared with the
untreated controls (Figure 3A). Quantitative analysis of
the cell-cycle distribution at 24 h post-exposure to
BO-1051 at different concentrations from 200 nM to
1200 nM is shown in Figure 3B-D, which shows that
G2/M phase arrest was caused by pre-treatment with
BO-1051 in a dose-dependent manner for all 3 glioma
cells (Figure 3A-D). Because the G2/M phase is known
as the cell cycle’s most radiosensitive phase [22,23], it
may in part account for the effects of BO-1051 on the
enhancement of radiosensitivity of glioma cell line. Immunofluorescent staining for g-H2AX As a result,
the radiation survival curves obtained by the clonogenic
assay showed that BO-1051 pretreatment sensitized
human glioma cells to the ionizing radiation. Besides,
Table 1 summarizes the relative reduction in SFs and
compares them with a virtual value, expected for each
of the combination of BO-1051 and irradiation dose. The actual SF measured for combinations is smaller
than that expected on the basis of the treatment effects
of each modality separately. It indicates a significant
synergistic interaction in all three glioma cells. The mean size of tumors receiving the combination
treatment was compared to the mean size of tumors in
mice from each of the other groups (receiving vehicle
control, radiation alone, or BO-1051 alone). The analysis
was done on day 42 after the treatment started because
this was the last day that all animals were still alive. Time
to treatment failure (TTF) was defined as the time from
the initiation of treatment (experimental or control) to
the time a tumor was severely necrotic or had reached a
volume > 1500 mm3. Normalized tumor growth delay is
defined as the time in days for tumors to reach 10 times
the initial volume in mice treated with the combination
of BO-1051 and radiation minus the time in days for the
tumors to reach 10 times the initial volume in mice trea-
ted with BO-1051 only, which was 6.7 days (i.e.,
16 minus 9.3 days). Statistical analysis The results are reported as mean ± SD. Statistical analy-
sis was performed using a Student’s t-test, one-way
ANOVA test or two-way ANOVA test followed by
Tukey’s test, as appropriate. A P < 0.05 was considered
to be statistically significant. Induction of a G2/M phase arrest in glioma cells exposed
to BO-1051 Table 1 Relative reduction in surviving fraction of three
glioma cells due to combination of irradiation and BO-
1051 treatment Table 1 Relative reduction in surviving fraction of three
glioma cells due to combination of irradiation and BO-
1051 treatment Table 1 Relative reduction in surviving fraction of three
glioma cells due to combination of irradiation and BO-
1051 treatment
Irradiation dose (Gy)
Relative reduction (%)
U87MG
U251MG
GBM-3
2
41.7
42.6
40.6
4
70.4
45.6
47.0
6
74.2
47.9
64.3
8
76.3
50.0
73.9
Percentage relative reduction of the observed surviving fraction (SF)
compared to the expected SF (calculated on the basis of combing individual
treatment component, each with respective SF value). Irradiation dose (Gy)
Relative reduction (%)
U87MG
U251MG
GBM-3
2
41.7
42.6
40.6
4
70.4
45.6
47.0
6
74.2
47.9
64.3
8
76.3
50.0
73.9 Enhancement of radiosensitivity by BO-1051 treatment is
not caused by apoptosis or mitotic catastrophes in
glioma cells Figure 2 The effect of BO-1051 on tumor cell radiosensitivity. Cultures of (A) U87MG, (B) U251MG and (C) GBM-3 cells were exposed to 200 nM
of BO-1051 or DMSO (IR only) for 24 h and irradiated with graded doses of g-rays, rinsed, and fed with fresh growth media. Colony-forming efficiency
was determined 10-14 days later, and survival curves were generated after normalizing for cell killing by BO-1051 alone. Points: mean survival fraction
from at least 3 independent experiments; bars: SD. (D) The survival fraction after 2 Gy (SF2), corrected for independent cytotoxic effect of BO-1051, of
human glioma cells treated with 200 nM of BO-1051 or control (DMSO) for 24 h pre-radiation was measured. Values are the mean survival fraction ±
SD of at least 3 independent experiments. * p < 0.05. (E) Sensitizer enhancement ratios (SER) of human glioma cells. SERs were calculated at 10% or
50% cell survival (0.1 or 0.5) by dividing the dose of radiation from the radiation-only surviving curve with the corresponding dose from the BO-1051
plus radiation curve. 5% of apoptosis (Figure 4). Moreover, treatment with
1200 nM BO-1051 significantly induced approximately
20% of apoptosis in all 3 cell lines, but the combined
protocol did not show obvious enhancement on the pro-
portion of apoptotic cell deaths (Figure 4). An increase
in radiosensitivity may be caused by radiation-induced
mitotic catastrophes. Nevertheless, no significant mitotic
catastrophes were detected in glioma cells treated with
both BO-1051 and irradiation up to 72 h (unpublished
data). These data indicate that the BO-1051-mediated
increase in radiosensitivity is not due to the apoptosis
and mitotic catastrophes. BO-1051 concentrations (from 200 to 1200 nM) for
24 h, and then were irradiated with 2 Gy of g-rays. The
Annexin V/PI staining was then determined with FACS
analysis. Cells treated with either 200 nM of BO-1051
alone or combined with irradiation exhibited less than Percentage relative reduction of the observed surviving fraction (SF)
compared to the expected SF (calculated on the basis of combing individual
treatment component, each with respective SF value). Enhancement of radiosensitivity by BO-1051 treatment is
not caused by apoptosis or mitotic catastrophes in
glioma cells 0
2
4
6
8
0.001
0.01
0.1
1
IR only
200 nM BO + IR
0
2
4
6
8
0.001
0.01
0.1
1
IR only
200 nM BO + IR
0
2
4
6
8
0.001
0.01
0.1
1
IR only
200 nM BO + IR
Surviving fraction
Radiation dose (Gy)
Surviving fraction
Radiation dose (Gy)
Surviving fraction
Radiation dose (Gy)
U87MG
U251MG
GBM-3 0
2
4
6
8
0.001
0.01
0.1
1
IR only
200 nM BO + IR
Surviving fraction
Radiation dose (Gy)
GBM-3 Surviving fraction Survival fraction at 2 Gy
Sensitizer enhancement ratio
1.0
1.2
1.4
1.6
1.8
2.0
0.0
0.2
0.4
0.6
0.8
1.0
Control
200 nM BO
U87MG
U251MG
GBM-3
Ύ
Ύ
Ύ
U87MG Survival fraction at 2 Gy
Sensitizer enhancement ratio
1.0
1.2
1.4
1.6
1.8
2.0
SF0.1
SF0.5
0.0
0.2
0.4
0.6
0.8
1.0
Control
200 nM BO
U87MG
U251MG
GBM-3
Ύ
Ύ
Ύ
U87MG
U251MG
GBM-3 Survival fraction at 2 Gy
0.0
0.2
0.4
0.6
0.8
1.0
Control
200 nM BO
U87MG
U251MG
GBM-3
Ύ
Ύ
Ύ
Sensitizer enhancement ratio
1.0
1.2
1.4
1.6
1.8
2.0
SF0.1
SF0.5
U87MG
U251MG
GBM-3 igure 2 The effect of BO-1051 on tumor cell radiosensitivity. Cultures of (A) U87MG, (B) U251MG and (C) GBM-3 cells we Figure 2 The effect of BO-1051 on tumor cell radiosensitivity. Cultures of (A) U87MG, (B) U251MG and (C) GBM-3 cells were exposed to 200 nM
of BO-1051 or DMSO (IR only) for 24 h and irradiated with graded doses of g-rays, rinsed, and fed with fresh growth media. Colony-forming efficiency
was determined 10-14 days later, and survival curves were generated after normalizing for cell killing by BO-1051 alone. Points: mean survival fraction
from at least 3 independent experiments; bars: SD. (D) The survival fraction after 2 Gy (SF2), corrected for independent cytotoxic effect of BO-1051, of
human glioma cells treated with 200 nM of BO-1051 or control (DMSO) for 24 h pre-radiation was measured. Values are the mean survival fraction ±
SD of at least 3 independent experiments. * p < 0.05. (E) Sensitizer enhancement ratios (SER) of human glioma cells. SERs were calculated at 10% or
50% cell survival (0.1 or 0.5) by dividing the dose of radiation from the radiation-only surviving curve with the corresponding dose from the BO-1051
plus radiation curve. Enhancement of radiosensitivity by BO-1051 treatment is
not caused by apoptosis or mitotic catastrophes in
glioma cells We next investigated whether BO-1051 enhanced radia-
tion sensitivity of glioma cells was associated with
increase of apoptosis. Cells were exposed to a range of Chu et al. Radiation Oncology 2011, 6:7
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2
4
6
8
0.001
0.01
0.1
1
IR only
200 nM BO + IR
0
2
4
6
8
0.001
0.01
0.1
1
IR only
200 nM BO + IR
0
2
4
6
8
0.001
0.01
0.1
1
IR only
200 nM BO + IR
Surviving fraction
Radiation dose (Gy)
Surviving fraction
Radiation dose (Gy)
Surviving fraction
Radiation dose (Gy)
Survival fraction at 2 Gy
Sensitizer enhancement ratio
1.0
1.2
1.4
1.6
1.8
2.0
SF0.1
SF0.5
0.0
0.2
0.4
0.6
0.8
1.0
Control
200 nM BO
U87MG
U251MG
GBM-3
Ύ
Ύ
Ύ
U87MG
U251MG
GBM-3
U87MG
U251MG
GBM-3
Figure 2 The effect of BO-1051 on tumor cell radiosensitivity. Cultures of (A) U87MG, (B) U251MG and (C) GBM-3 cells were exposed to 200 nM
of BO-1051 or DMSO (IR only) for 24 h and irradiated with graded doses of g-rays, rinsed, and fed with fresh growth media. Colony-forming efficiency
was determined 10-14 days later, and survival curves were generated after normalizing for cell killing by BO-1051 alone. Points: mean survival fraction
from at least 3 independent experiments; bars: SD. (D) The survival fraction after 2 Gy (SF2), corrected for independent cytotoxic effect of BO-1051, of
human glioma cells treated with 200 nM of BO-1051 or control (DMSO) for 24 h pre-radiation was measured. Values are the mean survival fraction ±
SD of at least 3 independent experiments. * p < 0.05. (E) Sensitizer enhancement ratios (SER) of human glioma cells. SERs were calculated at 10% or
50% cell survival (0.1 or 0.5) by dividing the dose of radiation from the radiation-only surviving curve with the corresponding dose from the BO-1051
plus radiation curve. BO-1051 combined with g-rays causes prolonged DNA
damage response in glioma cells Cells in exponential growth were sham treated (left panel), treated with BO-1051 (200 nM, right panel) and then harvested 24 h later. (B-D) Cell
cycle distributions of a panel of 3 human glioma cell lines (U87MG, U251MG and GBM-3) were exposed to the designated concentrations of BO-
1051 for 24 h. Data displayed by the DNA content profiles were analyzed, and the cell cycle phase information is represented graphically. markers for evaluating the level of DNA damage [24]. To investigate if BO-1051 can affect the extent of DNA
damage by g-rays, the formation of g-H2AX foci in cell
nuclei was determined. Cells were treated with or with-
out BO-1051 for 24 h prior to irradiation (2 Gy) and fed
with BO-1051-free medium, and the average number of
foci per cell was measured beginning at 1 h after irradia-
tion and followed thereafter for 24 h. The results
showed that exposure of glioma cells to either BO-1051
or irradiation (2 Gy) resulted in a significant increase of
g-H2AX foci at 1 h that was sustained for 6 h, and then
the g-H2AX foci declined to almost basal level at least
24 h after irradiation or drug removal (Figure 5A and
5B). The combined protocol resulted in a greater num-
ber of g-H2AX foci than either of the individual treat-
ments at 1 or 6 h. However, the number of residual
g-H2AX foci per cell 24 h post-irradiation was greater
in BO-1051 plus irradiation (19.9 ± 2.5 per cell) compared with the number of foci in cells treated with
either irradiation or BO-1051 alone (7.9 ± 2.8 and 11.2
± 1.9 per cell, respectively) (Figure 5A and 5B). Further-
more, the frequency of g-H2AX foci distribution at 24 h
post-irradiation showed that the percentage of > 30 foci
of g-H2AX was higher than additive in BO-1051 plus
irradiation (24.9%) compared with the percentage of foci
in cells treated with either irradiation or BO-1051 alone
(0.3% and 12.0%, respectively). These results suggest
that BO-1051 produces supra-additive and prolonged
effects of irradiation on glioma cells. BO-1051 combined with g-rays causes prolonged DNA
damage response in glioma cells DNA damage is the most important biological effects
caused by ionizing radiation. It has been reported that
the nuclear foci of g-H2AX is one of the canonical Chu et al. Radiation Oncology 2011, 6:7
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20
40
60
80
100
G1
S
G2/M
Control
0
20
40
60
80
100
G1
S
G2/M
0
20
40
60
80
100
G1
S
G2/M
200
400
600
0
1200
BO-1051 (nM)
200 nM BO
200
400
600
0
1200
BO-1051 (nM)
B U87MG
C U251MG
D GBM-3
200
400
600
0
1200
BO-1051 (nM)
Cell Cycle Distribution (%)
Cell Cycle Distribution (%)
Cell Cycle Distribution (%)
DNA content
Cell count
2n
4n
2n
4n
Figure 3 Effect of BO-1051 on cell cycle profile in human glioma cells. Cultures were exposed to BO-1051 for 24 h before collection and
FACS analysis of the propidium iodide-stained cells. (A) The DNA histograms depict cell cycle phase distributions of U87MG 24 h post-treatment. Cells in exponential growth were sham treated (left panel), treated with BO-1051 (200 nM, right panel) and then harvested 24 h later. (B-D) Cell
cycle distributions of a panel of 3 human glioma cell lines (U87MG, U251MG and GBM-3) were exposed to the designated concentrations of BO-
1051 for 24 h. Data displayed by the DNA content profiles were analyzed, and the cell cycle phase information is represented graphically. Control
0
20
40
60
80
100
G1
S
G2/M
200
400
600
0
1200
BO-1051 (nM)
200 nM BO
B U87MG
Cell Cycle Distribution (%)
DNA content
Cell count
2n
4n
2n
4n 0
20
40
60
80
100
G1
S
G2/M
0
20
40
60
80
100
G1
S
G2/M
BO 1051 (nM)
200
400
600
0
1200
BO-1051 (nM)
C U251MG
D GBM-3
200
400
600
0
1200
BO-1051 (nM)
Cell Cycle Distribution (%)
Cell Cycle Distribution (%) 0
20
40
60
80
100
G1
S
G2/M
(
)
D GBM-3
200
400
600
0
1200
BO-1051 (nM)
Cell Cycle Distribution (%) Figure 3 Effect of BO-1051 on cell cycle profile in human glioma cells. Cultures were exposed to BO-1051 for 24 h before collection and
FACS analysis of the propidium iodide-stained cells. (A) The DNA histograms depict cell cycle phase distributions of U87MG 24 h post-treatment. BO-1051 delays the growth of xenograft gliomas exposed
to irradiation For
each group, the time for tumors to grow from 150 to
1500 mm3 (i.e., a 10-fold increase in tumor size) was
calculated using tumor volumes from the individual
mice in each group (mean + SD). The time required for
tumors to reach 10-times the starting volume increased
from 20.2 days for control mice to 29.5 days for BO-
1051-treated mice. Irradiation treatment alone increased
the time to reach 10-times the initial volume to 23.6
days. However, in mice that received the combination
therapy, the time for tumors to reach 10-times the
initial volume increased to 36.2 days, which is signifi-
cantly greater than the individual treatment groups
(Figure 6A; Table 2, p > 0.05). Thus, the growth delay
after the combined treatment was more than the sum of
the growth delays caused by either BO-1051 or radiation
alone. To calculate an SER comparing the tumor radia-
tion responses in mice with and without the BO-1051
treatment, the normalized tumor growth delay was was used. Mice bearing s.c. xenografts (~150 mm3) were
stratified by size and randomized into 4 groups: control,
BO-1051 alone, irradiation alone (4 Gy), or combined
BO-1051 plus radiation. For the BO-1051 treatments,
mice were i.p. injected with dosage at 50 mg/kg on days
0, 3 and 6. The growth rates for the GBM-3 tumors
exposed to each treatment are shown in Figure 6A. For
each group, the time for tumors to grow from 150 to
1500 mm3 (i.e., a 10-fold increase in tumor size) was
calculated using tumor volumes from the individual
mice in each group (mean + SD). The time required for
tumors to reach 10-times the starting volume increased
from 20.2 days for control mice to 29.5 days for BO-
1051-treated mice. Irradiation treatment alone increased
the time to reach 10-times the initial volume to 23.6
days. However, in mice that received the combination
therapy, the time for tumors to reach 10-times the
initial volume increased to 36.2 days, which is signifi-
cantly greater than the individual treatment groups
(Figure 6A; Table 2, p > 0.05). Thus, the growth delay
after the combined treatment was more than the sum of
the growth delays caused by either BO-1051 or radiation
alone. BO-1051 delays the growth of xenograft gliomas exposed
to irradiation To determine if the enhanced radiosensitivity of
BO-1051 treated glioma cells could be translated into an
in vivo tumor model, a tumor growth delay assay using
GBM-3 cells grown s.c. in the hind leg of nude mice Chu et al. Radiation Oncology 2011, 6:7
http://www.ro-journal.com/content/6/1/7 Page 8 of 13 Figure 4 Apoptotic effects of BO-1051 in combination with irradiation in glioma cells. U87MG, U251MG and GBM-3 were exposed to
200 nM or higher concentration (1200 nM) of BO-1051 for 24 h and irradiated with 2 Gy, followed by FACS analysis of Annexin V-FITC and PI
staining 24 h later. Control: no treatment; IR: ionizing radiation at 2 Gy; BO: BO-1051; BO+IR: cells exposed to BO-1051 for 24 h and then
irradiation with 2 Gy of g-ray. Values are the means ± SD of 3 independent experiments. * p < 0.05. Figure 4 Apoptotic effects of BO-1051 in combination with irradiation in glioma cells. U87MG, U251MG and GBM-3 were exposed to
200 nM or higher concentration (1200 nM) of BO-1051 for 24 h and irradiated with 2 Gy, followed by FACS analysis of Annexin V-FITC and PI
staining 24 h later. Control: no treatment; IR: ionizing radiation at 2 Gy; BO: BO-1051; BO+IR: cells exposed to BO-1051 for 24 h and then
irradiation with 2 Gy of g-ray. Values are the means ± SD of 3 independent experiments. * p < 0.05. Figure 4 Apoptotic effects of BO-1051 in combination with irradiation in glioma cells. U87MG, U251MG and GBM-3 were exposed to
200 nM or higher concentration (1200 nM) of BO-1051 for 24 h and irradiated with 2 Gy, followed by FACS analysis of Annexin V-FITC and PI
staining 24 h later. Control: no treatment; IR: ionizing radiation at 2 Gy; BO: BO-1051; BO+IR: cells exposed to BO-1051 for 24 h and then
irradiation with 2 Gy of g-ray. Values are the means ± SD of 3 independent experiments. * p < 0.05. was used. Mice bearing s.c. xenografts (~150 mm3) were
stratified by size and randomized into 4 groups: control,
BO-1051 alone, irradiation alone (4 Gy), or combined
BO-1051 plus radiation. For the BO-1051 treatments,
mice were i.p. injected with dosage at 50 mg/kg on days
0, 3 and 6. The growth rates for the GBM-3 tumors
exposed to each treatment are shown in Figure 6A. BO-1051 delays the growth of xenograft gliomas exposed
to irradiation To calculate an SER comparing the tumor radia-
tion responses in mice with and without the BO-1051
treatment, the normalized tumor growth delay was measured to determine the role of BO-1051 on tumor
growth delay induced by the combination treatment. The SER of the xenograft gliomas was 1.97 with versus
without the combined treatment of BO-1051 and irra-
diation (Table 2). Thus, BO-1051 alone slows tumor
growth and enhances the effect of radiation, which is
similar to the results obtained in vitro. Finally, the
Kaplan-Meier survival curves of the combined treated
mice revealed a trend toward longer survival in mice
(Figure. 6B). We also noticed that the maximal toxicity
of these agents decreased with body weight, and there
was no more than a 15% weight reduction compared to
the pretreatment body weight. However after cession of
treatment, the body weight recovered (data not shown). Discussion Control
2Gy-1h
2Gy-6h
2Gy-24h
BO-1051
200nM, 24h
BO-1051
2Gy-1h
BO-1051
2Gy-6h
BO-1051
2Gy-24h
unirradiated
unirradiated
0
20
40
60
80
100
0-10
11-30
>30
0
5
10
15
20
25
30
35
vehicle
BO
ȖH2AX foci per cell
1h
6h
24h
0 Gy
1h
6h
24h
2 Gy
Control
2Gy-1h
2Gy-6h
2Gy-24h
BO-1051
200nM, 24h
BO-1051
2Gy-1h
BO-1051
2Gy-6h
BO-1051
2Gy-24h
Ctrl
BO
IR
BO/IR
unirradiated
unirradiated
Percent of cells with ȖH2AX foci
Ύ
Figure 5 Influence of BO-1051 on the repair of radiation-induced DSBs. GBM-3 cells growing on slides in 35-mm dishes were exposed to
200 nM of BO-1051 for 24 h, irradiated (2Gy), and then fixed at the specified times for immunofluorescent analysis of nuclear g-H2AX foci using
a confocal microscope. (A) Immunofluorescent microscopy images of GBM-3 cells untreated or treated with 200 nM BO-1051 24 h before
irradiation, fixed after 0, 1, 6, 24 h and then stained for g-H2AX foci. (B) Quantitative analysis of g-H2AX foci presented in irradiated cells following
the above treatments. Filled columns: data from vehicle-treated cells; open columns: data from cells exposed to BO-1051. Values are the means
± SD of 3 independent experiments. * p < 0.05. (C) Distribution of g-H2AX foci numbers per cell for one representative experiment at 24 h after
irradiation. Ctrl: no treatment; IR: ironing radiation at 2 Gy; BO: cells exposed to 200 nM BO-1051; BO/IR: cells exposed to 200 nM BO-1051 for 24 2Gy-1h 0
20
40
60
80
100
0-10
11-30
>30
Ctrl
BO
IR
BO/IR
Percent of cells with ȖH2AX foci
Figure 5 Influence of BO-1051 on the repair of radiation-induced DSBs. GBM-3 cells growing on slides in 35-mm dishes were exposed to
200 nM of BO-1051 for 24 h, irradiated (2Gy), and then fixed at the specified times for immunofluorescent analysis of nuclear g-H2AX foci using
a confocal microscope. (A) Immunofluorescent microscopy images of GBM-3 cells untreated or treated with 200 nM BO-1051 24 h before
irradiation, fixed after 0, 1, 6, 24 h and then stained for g-H2AX foci. (B) Quantitative analysis of g-H2AX foci presented in irradiated cells following
the above treatments. Filled columns: data from vehicle-treated cells; open columns: data from cells exposed to BO-1051. Values are the means
± SD of 3 independent experiments. * p < 0.05. Discussion Although human GBM is one of the most radio-resis-
tant tumors, radiotherapy remains routinely applied for
patient treatment. Lots of efforts are made to develop
methods for enhancing the radiosensitivity of GBM for
promising therapy. Previous studies have shown that
temozolomide (TMZ) combined with radiation exposure
results in an increase of survival rate in a subset of
human tumors [3,25,26]. Clinical studies also indicate
that delivery of TMZ during radiotherapy increases Chu et al. Radiation Oncology 2011, 6:7
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20
40
60
80
100
0-10
11-30
>30
0
5
10
15
20
25
30
35
vehicle
BO
ȖH2AX foci per cell
1h
6h
24h
0 Gy
1h
6h
24h
2 Gy
Control
2Gy-1h
2Gy-6h
2Gy-24h
BO-1051
200nM, 24h
BO-1051
2Gy-1h
BO-1051
2Gy-6h
BO-1051
2Gy-24h
Ctrl
BO
IR
BO/IR
unirradiated
unirradiated
Percent of cells with ȖH2AX foci
Ύ
Figure 5 Influence of BO-1051 on the repair of radiation-induced DSBs. GBM-3 cells growing on slides in 35-mm dishes were exposed to
200 nM of BO-1051 for 24 h, irradiated (2Gy), and then fixed at the specified times for immunofluorescent analysis of nuclear g-H2AX foci using
a confocal microscope. (A) Immunofluorescent microscopy images of GBM-3 cells untreated or treated with 200 nM BO-1051 24 h before
irradiation, fixed after 0, 1, 6, 24 h and then stained for g-H2AX foci. (B) Quantitative analysis of g-H2AX foci presented in irradiated cells following
the above treatments. Filled columns: data from vehicle-treated cells; open columns: data from cells exposed to BO-1051. Values are the means
± SD of 3 independent experiments. * p < 0.05. (C) Distribution of g-H2AX foci numbers per cell for one representative experiment at 24 h after
irradiation. Ctrl: no treatment; IR: ironing radiation at 2 Gy; BO: cells exposed to 200 nM BO-1051; BO/IR: cells exposed to 200 nM BO-1051 for 24
h and then irradiated with 2 Gy of g-rays. Foci were evaluated in 100 nuclei per treatment for each cell type. Values are the means at least of 3
independent experiments. Discussion (C) Distribution of g-H2AX foci numbers per cell for one representative experiment at 24 h after
irradiation. Ctrl: no treatment; IR: ironing radiation at 2 Gy; BO: cells exposed to 200 nM BO-1051; BO/IR: cells exposed to 200 nM BO-1051 for 24
h and then irradiated with 2 Gy of g-rays. Foci were evaluated in 100 nuclei per treatment for each cell type. Values are the means at least of 3
independent experiments. survival rates of GBM patients, which suggests that this
DNA alkylating agent can enhance the radiosensitivity
of GBM [2,27,28]. Based on these previous studies,
more efficient and safe DNA alkylating agents should be
developed to increase the radiosensitivity in human
GBM. Use of BO-1051 for cancer treatment has been
supported by in vitro and in vivo preclinical studies
[3,26]. The data presented here showed that the treat-
ment of primary glioma cells and established cell lines
with BO-1051 resulted in a dose-dependent induction of
clonogenic cell death. It is supposed that BO-1051 can
enhance the radiosensitivity via a synergistic effect since
the survival fractions of combined treatment are lower
than that of each individual treatment on glioma cell. However, additional studies are required to confirm
that BO-1051 plays a synergistic or additive role on
radiotherapy of gliomas. The anti-tumor and radiosensi-
tizing effects of BO-1051 are encouraging because drugs
showing efficacy against malignant glioma are still
uncommon. Bifunctional N-mustard alkylating agents, such as BO-
1051, exhibits anticancer activity due to its ability to
produce DNA interstrand and/or intrastrand cross-links
[29,30]. As has been known, bifunctional alkylating
agents induce collapsed replication forks that can lead
to either cell cycle arrest, DNA repair, or apoptosis [31]. For example, the new synthesized alkylating agent
BO-1012 shows anticancer activity on xenograft tumors Page 10 of 13 Chu et al. Radiation Oncology 2011, 6:7
http://www.ro-journal.com/content/6/1/7 Figure 6 The effects of BO-1051 on radiation-induced tumor growth delay and prolongation of TTF (time to treatment failure) in
nude mice bearing GBM-3 xenografts. When tumors reached 150 mm3, the nude mice with established GBM-3 flank xenografts were
randomized into 4 groups: control (black circle), radiation (white circle), BO-1051 (black triangle) or BO-1051 plus radiation (white triangle). BO-1051 (50 mg/kg) was delivered via i.p. injection on days 0, 3 and 6, where day 0 begins on the day of randomization. Discussion Radiation (4 Gy) was
delivered 24 h after the first injection of BO-1051 (day 1 after randomization), which corresponded to the same tumor size. Each group
contained at least 8 mice. (A) Tumor growth rates for each treatment group were plotted as the mean relative tumor volume ± SD. Arrows
indicate the time of BO-1051 and irradiation treatment. (B) Kaplan-Meier survival rates of nude mice with GBM-3 flank xenografts for each of the
four treatments is depicted. Survival analysis was monitored daily. Treatment failure was defined as tumor size greater than 1500 mm3 or the
development of severe necrosis requiring euthanasia. f BO-1051 on radiation-induced tumor growth delay and prolongation of TTF (time to treatment failure) in
3 Figure 6 The effects of BO-1051 on radiation-induced tumor growth delay and prolongation of TTF (time to treatment failure) in
nude mice bearing GBM-3 xenografts. When tumors reached 150 mm3, the nude mice with established GBM-3 flank xenografts were
randomized into 4 groups: control (black circle), radiation (white circle), BO-1051 (black triangle) or BO-1051 plus radiation (white triangle). BO-1051 (50 mg/kg) was delivered via i.p. injection on days 0, 3 and 6, where day 0 begins on the day of randomization. Radiation (4 Gy) was
delivered 24 h after the first injection of BO-1051 (day 1 after randomization), which corresponded to the same tumor size. Each group
contained at least 8 mice. (A) Tumor growth rates for each treatment group were plotted as the mean relative tumor volume ± SD. Arrows
indicate the time of BO-1051 and irradiation treatment. (B) Kaplan-Meier survival rates of nude mice with GBM-3 flank xenografts for each of the
four treatments is depicted. Survival analysis was monitored daily. Treatment failure was defined as tumor size greater than 1500 mm3 or the
development of severe necrosis requiring euthanasia. Figure 6 The effects of BO-1051 on radiation-induced tumor growth delay and prolongation of TTF (time to treatment failure) in
nude mice bearing GBM-3 xenografts. When tumors reached 150 mm3, the nude mice with established GBM-3 flank xenografts were
randomized into 4 groups: control (black circle), radiation (white circle), BO-1051 (black triangle) or BO-1051 plus radiation (white triangle). BO-1051 (50 mg/kg) was delivered via i.p. injection on days 0, 3 and 6, where day 0 begins on the day of randomization. only group to reach the same size.
# Normalized growth delay for the BO-1051+IR group divided by absolute growth delay for the radiation-only group. * Time for subcutaneous tumors to grow from the initial tumor volume to 10 times (see text). Discussion Radiation (4 Gy) was
delivered 24 h after the first injection of BO-1051 (day 1 after randomization), which corresponded to the same tumor size. Each group
contained at least 8 mice. (A) Tumor growth rates for each treatment group were plotted as the mean relative tumor volume ± SD. Arrows
indicate the time of BO-1051 and irradiation treatment. (B) Kaplan-Meier survival rates of nude mice with GBM-3 flank xenografts for each of the
four treatments is depicted. Survival analysis was monitored daily. Treatment failure was defined as tumor size greater than 1500 mm3 or the
development of severe necrosis requiring euthanasia. that are formed by various human lung and bladder
cancer cells [32]. BO-1051 and its analog(s) also exhibit
similar behavior, and several related synthetic bifunc-
tional N-mustards are under development [33]. Because
BO-1051 contains the inherent lipophilicity for penetra-
tion through blood-brain barrier, it has efficiently
demonstrated the ability to inhibit the growth of xeno-
graft glioma in nude mice. Compared to other clinically
used alkylating agents, such as melphalan and cisplatin,
BO-1051 induced a higher level of ICLs [14]. BO-1051 also enhances the radiosensitivity of human glioma cell
lines. Although repair mechanisms such as homologous
recombination and nonhomologous end-joining are
important mammalian responses to double-strand DNA
damage, cell cycle regulation is perhaps the most impor-
tant determinant of irradiation sensitivity [22,34]. The
cell cycle is strongly affected by DNA damage, and a
cell’s radiosensitivity depends on cell cycle position and
progression [22]. Conventionally, the G2/M phase is the Table 2 BO-1051-induced tumor growth delay in GBM-3 xenografts
Treatment group
Tumor growth period, days*
Absolute growth delay†
Normalized growth delay‡
Enhancement ratio#
Control
20.2
BO-1051
29.5
9.3
IR
23.6
3.4
BO-1051+IR
36.2
16
6.7
1.97
* Time for subcutaneous tumors to grow from the initial tumor volume to 10 times (see text). † The number of days for the treated tumors to reach 10 times the initial tumor volume minus the number of days for the control group to reach the same size. ‡ The number of days for the tumors in the BO-1051+IR group to reach 10 times the initial tumor volume minus the number of days for tumors in the BO-1051-
only group to reach the same size. # Normalized growth delay for the BO-1051+IR group divided by absolute growth delay for the radiation-only group. Time for subcutaneous tumors to grow from the initial tumor volume to 10 times (see text).
† The number of days for the treated tumors to reach 10 times the initial tumor volume minus the number of days for the control group to reach the same size.
‡ The number of days for the tumors in the BO-1051+IR group to reach 10 times the initial tumor volume minus the number of days for tumors in the BO-1051-
only group to reach the same size.
# Normalized growth delay for the BO 1051+IR group divided by absolute growth delay for the radiation only group y g
p
# Normalized growth delay for the BO-1051+IR group divided by absolute growth delay for the radiation-only group. Discussion Table 2 BO-1051-induced tumor growth delay in GBM-3 xenografts
Treatment group
Tumor growth period, days*
Absolute growth delay†
Normalized growth delay‡
Enhancement ratio#
Control
20.2
BO-1051
29.5
9.3
IR
23.6
3.4
BO-1051+IR
36.2
16
6.7
1.97
* Time for subcutaneous tumors to grow from the initial tumor volume to 10 times (see text). † The number of days for the treated tumors to reach 10 times the initial tumor volume minus the number of days for the control group to reach the same size. ‡ The number of days for the tumors in the BO-1051+IR group to reach 10 times the initial tumor volume minus the number of days for tumors in the BO-1051-
only group to reach the same size. # Normalized growth delay for the BO-1051+IR group divided by absolute growth delay for the radiation-only group. Table 2 BO-1051-induced tumor growth delay in GBM-3 xenografts
Treatment group
Tumor growth period, days*
Absolute growth delay†
Normalized growth delay‡
Enhancement ratio#
Control
20.2
BO-1051
29.5
9.3
IR
23.6
3.4
BO-1051+IR
36.2
16
6.7
1.97
* Ti
f
b
f
h
i i i l
l
0 i
(
) Table 2 BO-1051-induced tumor growth delay in GBM-3 xenografts y g
p
# Normalized growth delay for the BO-1051+IR group divided by absolute growth delay for the radiation-only group. Page 11 of 13 Chu et al. Radiation Oncology 2011, 6:7
http://www.ro-journal.com/content/6/1/7 Page 11 of 13 Page 11 of 13 However, theses phenomenon were not detected in
glioma cells exposed to BO-1051 following irradiation. Recently, we found that BO-1051 can induce autophagy
in glioma cell lines (unpublished observation), and sev-
eral lines of evidence have supported that autophagy is
one of the causes of radiosensitization instead of apopto-
sis [51-53]. Therefore, the correlation between autophagy
and radiosensitivity needs to be further investigated. most radiosensitive phase compared to others. Several
chemotherapeutic agents have been reported to enhance
the radiosensitivity of cancer cells by accumulating the
G2/M population, such as paclitaxel, indomethacin,
2-methoxyestradiol and TMZ [3,22,25,35-37]. From the
results in this study, BO-1051 works by partially synchro-
nized glioma cells in the most radiosensitive phase of the
cell cycle, and it is suggested that BO-1051 may be a use-
ful agent for adjuvant therapy on the glioma. Given that human GBM usually exhibits high radiore-
sistance, it is necessary to search for a specific radiosen-
sitizer to enhance the radiosensitivity of GBM during
radiotherapy. Discussion Kil et al. have demonstrated that TMZ
may be used as a radiosensitizer because it can enhance
the radiosensitivity of U251MG cells formed xenograft
tumors [26]. Nevertheless, we found that TMZ was
neither able to increase the radiosensitivity of xenograft
tumors derived from GBM-3 cells nor able to delay
tumor growth and improve animal survival after treat-
ment (unpublished observation). Therefore, TMZ may
exhibit cell specific effects for the treatment of different
sources of human GBM. However, BO-1051 enhances
the radiosensitivity of various glioma cell lines, as well
as that of the corresponding xenograft tumors formed
by GBM-3 cells. These results suggest that BO-1051 is a
radiosensitizer with broader effects on different human
GBM, and it may possess a clinical potential in the ther-
apeutic strategy for treating malignant gliomas. The phosphatidyl-inositol kinase-related protein ATM
(ataxia-telangiectasia mutated), the most proximal signal
transducer initiating cell cycle changes after the DNA
damage/genomic stress [38], can be activated by BO-1051
in a dose-dependent manner in SAS cell line. It also acti-
vates the checkpoint kinase 2 (Chk2) in squamous cell car-
cinoma cell line after exposure to BO-1051 (unpublished
observation). Chk2 activity is necessary for the phosphory-
lation of the dual-specificity phosphatases Cdc25A/C,
which inactivates the enzymes, blocks CDK1 activation
and causes a G2 arrest [39]. Furthermore, ATM’s essential
role in DNA damage and repair is highlighted by the
extreme sensitivity to ionizing radiation of cells with defec-
tive ATM [40,41]. It, together with DNA-dependent pro-
tein kinase, phosphorylate the histone g-H2AX foci, which
can be visualized by immunofluorescence microscope as a
discrete nuclear foci reflecting sites of DNA DSBs [42,43]. Although the specific relationship between the appearance
of g-H2AX foci and the repair of DSBs has not been com-
pletely defined, the reduction in the number of g-H2AX
foci in irradiated cells correlates with DNA repair, which
is associated with the radiosensitivity [44-46]. It is also
known that g-H2AX is present in focal aggregates at sites
of double-strand DNA damage and complex with other
important repair molecules. g-H2AX is required for foci
formation for numerous factors including p53, MRN com-
plex (MRE-11, RAD50, and NBS1), and BRCA1 [47]. MRN complex has also been implicated in the repair of
small fraction of DSBs detectable as g-H2AX foci that
remain 24 h post-irradiation [48] Therefore, the observa-
tion that combined BO-1051 plus radiation significantly
increased the levels of g-H2AX foci. BO-1051: 1-{4-[Bis(2-chloroethyl)amino]phenyl}-3-[2-methyl-5-(4-methylacridin-
9-ylamino)phenyl]urea; DBSs: Double-strand breaks; GBM: Glioblastoma
multiforme; ICLs: Interstrand cross-linking; PE: Plating efficacy; SER: Sensitizer
enhancement ratio; SF: Surviving fraction; TMZ: Temozolomide; TTF: Time to
treatment failure. Abbreviations
BO 0
{ [B BO-1051: 1-{4-[Bis(2-chloroethyl)amino]phenyl}-3-[2-methyl-5-(4-methylacridin-
9-ylamino)phenyl]urea; DBSs: Double-strand breaks; GBM: Glioblastoma
multiforme; ICLs: Interstrand cross-linking; PE: Plating efficacy; SER: Sensitizer
enhancement ratio; SF: Surviving fraction; TMZ: Temozolomide; TTF: Time to
treatment failure. Radiation sensitization could occur through any one of
multiple modes of cell death. Zou et al. observed radio-
sensitization through the promotion of apoptosis [35],
while another research group reported radiosensitization
through a mitotic catastrophe [26,49] or senescence [50]. Conclusions GBM is the most malignant primary brain tumor in
adults, but the effective therapeutic strategies remain
under investigation. BO-1051 has been shown to inhibit
the growth of gliomas. Here we further demonstrate
that BO-1051 can significantly enhance the radiosensi-
tivity. The enhanced radiosensitivity was found to be
associated with G2/M phase arrest as well as the sus-
tained DNA damage. In vivo studies further demon-
strated that BO-1051 enhanced the radiotherapeutic
effects on GBM-3-beared xenograft tumors. In this
model, the combination of BO-1051 plus radiation pro-
duced the best response in terms of both local control
and survival. These data suggest that BO-1051 provides
a new strategy to improve therapeutic gain for radiation
therapy. Discussion Because the pro-
longed expression of radiation-induced g-H2AX foci may
reflect the end result of disparate processes and events
leading to maintenance of unrepaired DSBs, a distinctly
different mechanism may be involved. Whereas the
mechanism of this repair inhibition is not revealed in this
investigation, additional investigations are required to
define the molecular processes responsible for BO-1051-
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Trabalho paralelo e trabalho doméstico: um diálogo teórico possível?
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Trabalho fora do trabalho: uma
etnografia das percepções. de me tornar etnógrafa, e este livro guarda tais
vestígios [...]” (p. 272-273). O livro se divide em três partes: “O ofício de
etnógrafo”, que trata do método e da teoria da
pesquisa; “Formas e funções do trabalho
paralelo”, que busca explorar a prática do
trabalho paralelo em si e suas valorações por
parte dos trabalhadores observados; e
“Pertencimentos sociais, pertencimentos locais”,
que explora as relações nas quais as valorações
desse tipo de atividade se constroem. WEBER, Florence. Tradução de Roberta Ceva. Rio de Janeiro: Garamond, 2009. 384 p. Tradução de Roberta Ceva. Rio de Janeiro: Garamond, 2009. 384 p. Tradução de Roberta Ceva. Rio de Janeiro: Garamond, 2009. 384 p. A análise é cuidadosa. As pessoas em geral
são observadas em diferentes aspectos, não só
pelo pertencimento àquilo que a autora chama
de operariado, mas a descendência, o sexo, a
renda, as relações políticas e a faixa etária são
aspectos levados em conta. Considerando que,
para ter acesso a informações que dados
estatísticos não resolveriam sobre o trabalho
realizado fora do ambiente formal (que muitas
vezes não era declarado por medo de uma
fiscalização governamental sobre sua irregula-
ridade ou até pelo caráter de lazer atribuído pelos
pesquisados a esse trabalho), a pesquisadora se
insere no ambiente estudado para obter as
informações. Assim, em muitos momentos, ela
coloca suas percepções encarando-as como
sendo parte do meio, apesar de trabalhar com
a ideia de distanciamento entre pesquisador e
objeto de pesquisa. Apesar de os estudos que têm como objeto
o operariado, nas Ciências Humanas, serem
inquestionavelmente extensos, de forma geral
esses estão ligados a uma identidade operária
construída na relação com a fábrica – ou outro
que seja seu ambiente de trabalho formal – ou
então construída na resistência a ela. Nas
pesquisas que se preocupam em observar essas
questões mais de perto, o ideal de bom operário
ou o de bom militante não aparece puro, estando
circulando e se relacionando de forma não
estática, não engessada. Florence Weber, em
sua pesquisa realizada entre trabalhadores/as de
uma área rural francesa entre 1983 e 1985,
buscou observar exatamente o que não estava
nem na fábrica nem no sindicato, questionando-
se sobre o que faziam os operários fora de seu
trabalho em uma fábrica metalúrgica. TTTTTrabalho paralelo e trabalho doméstico:
rabalho paralelo e trabalho doméstico:
rabalho paralelo e trabalho doméstico:
rabalho paralelo e trabalho doméstico:
rabalho paralelo e trabalho doméstico:
um diálogo teórico possível?
um diálogo teórico possível?
um diálogo teórico possível?
um diálogo teórico possível?
um diálogo teórico possível? Trabalho fora do trabalho: uma
etnografia das percepções. Trabalho fora do trabalho: uma
etnografia das percepções. Quer dizer, é possível que
na época da pesquisa ela intencionalmente não
tenha se apropriado dessas questões feministas
por razões políticas, pessoais ou até de
legitimidade acadêmica. operárias? Mesmo que representando uma
minoria faziam parte do quadro da fábrica. O
que elas faziam com seu tempo livre? Estando
convivendo na cidade por mais de um ano e
tendo relações próximas com uma operária que
inclusive a ajudou na pesquisa, essas perguntas
poderiam ter sido feitas. Leituras de teóricas
feministas sobre como o amor à família e a ideia
de lazer2 se entrelaçam com o trabalho
doméstico poderiam inclusive ajudar nas
observações sobre a realização pessoal
imbricada no “trabalho paralelo”. Os debates sobre os lugares de produção
e lugares de reprodução continuam atuais,3
ainda que transformados em muitos aspectos. Talvez isso justifique a retomada desta obra (de
1989 e republicada vinte anos depois), a qual
no século XXI pode oferecer subsídios para se
pensarem as relações de trabalho diluídas na
vida privada, a constituição de identidades
construídas sobre práticas laborais e a não fixidez
das relações que se dão nesses espaços; este
último ponto inclusive ressaltado por Florence
Weber em sua escrita. Para os estudos feministas
e de gênero, talvez a maior contribuição da
pesquisadora tenha sido, paradoxalmente, o
que ela considerou o resultado não feminista
de sua pesquisa: a conclusão de que não só as
mulheres produzem fora dos circuitos de
mercado. Essa conclusão tanto pode contribuir
para estudos de masculinidades e identidades
laborais, de trabalho reprodutivo e de relações
de gênero quanto para dar mais consistência
às conceituações sobre trabalho reprodutivo
para se questionar a constituição hierárquica
da noção de trabalho produtivo versus trabalho
reprodutivo, entre outros debates afins. Outro ponto, que nem ao menos no
posfácio é tratado, é sobre as mulheres operárias. A obra é problemática na medida em que utiliza
o tempo todo “os operários” como sujeito
universal, e nunca especifica com clareza que
se escolheu trabalhar apenas com homens. De
repente, aparece uma operária que a ajuda na
pesquisa, uma açougueira é citada. Mas o
trabalho paralelo analisado é apenas masculino. Trabalho fora do trabalho: uma
etnografia das percepções. Socióloga e antropóloga francesa,
Florence Weber se desloca na década de 1980
de Paris para Montbard, pequena cidade na qual
residiu durante a infância, para, através da
pesquisa etnográfica, buscar “descobrir, a partir
da observação dos comportamentos operários
fora da fábrica, valores e gostos positivos que
permitem explicar certas práticas operárias sem
reduzi-las a uma simples submissão à
necessidade” (p. 254). O foco principal da
pesquisadora é o “trabalho paralelo”, um termo
que ela classifica como nativo (próprio dos
pesquisados) e que designa uma série de
atividades que produzem bens de uso que não
são voltados, ou especificamente voltados, para
a mercantilização. No posfácio de 2009,
acrescido à edição original de 1989, a autora
faz duas afirmações que podem nos ajudar na
leitura de seu trabalho: “Sem dúvida nenhuma,
eu era marxista e feminista (nesta ordem), antes Nota-se que ela se preocupa em marcar
seu afastamento de abordagens que carregam
a alcunha de “pós-modernas”, percepção que
se completa com a ideia de que “uma interpreta-
ção aprofundada destes acontecimentos
microscópicos [atividades reconhecidas como
trabalho paralelo] pode torná-los exemplares” (p. 15). Mas lembra, em seu posfácio datado de
2009, que seus resultados não foram marxistas
porque os operários que descreve não são
proletários inteiramente presos à ordem industrial
capitalista. Tais afirmações podem gerar debates. De qualquer forma, em meio a essa discussão
sobre ser ou não marxista ou pós-estruturalista, a
ideia de “trabalho paralelo” como revanche ao
trabalho na fábrica, como uma prática de resis-
tência, uma das hipóteses iniciais da abordagem,
foi abandonada no decorrer de sua pesquisa. Sua posição perante o feminismo chama
especial atenção, principalmente, quando ela 335 Estudos Feministas, Florianópolis, 20(1): 313-337, janeiro-abril/2012 335 mesma reconhece, em 2009, e talvez após
receber algumas críticas nesse sentido, que sua
preocupação em descrever a vida cotidiana,
a produção doméstica e trocas não mercantis
aproximá-la-ia, nesses aspectos, aos trabalhos
feministas que se interessam pela parte invisível
da economia. Uma primeira questão que tais
afirmações podem gerar é de que essas
discussões sobre a economia invisível, por parte
dos feminismos, já existiam e tinham razoável
circulação quando ela escreveu seu livro, em
meados para o final da década de 1980.1 Mas
o trabalho paralelo das mulheres é sucintamente
citado no livro, em duas ou três linhas, apenas
no posfácio de 2009. Notas
Notas
Notas
Notas
Notas 1 Exemplos de trabalhos europeus neste sentido em Maria
Angeles DURAN, 1983; e Andrée KARTCHEVSKY-BULPORT et
al., 1986. 1 Exemplos de trabalhos europeus neste sentido em Maria
Angeles DURAN, 1983; e Andrée KARTCHEVSKY-BULPORT et
al., 1986. 2 Referência em DURAN, 1983. 2 Referência em DURAN, 1983. E o trabalho paralelo feminino? Esse, ela
qualifica em geral como trabalho doméstico, por
seu caráter de obrigatoriedade. Mas a partir do
momento em que ao trabalho paralelo que ela
analisa e conceitua são agregados valores de
satisfação pessoal e até lazer que os diferenciariam
de uma dupla jornada de trabalho, com
afirmações como “Caso se trabalhasse para um
patrão, faria-se muito menos” (p. 67), não
entendo a dificuldade em utilizar seu próprio
conceito para uma análise voltada também às
mulheres. É claro que as donas de casa, esposas
de operários, não eram seu objeto, apesar de se
terem feito incursões sobre elas. Mas e as mulheres 3 Exemplos em Cristina BRUSCHINI, 2007; e Cristina
CARRASCO, 2008. 3 Exemplos em Cristina BRUSCHINI, 2007; e Cristina
CARRASCO, 2008. Trabalho fora do trabalho: uma
etnografia das percepções. Se ao ler a obra publicada em 1989 a crítica
construir-se-ia no sentido de problematizar como
uma pesquisa voltada ao trabalho invisível se
esquece das mulheres que fazem parte de seu
objeto (sendo o trabalho invisível de forma geral
referido às mulheres), após ler o posfácio de 2009
e a identificação da pesquisadora como femi-
nista (ainda que seja uma identificação secundá-
ria em relação à de marxista), pode-se questionar
como uma feminista, que tomou cuidado com
tantas questões, invisibilizou as mulheres em sua
obra, que quando aparecem é pelo caráter de
“colaboração” – termo da autora – das mulheres
nesses trabalhos paralelos masculinos. DURAN, Maria Angeles. A dona de casa: crítica
política da economia doméstica. Rio de
Janeiro: Edições Graal, 1983. Lucia da; TITO, Neuza. Trabalho doméstico
e de cuidados: por outro paradigma de
sustentabilidade da vida humana. São
Paulo: SOF, 2008. p. 91-104. KARTCHEVSKY-BULPORT, Andrée et al. O sexo do
trabalho. Rio de Janeiro: Paz e Terra, 1986. KARTCHEVSKY-BULPORT, Andrée et al. O sexo do
trabalho. Rio de Janeiro: Paz e Terra, 1986.
Soraia Carolina de Mello
Universidade Federal de Santa Catarina Referências
Referências
Referências
Referências
Referências BRUSCHINI, Cristina. “Trabalho doméstico: inativi-
dade econômica ou trabalho não remune-
rado?”. In: ARAÚJO, Clara; PICANÇO, Felícia;
SCALON, Celi (Org.). Novas conciliações e
antigas tensões? Gênero, família e trabalho
em perspectiva comparada. Bauru: EDUSC,
2007. p. 21-58. CARRASCO, Cristina. “Por uma economia não
androcêntrica: debates e propostas a partir
da economia feminista”. In: SILVEIRA, Maria CARRASCO, Cristina. “Por uma economia não
androcêntrica: debates e propostas a partir
da economia feminista”. In: SILVEIRA, Maria 336 36 Estudos Feministas, Florianópolis, 20(1): 313-337, janeiro-abril/2012 KARTCHEVSKY-BULPORT, Andrée et al. O sexo do
trabalho. Rio de Janeiro: Paz e Terra, 1986. Soraia Carolina de Mello
Universidade Federal de Santa Catarina DURAN, Maria Angeles. A dona de casa: crítica
política da economia doméstica. Rio de
Janeiro: Edições Graal, 1983. Estudos Feministas, Florianópolis, 20(1): 313-337, janeiro-abril/2012 337
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Supplementary Figure 2 from α-Catulin Marks the Invasion Front of Squamous Cell Carcinoma and Is Important for Tumor Cell Metastasis
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S Figure 1 S Figure 1 A
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https://www.researchsquare.com/article/rs-15911/v1.pdf?c=1585630500000
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Clinical significance of peripheral blood-derived inflammation markers in advanced gastric cancer after radical resection
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BMC surgery
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cc-by
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Clinical significance of peripheral blood-derived
inflammation markers in advanced gastric cancer
after radical resection Lihu Gu
Ningbo No 2 Hospital
Bangsheng Chen
Ningbo Yinzhou No 2 Hospital
Dingcheng Zheng
Ningbo No 2 Hospital
Jiahang Mo
Zhejiang Chinese Medical University
Lingling Yuan
Ningbo No 2 Hospital
Feiyan Mao
Ningbo No 2 Hospital
Kang Zhang
Ningbo University Medical School
Derry Minyao Ng
Ningbo University Medical School
Ping Chen Feiyan Mao
Ningbo No 2 Hospital Research article Keywords: Gastric cancer, Systemic inflammatory marker, Platelet lymphocyte ratio, Prognosis,
Ch
th Keywords: Gastric cancer, Systemic inflammatory marker, Platelet lymphocyte ratio, Prognosis,
Chemotherapy Posted Date: March 4th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-15911/v1 Version of Record: A version of this preprint was published at BMC Surgery on October 2nd, 2020. See the
published version at https://doi.org/10.1186/s12893-020-00884-8. DOI: https://doi.org/10.21203/rs.3.rs-15911/v1 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 1/20 Page 1/20 Version of Record: A version of this preprint was published at BMC Surgery on October 2nd, 2020. See the
published version at https://doi.org/10.1186/s12893-020-00884-8. Version of Record: A version of this preprint was published at BMC Surgery on October 2nd, 2020. See the
published version at https://doi.org/10.1186/s12893-020-00884-8. Page 2/20 Abstract Background and objective
The prognostic significance of peripheral blood-derived inflammation markers
in patients with gastric cancer (GC) has not been elucidated. This study aimed to investigate the
relationship between systemic inflammatory markers and GC prognosis. Methods
A prospective
observational cohort study involving 598 patients was conducted to analyze the prognosis of GC based
on systemic inflammatory markers. The following peripheral blood-derived inflammation markers were
evaluated: the neutrophil-lymphocyte ratio (NLR), platelet lymphocyte ratio (PLR), systemic immune-
inflammation index (SII), C-reactive protein/albumin (CRP/Alb) ratio, Glasgow Prognostic Score (GPS),
modified Glasgow Prognostic Score (mGPS), prognostic nutrition index (PNI), and prognostic index (PI). The receiver operating characteristics (ROC) curve and the Youden index were used to determine the
optimal cutoff values. Univariate and multivariate analysis of prognostic factors was conducted
accordingly. Results
The optimal cutoff values of the PNI, fibrinogen, NLR, PLR, SII, and CRP/Alb were
49.5, 397ng/dl, 2.5, 154, 556, and 0.05, respectively. Multivariate analysis showed that age, PLR, TNM
stage, and chemotherapy were the independent prognostic factors for advanced gastric cancer (AGC). Adjuvant chemotherapy improved the long-term prognosis of patients with PLR ≥154, but chemotherapy
had no significant effect on the survival of patients with PLR <154. Conclusions
Our findings show that
higher PLR (≥154) is an independent risk factor for poor prognosis in GC patients. Besides, PLR can
predict adjuvant chemotherapy (oxaliplatin/5-fluorouracil combination) response in patients with GC
after surgery. Study population This was a prospective observational cohort study involving patients who underwent radical gastrectomy
from January 2013 to December 2016 at HwaMei Hospital, University of Chinese Academy of Sciences. And was approved by the Ethics Committee of the HwaMei Hospital and the University of Chinese
Academy of Sciences (approval NO. PJ-NBEY-KY-2019-153-01). Written consent was obtained from all
patients before enrollment. The inclusion criteria were: (1) patients with histologically proven primary
adenocarcinoma of the stomach; (2) patients without a history of gastrectomy or other malignant tumor;
(3) patients without inflammatory or hematological diseases; (4) patients with pathologically negative
resection margins (R0); (5) patients without neoadjuvant chemoradiotherapy; (6) follow-up of at least 36
months. Treatment with 5-fluorouracil (5-FU)-based or platinum-based adjuvant chemotherapy was
recommended for all patients with stage II-III of the disease[13]. Introduction Gastric cancer (GC) is one of the most common malignant tumors and that poses a serious threat to
human health, especially in Asia. Approximately 300,000 deaths and 400,000 new cases of GC occur in
China every year[1]. Despite the advancement in diagnostic and treatment methods, the prognosis of
advanced gastric cancer (AGC) patients has remained poor[2]. Tumor, Node, Metastasis (TNM) staging
based on the International Union Against Cancer (UICC)/American Joint Committee on Cancer (AJCC)
guidelines is currently the standard approach of determining the prognosis of GC patients[3]. However,
several prognostic factors related to GC have been proposed, which include peripheral blood-derived
inflammation markers, such as neutrophil-lymphocyte ratio (NLR), platelet lymphocyte ratio (PLR),
systemic immune-inflammation index (SII), C-reactive protein/albumin (CRP/Alb) ratio, and Glasgow
Prognostic Score (GPS)[4–6]. Some studies have combined the TNM staging system with GC related risk factors to improve the
accuracy of the long-term prognosis of the disease[7]. Routine peripheral blood-derived inflammation
markers are closely associated with the pathogenesis of GC[8]. Moreover, the use of these markers as
prognostic factors is advantageous because most of the peripheral blood-derived inflammation markers
belong to the routine test items; the test cost is cheap and does not require special equipment. Page 3/20 Page 3/20 Park indicated that preoperative body mass index (BMI) and prognostic nutritional index (PNI), as well as
their postoperative changes, are related to the prognosis of stage II/III GC[9]. Also, Jagadesham reported
that the combination of one or more markers of systemic inflammation could precisely predict the
prognosis of advanced adenocarcinoma of the esophagus[10]. Studies have suggested that combining
NLR and PLR could significantly improve the accuracy of predicting the first-line chemosensitivity in
AGC[11]. Contrarily, Xu et al. indicated that CRP/Alb might be a promising predictor of early recurrence
(recurrence within 12 months after radical gastrectomy) and postoperative chemotherapy in stage III
GC[12]. Unfortunately, most of these findings are based on small sample retrospective studies with insufficient
evidence, which could be the reason for the inconsistencies among the various reports. Herein, we
designed a prospective observational cohort to examine the relationship between peripheral blood-derived
inflammation markers and the prognosis of GC. Also, we hypothesized that derangements in one or more
systemic inflammation markers may be associated with poor disease outcomes and the ineffectiveness
of chemotherapy. Follow-up All patients were followed up every 3–6 months for the first two years and annually thereafter until death
or at least five years after surgery. Disease-free survival (DFS) was defined as the time from surgery to
death, loco-regional recurrence or distant recurrence. Disease-specific survival (DSS) was defined as the
time from surgery to death as a result of GC. Patients for whom none of these events was recorded were
censored at the date of their last known contact. The median follow-up time for the entire cohort was
50 months (range 4–83 months), and follow-up of all patients was stopped in December 2019. All patients were followed up every 3–6 months for the first two years and annually thereafter until death
or at least five years after surgery. Disease-free survival (DFS) was defined as the time from surgery to
death, loco-regional recurrence or distant recurrence. Disease-specific survival (DSS) was defined as the
time from surgery to death as a result of GC. Patients for whom none of these events was recorded were
censored at the date of their last known contact. The median follow-up time for the entire cohort was
50 months (range 4–83 months), and follow-up of all patients was stopped in December 2019. Statistical analysis Continuous variables were compared using the independent sample t-test or Wilcoxon rank-sum test,
whereas categorical variables were compared using Pearson’s chi-squared test or Fisher’s exact test, as
appropriate. The receiver operating characteristics (ROC) curve was calculated, and the Youden index
(maximum = sensitivity + specificity − 1) was used to determine the optimal cutoff value for the number
of lymph nodes retrieved, PNI, fibrinogen, NLR, PLR, SII, and CRP/Alb. The potentially relevant factors
obtained from the univariate analysis were assessed in the multivariate model using Cox’s regression. Hazard ratios (HR) and 95% confidence intervals (CI) were calculated. The DFS and DSS were calculated
using the Kaplan-Meier method, and the log-rank test was employed to determine the significance. All
statistical tests were performed 2-sided, and a p < 0.05 was considered statistically significant. All the
analyses were performed using SPSS software (version 25.0, SPSS Inc. IL, USA). Systemic inflammatory markers and histological examination The whole blood and clinicopathological data were obtained one week before initial treatment. Blood
samples were collected for routine laboratory tests, which included complete blood count, CRP, albumin,
fibrinogen, and tumor markers, such as carcinoembryonic antigen (CEA). The following common
peripheral blood-derived inflammation markers were included based on previous studies: NLR, PLR, SII,
CRP/Alb, GPS, modified Glasgow Prognostic Score (mGPS), PNI, and prognostic index (PI). All surgical resection specimens were assessed according to the handling guideline of the third edition of
the Japanese classification of gastric carcinoma. And the staging was conducted by pathologists using
the 8th edition of the UICC/AJCC TNM staging system[3]. Page 4/20 Results A total of 598 patients were recruited from January 2013 to December 2016. The 5-year DFS and DSS
rates of all patients were 72.6% and 75.4%, respectively. Male patients were about twice as many as
female patients, and the tumors in the distal stomach accounted for 77% of all the tumors. Concerning
the GC staging, patients with GC stage I, II, and III were 119, 113, and 366, respectively. The 5-year DFS
and DSS rates after surgery for stage I patients were 97% and 98%, respectively, whereas, for stage II
patients, the rates were 81.4% and 85.8%, respectively. For stage III patients, the rates were 52.1% and
55.6%, respectively (Supplementary Table 1). A total of 376 patients received adjuvant chemotherapy, of which 239 received SOX regimen [14] and 112
XELOX[15]. The remaining 25 patients received other chemotherapy treatments[16]. Optimal cutoff analysis The optimal cutoff value of the number of lymph nodes retrieved, PNI, fibrinogen, NLR, PLR, SII, and
CRP/Alb were 30, 49.5, 397ng/dl, 2.5, 154, 556, and 0.05, respectively. Risk factors for advanced gastric cancer Considering the close correlation between the TNM stage and the 5-year DFS, the number of lymph nodes
retrieved may be related to the prognosis. Lymph nodes less than 16 is considered inadequate lymph
node dissection, especially for AGC. Therefore, only patients with the AGC lymph nodes ≥16 were
included in the analysis. Multivariate analysis showed that age, PLR, and TNM stages were associated
with 5-year DFS. Also, age, PLR, TNM stage, and chemotherapy were associated with 5-year DSS (Table
2). Clinicopathological factors and survival analysis Clinicopathological factors and survival analysis Page 5/20 Page 5/20 A total of 23 potential risk factors were selected. According to univariate analysis, the following 17
clinicopathological characteristics were found to be significantly associated with the 5-year DFS rate in
all enrolled patients: age, tumor location, type of gastrectomy, tumor size, perineural invasion,
lymphovascular invasion, T stage, N stage, chemotherapy, PNI, fibrinogen, NLR, PLR, SII, GPS, CRP/Alb,
and CEA (Table 1). Multivariate Cox proportional hazards model analysis showed that age, T stage, N
stage, number of lymph nodes retrieved, and PLR were independent prognostic factors for GC (Table 1). Risk factors associated with stage II and III gastric cancer Kaplan-Meier curves were used to determine the long-term disease outcomes of GC patients in stage II
and stage III. In GC stage II, the 5-year DFS rates were similar among patients with age ≤60 and age >60
(p=0.213). However, in stage III, the 5-year DFS rate of GC patients aged >60 was worse than those aged
≤60 (p=0.016). Similarly, in GC stage III, the 5-year DFS rate of patients with PLR ≥154 was worse than
those with PLR <154 (p<0.001). But this phenomenon was not observed in GC stage II (p=0.153). The
effect of chemotherapy on the prognosis of patients with stage II GC was not statistically significant
(p=0.260). In contrast, chemotherapy significantly improved the 5-year DFS in patients with stage III GC
(p=0.017) [Figure 1]. With the 5-year DSS as a long-term prognostic index, age, PLR, and chemotherapy had the same effect on
the prognosis of stage II and III GC patients as 5-year DFS, except that chemotherapy also improved the 5-
year DSS of stage II GC patients (p=0.033) [Figure 2]. Combined with the above observations, PLR was found to be an independent risk factor for the prognosis
of AGC. Patients with AGC were divided into the PLR <154 and PLR ≥154 subgroups. Further analysis of
the clinicopathological factors between the two groups revealed that tumor lesions in the PLR <154 group
were smaller than those in the PLR ≥154 group. But, there was no other difference between the groups
(Table 3). Further analysis of the relationship between chemotherapy and PLR showed that
chemotherapy improved the long-term prognosis of patients in the PLR ≥154 group, including 5-year DFS
and DSS (p=0.026, p=0.014, respectively). Notably, chemotherapy had no significant effect on the long-
term prognosis of patients in the PLR <154 group (Figure 3). Discussion Page 6/20 Many studies have been conducted to investigate the correlation between peripheral blood-derived
inflammation markers and tumor prognosis[17, 18]. Liu showed that CRP/Alb was an independent
prognostic marker for patients with ovarian cancer[19]. Also, the NLR and PLR are prognostic factors in
patients with non-small cell lung cancer after stereotactic radiation therapy[20]. The independent risk
factors for poor GC prognosis include NLR, PLR, fibrinogen, PNI, GPS, CRP/Alb, among others. Also, some
studies have combined these systemic inflammatory markers with or without TNM stage to provide new
prognostic tools[21–23]. However, most of these studies were retrospective, and reported inconsistent
results, particularly on the significance of each inflammatory index and the threshold value. Therefore, in addition to the peripheral blood-derived inflammation markers reported in previous studies,
this study further explored the prognostic value of some conventional systemic inflammatory marker in
patients with GC. We prospectively analyzed 598 GC patients after radical surgery and found that
independent risk factors for poor prognosis of GC included age, T stage, N stage, number of lymph nodes
retrieved, and PLR. Currently, TNM staging is the standard prognostic tool for GC. Given the excellent
prognosis of early gastric cancer, our focus was to analyze the prognosis of patients with AGC (stage
II/III). Many studies have demonstrated that the number of lymph node dissection has an impact on the
prognosis of GC; therefore, we further excluded patients with an inadequate amount of lymph node
dissection (less than 16)[24–26]. Independent risk factors for AGC included age, PLR, TNM stages, and chemotherapy. Previous studies
have investigated the effect of age on GC prognosis; however, most of these studies found no significant
association between age and GC prognosis[27–29]. Takatsu analyzed 5000 GC cases and found that
early-onset GC (age < 40 years) was likely to present lymph node metastases. But the survival rate of
young GC patients was equivalent to that of older GC patients (age ≥ 60 years)[30]. In the present study,
older GC patients (age > 60 years) had a worse prognosis, which was closely related to tumor recurrence. Nutritional status is associated with survival in patients with malignant tumors, including GC. Nutritional status is associated with survival in patients with malignant tumors, including GC. Preoperative underweight and low PNI are considered poor prognostic factors. Park suggested that
careful nutritional intervention after surgery could improve the survival rate[9]. Discussion Besides, a meta-analysis
concluded that a low PNI is significantly associated with poor overall survival except for stage IV GC[31]. However, consistent with the results of Li et al.[32], we observed that PNI was not associated with
prognosis. Also, our results did not show a correlation between fibrinogen and the prognosis of GC. Recent studies have shown that fibrinogen is one of the risk factors for poor prognosis in upper gastric
cancer[33]. Fibrinogen is the primary acute-phase protein, and as a critical component of the hemostatic
system, it regulates the systemic inflammatory state and cancer progression. However, its clinical
significance in the prognosis of GC has not been elucidated. The NLR and PLR are the most extensively studied markers of peripheral blood-derived inflammation,
which are associated with the prognosis of GC. Accumulating evidence has shown that NLR and PLR are
associated with distant metastases during GC progression[34, 35]. Kim reported that although both the
PLR and NLR could reflect the prognosis, the NLR was more predictive of overall survival than the PLR in Page 7/20 Page 7/20 GC[36]. Also, they suggested that NLR and PLR might be associated with lymph node metastasis in early
gastric cancer[37]. On the contrary, Zhu et al. indicated that NLR and PLR could not predict lymph node
metastasis and prognosis in early gastric cancer[38]. In the present study, PLR was significantly
correlated with the prognosis of GC, but there was no statistical difference between NLR and prognosis of
GC. This observation seems to be inconsistent with previous studies, but the exact mechanism is still
unclear. However, the inconsistencies could be because most previous studies focused on overall survival
as the primary outcome, whereas herein, we used tumor recurrence and tumor-related mortality as
observational indicators, which seem to be more accurate. In addition, the clinicopathological
characteristics were similar between the PLR elevating group (PLR < 154) and the PLR decreasing group
(PLR ≥ 154), except for tumor size, which further suggested that PLR might influence the prognosis of
tumor through other mechanisms. A recent meta-analysis has revealed that PLR is associated with
prognosis of GC[39]. Abnormal levels of CRP and Alb have been related to poor prognosis of tumor patients. It is noteworthy
that the combinations of these two indicators can enhance the accuracy to predict the recurrence of
multiple tumors. Among them, the most common evaluation criteria after combination include GPS and
CRP/Alb. Discussion Besides, many studies have used GPS to predict the prognosis of various tumors, including GC. Hsueh recently recommended the use of GPS as a predictive and prognostic factor in patients with GC. A
significant correlation was observed between the GPS, short-term postoperative complications, and long-
term survival outcomes in patients with GC undergoing D2 gastrectomy[40]. Some studies have indicated
that GPS and mGPS, used either alone or in combination, represent an independent prognostic factor for
long-term outcome in resected GC[41, 42]. However, Walsh’s results did not show a correlation between
prognosis of patients and mGPS levels, although mGPS was associated with advanced GC stage[17]. Liu
et al. retrospectively analyzed 455 patients with resectable GC and showed that CRP/Alb, rather than GPS
and mGPS, was associated with overall survival[43]. Similarly, Xu[12] and Lu[44] et al. also observed that
CRP/Alb and CRP/prealbumin were associated with recurrence of GC based on the data from a phase III
randomized clinical trial. On the contrary, our results showed that although CRP/Alb and GPS were
associated with prognosis in the univariate analysis, the multivariate analysis showed that both were not
related to long-term survival. Consistent with the results of previous clinical trials, our findings showed that chemotherapy could
significantly improve the prognosis of AGC, especially for patients with stage III GC[45, 46]. Moreover,
many studies have investigated the correlation between peripheral blood-derived inflammation markers
and the effects of chemotherapy, to guide the selection of chemotherapy-sensitive patients. A study
suggested that the sensitivity of chemotherapy (oxaliplatin/5-fluorouracil combination) might be closely
related to NLR, PLR, and their changes in metastatic gastric cancer[47]. Hirahara believed that the
combination of NLR and PLR might be more effective in predicting the chemotherapy response in
patients with metastatic gastric cancer[48]. Besides, Tang[49] and Chen[50] concluded that PLR could
predict the efficacy of neoadjuvant chemotherapy of GC patients treated with oxaliplatin and
capecitabine regimens. In the current study, PLR effectively predicted adjuvant chemotherapy
(oxaliplatin/5-fluorouracil combination) response in patients with AGC after surgery. For patients with Page 8/20 Page 8/20 PLR ≥ 154, chemotherapy significantly improved long-term survival, including DFS and DSS; however,
patients with PLR < 154 did not benefit from adjuvant chemotherapy. Thus, we recommend that AGC
patients with PLR ≥ 154 should actively receive adjuvant chemotherapy (oxaliplatin/5-fluorouracil
combination) after surgery, whereas patients with PLR < 154 need to be cautious when choosing adjuvant
chemotherapy. Conclusion Our findings show that PLR is significantly correlated with the prognosis of GC, especially for stage III. That is, higher PLR (≥154) is an independent risk factor for poor long-term survival rate of GC patients. Moreover, PLR can be used to predict adjuvant chemotherapy (oxaliplatin/5-fluorouracil combination)
response in patients with GC after surgery. Discussion PLR ≥ 154, chemotherapy significantly improved long-term survival, including DFS and DSS; however,
patients with PLR < 154 did not benefit from adjuvant chemotherapy. Thus, we recommend that AGC
patients with PLR ≥ 154 should actively receive adjuvant chemotherapy (oxaliplatin/5-fluorouracil
combination) after surgery, whereas patients with PLR < 154 need to be cautious when choosing adjuvant
chemotherapy. This study systematically evaluated the relationship between peripheral blood-derived inflammation
markers and the prognosis of GC. Notably, the potential ability of inflammatory markers to predict the
effects of chemotherapy was further demonstrated. However, this study had a few limitations. Importantly, this was an observational study and was therefore influenced by other confounding factors. Also, the sample size was relatively small, and this may have reduced the reliability of the findings. Thus,
these results need to be further validated by large multicenter randomized clinical trials. Conflict of interest Conflict of interest The authors declare no conflict of interest. Abbreviations GC, gastric cancer; AGC, advanced gastric cancer; UICC, International Union Against Cancer; AJCC,
American Joint Committee on Cancer; NLR, neutrophil-lymphocyte ratio; PLR, platelet lymphocyte ratio;
SII, systemic immune-inflammation index; CRP/Alb, C-reactive protein/albumin ratio; GPS, Glasgow
Prognostic Score; mGPS, modified Glasgow Prognostic Score; BMI, body mass index; PNI, prognostic
nutritional index; PI, prognostic index; DFS, disease-free survival; DSS, disease-specific survival; ROC,
receiver operating characteristics; HR, hazard ratios; CI, confidence intervals Financial support This study was funded by the Key Laboratory of Diagnosis and Treatment of Digestive System Tumors of
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PLR<154
PLR≥154
p value
Age (year)
0.223
≤60
78
55
>60
110
103
Gender
0.818
Male
128
105
Female
60
53
Tumor location
0.941
Upper third
26
47
Middle third
23
17
Lower third
133
116
Two-thirds or more
6
4
Tumor size
<0.001
≤5cm
135
73
>5cm
53
85
Histologic type
0.156
Differentiated
74
75
Undifferentiated
114
83
Perineural invasion
0.194
Absence
79
78
Presence
109
80
Lymphovascular invasion
0.509
Absence
77
59
Presence
111
99
T category
0.835
T1
7
4
T2
15
10
T3
7
6
T4a
153
135
T4b
6
3
N category
0.416
N0
35
28
N1
39
29
N2
58
41
N3a
45
43
N3b
11
17
CEA
0.275
≤5 ng/mL
166
133
>5 ng/mL
22
25
PLR Platelet-lymphocyte ratio patients characteristics
PLR<154
PLR≥154
p value
Age (year)
0.223
≤60
78
55
>60
110
103
Gender
0.818
Male
128
105
Female
60
53
Tumor location
0.941
Upper third
26
47
Middle third
23
17
Lower third
133
116
Two-thirds or more
6
4
Tumor size
<0.001
≤5cm
135
73
>5cm
53
85
Histologic type
0.156
Differentiated
74
75
Undifferentiated
114
83
Perineural invasion
0.194
Absence
79
78
Presence
109
80
Lymphovascular invasion
0.509
Absence
77
59
Presence
111
99
T category
0.835
T1
7
4
T2
15
10
T3
7
6
T4a
153
135
T4b
6
3
N category
0.416
N0
35
28
N1
39
29
N2
58
41
N3a
45
43
N3b
11
17
CEA
0.275
≤5 ng/mL
166
133
>5 ng/mL
22
25 PLR, Platelet-lymphocyte ratio PLR, Platelet-lymphocyte ratio Tables Table 1-1 Univariate and multivariate analysis of prognostic factors in patients with gastric cancer Page 14/20 Page 14/20 Page 14/20 Page 14/20 Clinicopathological
Univariate analysis
Multivariate analysis
feature
HR
95% CI
p value
HR
95% CI
p value
Age (years)
≤60
1
1
>60
1.62
1.16-2.25
0.005
1.63
1.15-2.31
0.007
Gender
Male
1
Female
1.01
0.73-1.40
0.967
BMI
≤24 kg/m2
1
>24 kg/m2
0.77
0.52-1.13
0.175
Tumor location
Upper third
1
Middle third
0.53
0.29-0.98
0.041
Lower third
0.47
0.31-0.71
<0.001
Two-thirds or more
0.76
0.27-2.15
0.600
Gastrectomy
Distal
1
Total
1.57
1.10-2.23
0.013
Proximal
NA
Tumor size
≤5cm
1
>5cm
2.60
1.90-3.54
<0.001
Histologic type
Differentiated
1
Undifferentiated
1.29
0.95-1.76
0.106
Perineural invasion
Absence
1
Presence
2.73
2.00-3.72
<0.001
Lymphovascular invasion
Absence
1
Presence
2.78
2.02-3.83
<0.001
T category
T1
1
1
T2
4.30
1.56-11.83
0.005
3.04
1.08-8.57
0.036
T3
2.04
0.25-16.92
0.510
1.69
0.20-14.50
0.633
T4a
15.79
6.97-35.76
<0.001
8.22
3.26-20.75
<0.001
T4b
28.21
9.78-81.36
<0.001
13.05
3.98-42.82
<0.001
N category
N0
1
1
N1
3.22
1.76-5.90
<0.001
1.70
0.88-3.26
0.112
N2
4.71
2.75-8.05
<0.001
2.09
1.15-3.81
0.016
N3a
11.35
6.82-18.89
<0.001
5.38
3.00-9.65
<0.001
N3b
24.19
13.27-44.07
<0.001
11.01
5.50-22.04
<0.001
lti
i t
l
i
f
ti
f
t
i
ti
t
ith
t i Page 15/20 Clinicopathological
Univariate analysis
Multivariate analysis
feature
HR
95% CI
p value
HR
95% CI
p value
Chemotherapy
No
1
1
Yes
1.58
1.12-2.22
0.009
0.70
0.48-1.01
0.059
Number of lymph nodes retrieved
≤15
1
1
16-29
0.75
0.50-1.13
0.169
0.46
0.30-0.70
<0.001
≥30
1.36
0.88-2.11
0.166
0.52
0.32-0.84
0.008
PI
0
1
1
1.54
0.96-2.49
0.075
PNI
<49.5
1
≥49.5
0.40
0.27-0.59
<0.001
Fibrinogen
<397 ng/dl
1
≥397 ng/dl
2.06
1.51-2.81
<0.001
NLR
<2.5
1
1
≥2.5
1.69
1.24-2.31
0.001
0.70
0.48-1.01
0.059
PLR
<154
1
1
≥154
2.21
1.62-3.01
<0.001
1.70
1.20-2.42
0.003
SII
<556
1
≥556
1.94
1.43-2.64
<0.001
GPS
0
1
1
1.76
1.24-2.49
0.002
2
1.89
1.04-3.43
0.037
mGPS
0
1
1
1.53
0.68-3.46
0.307
2
1.66
0.92-3.00
0.091
CRP/Alb
<0.05
1
≥0.05
1.91
1.40-2.60
<0.001
CEA
≤5 ng/mL
1
>5 ng/mL
1.76
1.16-2.68
0.008 HR, hazard ratios; CI, confidence interval; BMI, Body mass index; PNI, Prognostic nutritional index; PI, Prognostic index; GPS, Glasgow Prognostic
Score; mGPS, Modified Glasgow Prognostic Score; NLR, Neutrophil-lymphocyte ratio; PLR, Platelet-lymphocyte ratio; CRP/Alb, C-reactive
protein/albumin; SII, Systemic immune-inflammatory index Page 16/20
Table 2 Multivariate analysis of prognostic factors in patients with advanced gastric cancer
Clinicopathological
Multivariate analysis DFS
Multivariate analysis DSS
feature
HR
95% CI
p value
HR
95% CI
p value
Age (years)
≤60
1
1
>60
1.61
1.10-2.35
0.014
1.61
1.06-2.42
0.024
PLR
<154
1
1
≥154
2.00
1.40-2.84
<0.001
2.09
1.43-3.04
<0.001
TNM
II
1
1
III
4.46
2.51-7.91
<0.001
5.94
3.01-11.74
<0.001
Chemotherapy
No
1
1
Yes
0.70
0.48-1.04
0.074
0.66
0.45-0.99
0.043
DFS, disease-free survival; DSS, disease-specific survival; HR, hazard ratios; CI, confidence interval; PLR, platelet-lymphocyte ratio Table 2 Multivariate analysis of prognostic factors in patients with advanced gastric cancer Page 16/20
DFS, disease-free survival; DSS, disease-specific survival; HR, hazard ratios; CI, confidence interval; PLR, platelet-lymphocyte ratio P
16/20
DFS, disease-free survival; DSS, disease-specific survival; HR, hazard ratios; CI, confidence interval; PLR, platelet-lymphocyte ratio Table 3. Figure 3 Comparison of survival curves between patients with or without chemotherapy in advanced gastric
cancer. (A) PLR <154; (B) PLR ≥154. SupplementaryTable1.docx Figures Page 17/20 Page 17/20 Page 17/20 Figure 1
Disease-free survival (DFS) of patients with advanced gastric cancer according to the risk factors. (A)
stage II; (B) stage III. Figure 1 Disease-free survival (DFS) of patients with advanced gastric cancer according to the risk factors. (A)
stage II; (B) stage III. (A) Page 18/20 Figure 2
Disease-specific survival (DSS) of patients with advanced gastric cancer according to the risk factors. (
stage II; (B) stage III. Figure 2 Disease-specific survival (DSS) of patients with advanced gastric cancer according to the risk factors. (A)
stage II; (B) stage III. (A) Page 19/20 Figure 3
Comparison of survival curves between patients with or without chemotherapy in advanced gastric
cancer. (A) PLR <154; (B) PLR ≥154. Figure 3 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryTable1.docx Page 20/20
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https://openalex.org/W2069342665
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https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0002451&type=printable
|
English
| null |
Inducible Ablation of Melanopsin-Expressing Retinal Ganglion Cells Reveals Their Central Role in Non-Image Forming Visual Responses
|
PloS one
| 2,008
|
cc-by
| 9,780
|
Abstract * E-mail: satchin@salk.edu ¤ Current address: Kyorin University, Tokyo, Japan ¤ Current address: Kyorin University, Tokyo, Japan completely abolished in mice that lack both melanopsin and
functional
outer
retina
photoreceptors,
thus
establishing
a
dominant role of melanopsin photopigment in these processes
[12,13]. Yet, the AOPs are attenuated, not eliminated, in
melanopsin deficient (Opn42/2) mice, an observation that suggests
a role for rod/cone photoreceptors in AOPs [14–16]. The
mRGCs develop normally in Opn42/2 mice and make normal
monosynaptic projections to the suprachiasmatic nucleus (SCN)
and olivary pretectal nucleus (OPN) which regulate the circadian
behavior and pupillary constriction, respectively [14]. Both the
SCN and OPN also receive direct and indirect projections from
RGCs that do not express melanopsin [17]. Therefore, it is
unclear whether rod/cone-initiated light signal is transmitted
primarily via the mRGCs or via other RGCs to brain regions that
regulate
AOPs. Additionally,
the
mRGCs
have
also
been
suggested to play a role in modulating rod/cone initiated image-
forming functions [18,19]. Megumi Hatori1, Hiep Le1, Christopher Vollmers1, Sheena Racheal Keding1, Nobushige Tanaka1¤,
Christian Schmedt2, Timothy Jegla3, Satchidananda Panda1* 1 The Salk Institute for Biological Studies, La Jolla, California, United States of America, 2 Genomics Institute of Novartis Research Foundation, San Diego, California, United
States of America, 3 Department of Cell Biology and Institute for Childhood and Neglected Diseases, The Scripps Research Institute, La Jolla, California, United States of
America PLoS ONE | www.plosone.org Abstract Rod/cone photoreceptors of the outer retina and the melanopsin-expressing retinal ganglion cells (mRGCs) of the inner
retina mediate non-image forming visual responses including entrainment of the circadian clock to the ambient light, the
pupillary light reflex (PLR), and light modulation of activity. Targeted deletion of the melanopsin gene attenuates these
adaptive responses with no apparent change in the development and morphology of the mRGCs. Comprehensive
identification of mRGCs and knowledge of their specific roles in image-forming and non-image forming photoresponses are
currently lacking. We used a Cre-dependent GFP expression strategy in mice to genetically label the mRGCs. This revealed
that only a subset of mRGCs express enough immunocytochemically detectable levels of melanopsin. We also used a Cre-
inducible diphtheria toxin receptor (iDTR) expression approach to express the DTR in mRGCs. mRGCs develop normally, but
can be acutely ablated upon diphtheria toxin administration. The mRGC-ablated mice exhibited normal outer retinal
function. However, they completely lacked non-image forming visual responses such as circadian photoentrainment, light
modulation of activity, and PLR. These results point to the mRGCs as the site of functional integration of the rod/cone and
melanopsin phototransduction pathways and as the primary anatomical site for the divergence of image-forming and non-
image forming photoresponses in mammals. Citation: Hatori M, Le H, Vollmers C, Keding SR, Tanaka N, et al. (2008) Inducible Ablation of Melanopsin-Expressing Retinal Ganglion Cells Reveals Their Central
Role in Non-Image Forming Visual Responses. PLoS ONE 3(6): e2451. doi:10.1371/journal.pone.0002451 Editor: Michael Hendricks, Temasek Life Sciences Laboratory, Singapore Received April 25, 2008; Accepted May 14, 2008; Published June 11, 2008 ori 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. pyright: 2008 Hatori 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. Funding: MH, HL, CV, SK, and SP were supported by Pew Scholars Program in Biomedical Sciences, Dana Foundation, Whitehall Foundation and NIH grant
EY016807. CS and TJ were supported by funding from GNF. The funding agencies had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Inducible Ablation of Melanopsin-Expressing Retinal
Ganglion Cells Reveals Their Central Role in Non-Image
Forming Visual Responses Megumi Hatori1, Hiep Le1, Christopher Vollmers1, Sheena Racheal Keding1, Nobushige Tanaka1¤,
Christian Schmedt2, Timothy Jegla3, Satchidananda Panda1* Introduction The rod/cone photoreceptors of the outer retina signal via
multisynaptic pathways to the retinal ganglion cells (RGCs) of the
inner retina. The RGCs, in turn, transmit the visual information to
the brain via their axonal projections. A small subset of RGCs
exclusively expresses the functional photopigment melanopsin
(OPN4) and is intrinsically photosensitive, but also receives rod/
cone inputs (Figure 1A) [1-5]. These mRGCs, along with the rod/
cone photoreceptors, mediate several non-image forming, or
adaptive ocular photoresponses (AOPs), which help organisms
optimize their physiological performance in variable ambient light
conditions. These AOPs include rapid adjustment of pupil size,
modulation of general activity and endocrine function, and tuning
of the phase and period length of the circadian clock to adapt to
the light environment (reviewed in [6]). Mouse genetics has established the complementary roles of both
rod/cone and melanopsin in these AOPs. Mice with outer retinal
degeneration (rd/rd) retain functional mRGCs and exhibit almost
intact AOPs [7–11]. However, these adaptive responses are To understand the specific role of mRGCs in both image-
forming and adaptive ocular photoresponses, we generated mice
expressing Cre-inducible diphtheria toxin receptor exclusively in PLoS ONE | www.plosone.org June 2008 | Volume 3 | Issue 6 | e2451 1 June 2008 | Volume 3 | Issue 6 | e2451 Melanopsin RGC Ablation Figure 1. Strategy for fluorescent labeling or inducible ablation of mRGC lineage in the mouse retina. (A) The cellular circuitry
underlying the rod/cone and mRGC contribution to visual responses. Thickness of the filled arrows roughly highlights the relative strength of
information flow. (B) Strategy to fluorescently label mRGCs by breeding a mouse carrying a Cre recombinase ‘‘knocked-in’’ to the melanopsin
promoter to Z/EG mouse that allows Cre-dependent expression of GFP from chicken beta-actin promoter. (C) Strategy to achieve inducible and
specific ablation of mRGCs. Opn4Cre/+ mouse was bred to a mouse expressing Cre-dependent expression of simian diphtheria toxin receptor. The
resulting progeny develop normal mRGCs expressing DTR, which allows specific ablation of these cells by DT administration. Schematic of two
targeting vectors used to achieve inducible mRGC ablation are shown in (D). The Cre knock-in cassette for targeted insertion to melanopsin locus also
carried coding sequences for CRE dependent expression of bTau-eYFP. However, fluorescence from bTau:eYFP was undetectable in retina from
Opn4Cre/+ mice (data not shown). The targeting vector and generation of R26iDTR/+ mice are described in [20]. Introduction A schematic of the targeting vector is
shown here. doi:10.1371/journal.pone.0002451.g001 Figure 1. Strategy for fluorescent labeling or inducible ablation of mRGC lineage in the mouse retina. (A) The cellular circuitry
underlying the rod/cone and mRGC contribution to visual responses. Thickness of the filled arrows roughly highlights the relative strength of
information flow. (B) Strategy to fluorescently label mRGCs by breeding a mouse carrying a Cre recombinase ‘‘knocked-in’’ to the melanopsin
promoter to Z/EG mouse that allows Cre-dependent expression of GFP from chicken beta-actin promoter. (C) Strategy to achieve inducible and
specific ablation of mRGCs. Opn4Cre/+ mouse was bred to a mouse expressing Cre-dependent expression of simian diphtheria toxin receptor. The
resulting progeny develop normal mRGCs expressing DTR, which allows specific ablation of these cells by DT administration. Schematic of two
targeting vectors used to achieve inducible mRGC ablation are shown in (D). The Cre knock-in cassette for targeted insertion to melanopsin locus also
carried coding sequences for CRE dependent expression of bTau-eYFP. However, fluorescence from bTau:eYFP was undetectable in retina from
Opn4Cre/+ mice (data not shown). The targeting vector and generation of R26iDTR/+ mice are described in [20]. A schematic of the targeting vector is
shown here. doi:10 1371/journal pone 0002451 g001 doi:10.1371/journal.pone.0002451.g001 the mRGC lineage. Diphtheria toxin (DT) crosses the blood-brain
barrier after systemic injection and has been shown to trigger cell
death in neurons expressing a primate DTR without triggering a
significant immune response [20,21]. This strategy allows normal
embryonic and postnatal differentiation and development of the
target cell type which can be verified in adult mice. Subsequent acute
cell ablation with local or systemic DT administration circumvents
any potential developmental compensation. Systemic DT injection
in the adult mice with DTR expressing mRGCs triggers a profound
loss of mRGCs. The mRGC-ablated mice lose non-image forming
visual responses while maintaining largely intact image-forming
functions. This demonstrates a central role of the mRGCs as the site
of integration of melanopsin and rod/cone initiated photoresponses
for generating adaptive light responses in mammals. (GFP) from a strong b-actin promoter, such that GFP is uniformly
expressed in all mRGCs irrespective of the heterogeneity in the
level of transcription from the native melanopsin locus or in the level
of immunologically detectable melanopsin protein. In the retina of
adult Opn4Cre/+;Z/EG mice, GFP expressing cells were mostly
found in the RGC sub-layer, and these cells had extensive
dendritic arborization characteristic of the mRGCs. Introduction An average of
131 GFP expressing cells/mm2 (625.4, SD, n = 3) were found in
these retina, 42.6% of which also expressed immunologically
detectable levels of melanopsin (Figures 2A–2C). These cells with
detectable levels of melanopsin protein likely represent the M1
type mRGCs [23]. The second group of GFP positive mRGCs
may express very low level of melanopsin representing the M2 type
of mRGCs [23], or may represent cells where melanopsin
promoter is almost silent in adulthood. A small number of RGCs
stained positive for melanopsin, but showed no detectable level of
GFP fluorescence. This may represent cells with insufficient Cre
expression, Cre activity, or GFP level. In summary, GFP
expression
pattern
in
Opn4Cre/+;Z/EG
mice
established
(a)
restricted expression of melanopsin in RGC layer and (b) sufficient
CRE activity in vast majority of both M1 and M2 type mRGCs. Fluorescent tagging of mRGCs To comprehensively tag mRGCs with transcriptionally active
melanopsin locus, we generated a mouse line which carries a Cre
recombinase and a Cre-dependent bTau-Yellow Fluorescent
Protein (YFP) expression cassette knocked-in to the native
melanopsin locus (Figure 1). However, no detectable YFP expression
was found in the retina of Opn4Cre/+ mouse, which may be due to
weak transcriptional activity from the native melanopsin promoter
and/or low expression of the second transcript downstream of an
internal ribosomal entry site (IRES) cassette. Next, we bred the
Opn4Cre/+ mouse with Z/EG mouse (Figure 1B) [22]. This strategy
allows Cre-dependent expression of green fluorescent protein Specific ablation of mRGCs in adult mice (F) Average melanopsin immunoreactive cell density in
WT and Opn4Cre/+;R26iDTR/+ retina was comparable. Two weeks after DT administration, the number of cells in WT retina remained unchanged, while
that in Opn4Cre/+;R26iDTR/+ retina reduced from 62.3 mm22 to 2.8 mm22. No melanopsin immunostaining was observed in Opn42/2 mice. It is
important to note that retina from all DT-treated mice tested by immunostaining still retained a few melanopsin staining cells (arrows), implying
incomplete expression of Cre in all mRGCs and/or insufficient level of bioavailable DT. Average cell counts (+SEM, n = 3 to 5 retinas) from each
genotype/treatment group are shown. Significant difference in cell numbers (Student’s t test, p,0.05) between mRGCs without and with DT was
highlighted with an asterisk. doi:10.1371/journal.pone.0002451.g002 Specific ablation of mRGCs in adult mice To achieve inducible ablation of mRGC lineage, we bred the
Opn4Cre/+ mouse with a mouse strain in which the inducible
diphtheria toxin receptor (simian Hbegf) (iDTR) is knocked-in to
the ROSA26 locus (R26iDTR). The iDTR can only be expressed
after Cre-mediated excision of a transcriptional STOP cassette
[20] (Figures 1C and 1D). As melanopsin function is haplosuffi- PLoS ONE | www.plosone.org June 2008 | Volume 3 | Issue 6 | e2451 2 Melanopsin RGC Ablation Figure 2. Cre-dependent GFP labeling and inducible ablation of mRGCs. Retina of adult Opn4Cre/+;Z/EG mouse probed with (A) anti-OPN4 or
(B) anti-GFP antibodies show staining of a small subset of cells. (C) Only a subset of GFP positive cells also stained with anti-OPN4 antibody. A section
of the flat mount retina containing a cell (marked with an arrow) that stained with melanopsin antibody, but did not express detectable level of GFP
is shown. (D) Retina of Opn4Cre/+;R26iDTR/+ showed normal melanopsin immunostaining in a small fraction of RGCs. (E) Two weeks after DT
administration, the number of melanopsin-immunoreactive cells were significantly reduced. (F) Average melanopsin immunoreactive cell density in
WT and Opn4Cre/+;R26iDTR/+ retina was comparable. Two weeks after DT administration, the number of cells in WT retina remained unchanged, while
that in Opn4Cre/+;R26iDTR/+ retina reduced from 62.3 mm22 to 2.8 mm22. No melanopsin immunostaining was observed in Opn42/2 mice. It is
important to note that retina from all DT-treated mice tested by immunostaining still retained a few melanopsin staining cells (arrows), implying
incomplete expression of Cre in all mRGCs and/or insufficient level of bioavailable DT. Average cell counts (+SEM, n = 3 to 5 retinas) from each
genotype/treatment group are shown. Significant difference in cell numbers (Student’s t test, p,0.05) between mRGCs without and with DT was
highlighted with an asterisk. doi:10.1371/journal.pone.0002451.g002 Figure 2. Cre-dependent GFP labeling and inducible ablation of mRGCs. Retina of adult Opn4Cre/+;Z/EG mouse probed with (A) anti-OPN4 or
(B) anti-GFP antibodies show staining of a small subset of cells. (C) Only a subset of GFP positive cells also stained with anti-OPN4 antibody. A section
of the flat mount retina containing a cell (marked with an arrow) that stained with melanopsin antibody, but did not express detectable level of GFP
is shown. (D) Retina of Opn4Cre/+;R26iDTR/+ showed normal melanopsin immunostaining in a small fraction of RGCs. (E) Two weeks after DT
administration, the number of melanopsin-immunoreactive cells were significantly reduced. PLoS ONE | www.plosone.org Normal image-forming visual responses in mRGC ablated
mice cient in mice [14], this approach allows normal differentiation and
function of the melanopsin cell lineage in the double heterozygote
mice (Opn4Cre/+;R26iDTR/+) and their specific ablation upon DT
administration. Image-forming vision requires normal retina architecture, light-
induced electrical activities in the retina and RGC-mediated
transmission of the light information to the image processing brain
centers. As shown in Figure 3A, DT-induced mRGC ablation had
minimal impact on the overall retina architecture. These mice also
exhibited normal electroretinogram (ERG) (Figure 3B). Rod-
mediated
responses
to
scotopic
illumination,
cone-mediated
responses to photopic illumination after photobleaching of rods
and maximal rod/cone combined responses were comparable in
WT and Opn4Cre/+;R26iDTR/+ mice injected with DT. Finally, to
evaluate pattern forming visual responses, we tested the perfor-
mance of mice in a visual cliff test (Figure 3C). The test evaluates
the ability of the mice to visually discriminate between a ‘‘safe’’
and an ‘‘unsafe’’ landing space and accordingly step down from a
slightly raised platform to the safe area. Typically, mice with intact The retina of 8 week old adult Opn4Cre/+;R26iDTR/+ mice
showed normal stratification and density of melanopsin-immuno-
staining (Figures 2D and 3A). Two weeks after DT administration
(intraperitoneal injections of DT at 50 mg/kg body weight, 2–3
times at 3 d apart) there was a dramatic reduction of mRGCs by
over 90% in Opn4Cre/+;R26iDTR/+(Figure 2E), but not in wild type
(WT) mice carrying only one or no copy of either of the two
transgenes (Figure 2F). The DT administration did not cause any
widespread cell death in the retina as the normal stratification of
the
major
cell
layers
remained
intact
(Figure
3A). This
demonstrated the accessibility of DT across the blood-retina
barrier and the success of the iDTR system in specific ablation of
differentiated RGC subtypes in live mice. PLoS ONE | www.plosone.org June 2008 | Volume 3 | Issue 6 | e2451 June 2008 | Volume 3 | Issue 6 | e2451 3 Melanopsin RGC Ablation Figure 3. mRGC ablation does not alter the normal retina
architecture and image-forming responses. (A) Hematoxylin and
Eosin staining of 5 mm thick paraffin embedded sections of retina from
Opn4Cre/+;R26iDTR/+ mice without and with DT injection. DT application
had no detectable adverse effect on the normal stratification of the
retina (outer segment (OS), outer nuclear layer (ONL), outer plexiform
layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and
ganglion cell layer (GCL)). Normal image-forming visual responses in mRGC ablated
mice (B) Representative full-field ERG of WT and
DT-treated Opn4Cre/+;R26iDTR/+ mice showing rod, cone and maximal
combined responses. Responses from both eyes were simultaneously
measured and plotted. Quantitative analysis of magnitude and timing
of a-wave, b-wave and oscillatory potentials of these two genotype
(3
i
h)
h
d
i
ifi
diff
(d induced damage to the retina and thereby affect image-forming
responses. Loss of circadian photoentrainment in mRGC ablated (A) Hematoxylin and
Eosin staining of 5 mm thick paraffin embedded sections of retina from
Opn4Cre/+;R26iDTR/+ mice without and with DT injection. DT application
had no detectable adverse effect on the normal stratification of the
retina (outer segment (OS), outer nuclear layer (ONL), outer plexiform
layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and
ganglion cell layer (GCL)). (B) Representative full-field ERG of WT and
DT-treated Opn4Cre/+;R26iDTR/+ mice showing rod, cone and maximal
combined responses. Responses from both eyes were simultaneously
measured and plotted. Quantitative analysis of magnitude and timing
of a-wave, b-wave and oscillatory potentials of these two genotype
groups (3 mice each) showed no significant difference (data not
shown). (C) Image forming visual function as assessed by the visual cliff
test was unaffected by mRGC ablation. Average percentage (+SEM,
n = 5 to 13 mice) of positive choice in 10 trials for each mouse are
shown. Mice with outer retina degeneration (rd/rd) made random
choices while stepping down from the platform and were significantly
different (Student’s t test, p,0.05; red asterisk) from the other four
groups. No significant difference in test performance was found among
native or DT-treated WT or Opn4Cre/+;R26iDTR/+ mice. doi:10.1371/journal.pone.0002451.g003 To clearly establish the roles of retina photopigments and
photoreceptor cells in circadian entrainment, we compared the
temporal activity rhythms of DT-treated WT and DT-treated
Opn4Cre/+;R26iDTR/+ with those of rd/rd, Opn42/2 and Opn42/2;rd/rd
mice under three different lighting conditions: LD, DD and LL
(constant light). Mice with complete loss of photopigments or acute
loss of mRGC lineage in adulthood showed a DD period length that
was indistinguishable from that of WT mice, thus implying normal
function of the SCN oscillator sustains without tonic input from the
mRGCs. As shown before [8,15,16] under LD and LL conditions,
the circadian clock in rd/rd and in Opn42/2 mice showed signs of
light input. Both genotypes entrained normally to LD cycle; i.e. they
exhibited 24 h LD period length. As reported earlier [13,15,16],
under constant light, both rd/rd and Opn42/2 mice showed period
lengthening–although the effect was less pronounced in the latter
genotype. A parsimonious interpretation of circadian photoentrain-
ment in melanopsin photopigment deficient (Opn42/2) mice, but not pattern forming vision make correct decisions and step on the safe
side in .70% of trials, while mice lacking image-forming vision
make random choices and choose the safe side in ,50% of trials. Loss of PLR in mRGC ablated mice Both rod/cone photoreceptors and melanopsin play complemen-
tary roles in dynamically adapting the pupil size to ambient light. Specifically, rod/cone photoreceptors mediate response to low light
intensity, while melanopsin partly regulates pupil constriction under
high intensity light conditions [12–14,24]. The OPN, which
mediates PLR, receives a significant number of mRGC projections
[4,17]. To test the role of mRGCs in the non-image forming visual
responses, we measured PLR at 20 mW of monochromatic blue light
(470 nm, 10 nm half-peak width). This intensity of light triggers
,80% pupil constriction in WT mice. Both WT and the
Opn4Cre/+;R26iDTR/+ mice showed comparable constriction to this
light intensity (data not shown). After two doses of DT injection
(50 mg/kg, 3 d apart), WT mice maintain normal pupil constriction. However, during the course of a week following the first DT
injection, PLR in the Opn4Cre/+;R26iDTR/+ mice gradually lost
sensitivity (Figures 4A and 4B). The rate of loss in PLR was highly
variable among this group. Almost 90% sensitivity was lost in the first
week, and complete loss of PLR as in Opn42/2;rd/rd mice was
observed after the second week following DT injection (Figures 4C
and 4D). The loss in PLR, coupled with normal ERG in DT-treated
Opn4Cre/+;R26iDTR/+ mice implies a necessary role of mRGCs in
dynamic adaptation of pupil size to ambient light. Loss of circadian photoentrainment in mRGC ablated We next tested the effect of mRGC ablation on circadian
photoentrainment. The circadian wheel running activity of mice
has an intrinsic periodicity of less than 24 h. Photic input to the
SCN makes daily phase adjustments to the clock so that the
animal’s activity rhythm maintains a constant phase relation with
the ambient photoperiod (reviewed in [25]). The daily wheel
running activity of Opn4Cre/+;R26iDTR/+ and of WT mice entrained
normally to an imposed 12 h light:12 h dark (LD) cycle (Figures 5A
and 5B). The mice consolidated their activity with activity
commencement juxtaposed to the dark onset. After 10 days of
entrainment all mice were administered DT. The WT mice
continued to exhibit 24 h rhythm in activity-rest cycle with the
activity onset coincident with the dark onset. However, after one
week following DT injection, the Opn4Cre/+;R26iDTR/+ mice
phenocopied
the
Opn42/2;rd/rd
mice
showing
no
sign
of
functional light input to the circadian clock. Their activity rhythm
under LD conditions exhibited a period length of ,23.5 h which
is similar to that under constant dark (DD) conditions (Figure 5C). We next tested the effect of mRGC ablation on circadian
photoentrainment. The circadian wheel running activity of mice
has an intrinsic periodicity of less than 24 h. Photic input to the
SCN makes daily phase adjustments to the clock so that the
animal’s activity rhythm maintains a constant phase relation with
the ambient photoperiod (reviewed in [25]). The daily wheel
running activity of Opn4Cre/+;R26iDTR/+ and of WT mice entrained
normally to an imposed 12 h light:12 h dark (LD) cycle (Figures 5A
and 5B). The mice consolidated their activity with activity
commencement juxtaposed to the dark onset. After 10 days of
entrainment all mice were administered DT. The WT mice
continued to exhibit 24 h rhythm in activity-rest cycle with the
activity onset coincident with the dark onset. However, after one
week following DT injection, the Opn4Cre/+;R26iDTR/+ mice
phenocopied
the
Opn42/2;rd/rd
mice
showing
no
sign
of
functional light input to the circadian clock. Their activity rhythm
under LD conditions exhibited a period length of ,23.5 h which
is similar to that under constant dark (DD) conditions (Figure 5C). Loss of circadian photoentrainment in mRGC ablated To clearly establish the roles of retina photopigments and
photoreceptor cells in circadian entrainment, we compared the
temporal activity rhythms of DT-treated WT and DT-treated
Opn4Cre/+;R26iDTR/+ with those of rd/rd, Opn42/2 and Opn42/2;rd/rd
mice under three different lighting conditions: LD, DD and LL
(constant light). Mice with complete loss of photopigments or acute
loss of mRGC lineage in adulthood showed a DD period length that
was indistinguishable from that of WT mice, thus implying normal
function of the SCN oscillator sustains without tonic input from the
mRGCs. As shown before [8,15,16] under LD and LL conditions,
the circadian clock in rd/rd and in Opn42/2 mice showed signs of
light input. Both genotypes entrained normally to LD cycle; i.e. they
exhibited 24 h LD period length. As reported earlier [13,15,16],
under constant light, both rd/rd and Opn42/2 mice showed period
lengthening–although the effect was less pronounced in the latter
genotype. A parsimonious interpretation of circadian photoentrain-
ment in melanopsin photopigment deficient (Opn42/2) mice, but not Figure 3. mRGC ablation does not alter the normal retina
architecture and image-forming responses. (A) Hematoxylin and
Eosin staining of 5 mm thick paraffin embedded sections of retina from
Opn4Cre/+;R26iDTR/+ mice without and with DT injection. DT application
had no detectable adverse effect on the normal stratification of the
retina (outer segment (OS), outer nuclear layer (ONL), outer plexiform
layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and
ganglion cell layer (GCL)). (B) Representative full-field ERG of WT and
DT-treated Opn4Cre/+;R26iDTR/+ mice showing rod, cone and maximal
combined responses. Responses from both eyes were simultaneously
measured and plotted. Quantitative analysis of magnitude and timing
of a-wave, b-wave and oscillatory potentials of these two genotype
groups (3 mice each) showed no significant difference (data not
shown). (C) Image forming visual function as assessed by the visual cliff
test was unaffected by mRGC ablation. Average percentage (+SEM,
n = 5 to 13 mice) of positive choice in 10 trials for each mouse are
shown. Mice with outer retina degeneration (rd/rd) made random
choices while stepping down from the platform and were significantly
different (Student’s t test, p,0.05; red asterisk) from the other four
groups. No significant difference in test performance was found among
native or DT-treated WT or Opn4Cre/+;R26iDTR/+ mice. doi:10.1371/journal.pone.0002451.g003 Figure 3. mRGC ablation does not alter the normal retina
architecture and image-forming responses. Loss of circadian photoentrainment in mRGC ablated Both WT and Opn4Cre/+;R26iDTR/+ mice treated with or without
DT performed equally and made the safe choice in .70% of 10
repeated trials, while mice with outer retina degeneration (rd/rd)
made random choices. These results indicate that the mRGCs do
not play a major role in the overall image-forming and visual
responses in mammals, which is consistent with the limited
monosynaptic projections of the mRGCs to image processing
brain regions. Nonetheless, mRGCs may still play some roles in
visual responses. For instance, the loss of PLR may cause light- PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org June 2008 | Volume 3 | Issue 6 | e2451 4 Melanopsin RGC Ablation Figure 4. Necessity of mRGCs for PLR. DT injection severely attenuates pupil constriction in response to 20 mW of monochromatic blue light
(470 nm) in Opn4Cre/+;R26iDTR/+ mice (A), but does not affect in such PLR response in WT mice (B). Normalized pupil constriction (-SD; n = 3 mice)
measured one day prior to or every day following DT injection for up to 8 days are shown. There was variability in the rate of loss in PLR response
among Opn4Cre/+;R26iDTR/+ as reflected in the larger error bars. (C) Pupil constriction in response to varying irradiance levels over 5 log units shows the
necessity of mRGC for PLR. Average (+SEM, n = 5 to 6 mice) and fitted sigmoid curves for WT, Opn4Cre/+;R26iDTR/+, Opn42/2;rd/rd and DT-treated
Opn4Cre/+;R26iDTR/+ mice are shown. (D) Representative frozen video images showing dark adapted pupil and pupil under low (1011 photons.cm22.s21)
or high intensity light (1015 photons.cm22.s21) of 470 nm are shown. Notice the complete lack of pupil constriction in Opn42/2;rd/rd and in DT-
treated Opn4Cre/+;R26iDTR/+ mice. For each genotype representative images of the same eye under three different conditions are shown. doi:10.1371/journal.pone.0002451.g004 Figure 4. Necessity of mRGCs for PLR. DT injection severely attenuates pupil constriction in response to 20 mW of monochromatic blue light
(470 nm) in Opn4Cre/+;R26iDTR/+ mice (A), but does not affect in such PLR response in WT mice (B). Normalized pupil constriction (-SD; n = 3 mice)
measured one day prior to or every day following DT injection for up to 8 days are shown. There was variability in the rate of loss in PLR response
among Opn4Cre/+;R26iDTR/+ as reflected in the larger error bars. Loss of circadian photoentrainment in mRGC ablated (C) Pupil constriction in response to varying irradiance levels over 5 log units shows the
necessity of mRGC for PLR. Average (+SEM, n = 5 to 6 mice) and fitted sigmoid curves for WT, Opn4Cre/+;R26iDTR/+, Opn42/2;rd/rd and DT-treated
Opn4Cre/+;R26iDTR/+ mice are shown. (D) Representative frozen video images showing dark adapted pupil and pupil under low (1011 photons.cm22.s21)
or high intensity light (1015 photons.cm22.s21) of 470 nm are shown. Notice the complete lack of pupil constriction in Opn42/2;rd/rd and in DT-
treated Opn4Cre/+;R26iDTR/+ mice. For each genotype representative images of the same eye under three different conditions are shown. doi:10.1371/journal.pone.0002451.g004 Opn4Cre/+;R26iDTR/+mice
firmly
suggests
specific
ablation
of
mRGCs in these mice. in mRGC-ablated (DT-treated Opn4Cre/+;R26iDTR/+) mice is the
photic input to the circadian clock in Opn42/2 mice must initiate
from the outer-retina rod/cone photoreceptors and transmit through
the mRGCs (which are intact in these mice). Degeneration or
dysfunction of the outer retina photoreceptors along with lack of
melanopsin photopigment or acute ablation of mRGCs severs light
input to the SCN. mRGCs mediate light suppression of activity The wheel running activity of the WT mouse remained entrained to
the LD cycle even after DT injection and was lengthened under constant light. Daily wheel running activity profile of mice were binned in 6 min and
double plotted such that the activity from consecutive days are plotted to the right and beneath the data from previous day. Periods of darkness are
shown in shaded area. (C) Genotypes of mice and their respective average (6SEM, n = 3 to 6 mice) period length of wheel running activity rhythm
under conditions of LD, DD and LL as determined by periodogram analysis in Clocklab software [41] are shown. Within each lighting or treatment
group (separated by solid box) significant difference (Student’s t test, p,0.05) from WT group is shown by red asterisk. (D) Anterograde CTB-Alexa
Fluor 488 tracing in the optic tract (OT) is intact, but is completely abolished in the SCN of DT treated Opn4Cre/+;R26iDTR/+ mice. (E) Staining in both
regions are left intact in WT mice treated with DT. doi:10.1371/journal.pone.0002451.g005 Figure 5. mRGCs are necessary for light adaptation of circadian wheel running activity rhythm. Representative daily wheel running
activity profile of an (A) Opn4Cre/+;R26iDTR/+ and (B) WT mouse under 12 h light:12 h dark (LD), constant dark (DD) or constant light (LL) are shown. A
week after DT injection (red arrows) the wheel running activity of Opn4Cre/+;R26iDTR/+ began to ‘‘free run’’ with a periodicity similar to that under no
light. Constant light had no effect on the daily drift in activity onset in this mouse. The wheel running activity of the WT mouse remained entrained to
the LD cycle even after DT injection and was lengthened under constant light. Daily wheel running activity profile of mice were binned in 6 min and
double plotted such that the activity from consecutive days are plotted to the right and beneath the data from previous day. Periods of darkness are
shown in shaded area. (C) Genotypes of mice and their respective average (6SEM, n = 3 to 6 mice) period length of wheel running activity rhythm
under conditions of LD, DD and LL as determined by periodogram analysis in Clocklab software [41] are shown. Within each lighting or treatment
group (separated by solid box) significant difference (Student’s t test, p,0.05) from WT group is shown by red asterisk. mRGCs mediate light suppression of activity The general locomotor activity of nocturnal rodents is acutely
suppressed upon exposure to photopic light levels [26]. Such
activity masking by light is left almost intact in rd/rd mice [27],
mildly attenuated in Opn42/2 mice [28], and is abolished in
Opn42/2;rd/rd mice [13]. Multiple brain centers are involved in
this response, which requires photic input from the retina. To
assess the role of mRGCs in negative masking, we measured the
wheel running activity of mice subjected to a short photocycle of
4 h light and 4 h dark over several days (Figure 6). Such a short
photocycle suppresses the normal circadian nocturnal activity and
has been useful in uncovering the complementary roles of rod/
cone and melanopsin photopigments in negative masking [28]. Typically, activity during the light phase of the day is suppressed,
so that the total activity is largely distributed over the three 4 h
dark periods. The rd/rd, Opn42/2 and WT mice showed strong At the end of the circadian wheel running experiments, we
intravitreally injected Alexa Fluor 488 conjugated Cholera Toxin
subunit B (CTB) to the eyes of DT treated WT and Opn4Cre/
+;R26iDTR/+mice and monitored staining of the RGC axons and
terminals in the brain. Brain sections of both genotypes showed
normal staining in the optic tract (OT), which is known to consist
of projections from both mRGCs and other RGCs. However, the
SCN of Opn4Cre/+;R26iDTR/+mice showed very little staining, while
the SCN of WT mice showed significant amount of fluorescence
(Figures
5D
and
5E). Therefore,
the
loss
of
melanopsin
immunostaining in the retina along with the loss of a majority
of projection to the SCN, but not in the OT of DT treated PLoS ONE | www.plosone.org June 2008 | Volume 3 | Issue 6 | e2451 5 Melanopsin RGC Ablation Figure 5. mRGCs are necessary for light adaptation of circadian wheel running activity rhythm. Representative daily wheel running
activity profile of an (A) Opn4Cre/+;R26iDTR/+ and (B) WT mouse under 12 h light:12 h dark (LD), constant dark (DD) or constant light (LL) are shown. A
week after DT injection (red arrows) the wheel running activity of Opn4Cre/+;R26iDTR/+ began to ‘‘free run’’ with a periodicity similar to that under no
light. Constant light had no effect on the daily drift in activity onset in this mouse. mRGCs mediate light suppression of activity (D) Anterograde CTB-Alexa
Fluor 488 tracing in the optic tract (OT) is intact, but is completely abolished in the SCN of DT treated Opn4Cre/+;R26iDTR/+ mice. (E) Staining in both
regions are left intact in WT mice treated with DT. doi:10.1371/journal.pone.0002451.g005 negative masking, such that .80% of the daily activity was
partitioned into the three dark phases, while both Opn42/2;rd/rd
and DT-treated Opn4Cre/+;R26iDTR/+ mice continued to exhibit
strong circadian activity consolidation that was unperturbed by
periods of illumination (Figure 6C). They showed equal activity
during light and dark phases. of the mammalian circadian system, multiple cell types in the SCN
constitute a network of oscillators which is entrained to the
ambient light via ocular photoreceptors (reviewed in [30]). The
mRGCs have emerged as the predominant RGC sub-type making
monosynaptic connection to SCN oscillators. In this study we generated a mouse line that expresses functional
CRE recombinase from the native melanopsin locus. We used this
mouse line to (a) comprehensively tag mRGC lineage with GFP
and to (b) ablate the mRGCs after their normal differentiation. This approach revealed that the melanopsin promoter is active in a
large number of RGCs, of which ,40% produce immunologically
detectable level of melanopsin protein. Comparable heterogeneity Discussions Our approach of transgene tagging from a
non-native promoter now allows comprehensive identification of
all mRGCs and their classification into two distinct groups based
on the level of OPN4 expression. Such differences in melanopsin
protein levels along with possible heterogeneity in the relative
expression levels of genes defining morphological and functional
properties may further contribute to the functional diversity in
mRGC population. in mRGC morphology (Provencio, personal communication),
dendritic arborization [31], light induced calcium responses
[11,32], electrical activity [33] and immunostaining [23] has
already been reported. Our approach of transgene tagging from a
non-native promoter now allows comprehensive identification of
all mRGCs and their classification into two distinct groups based
on the level of OPN4 expression. Such differences in melanopsin
protein levels along with possible heterogeneity in the relative
expression levels of genes defining morphological and functional
properties may further contribute to the functional diversity in
mRGC population. We have used the acute cell ablation to understand the cellular
bases for light input to the circadian clock. Our results show a
central non-redundant role for mRGCs in the relay of photic input
to two brain regions that generate adaptive photoresponses. Rod/
cone and melanopsin initiated photo signals converge at the level
of the mRGCs before relaying to oscillator neurons in the SCN. Similarly, mRGCs appear to serve as the sole functional relay for
photic input to the OPN. These results highlight an important
cellular design feature of the mammalian circadian system. The
mammalian clock, like that of plants, invertebrates and lower
vertebrates, uses multiple, mutually compensating photopigments
to adapt to the ambient light [36]. However, in other organisms at
least some circadian photoreceptors and the clock are cell
autonomous and there appear to be multiple molecular nodes
for integration of light signal to the molecular clock [25]. The
mammalian circadian time keeping system recruits non-cell
autonomous multiple photoreceptors, including rod, cones and
melanopsin. The complete loss of light entrainment in mRGC The strategy to acutely ablate target cell types in mice with DT-
DTR specificity has been successful for many peripheral and
intracranial neuronal populations [20,21,34,35], but its efficacy for
acute ablation of retina cell types has never been tested. From
immunostaining, parallel loss of circadian photoentrainment and
other adaptive ocular photoresponses, it is clear that DT can
successfully cross the blood-retina barrier in sufficient quantity to
trigger ablation of DTR expressing cells. Discussions Comprehensive identification and perturbation of genes and
circuits are necessary to gain a systems level understanding of
circadian regulation of behavior [29]. In the current cellular model PLoS ONE | www.plosone.org June 2008 | Volume 3 | Issue 6 | e2451 June 2008 | Volume 3 | Issue 6 | e2451 6 Melanopsin RGC Ablation Figure 6. Lack of negative masking in mRGC ablated mice. Representative wheel running activity profile of DT-treated (A) Opn4Cre/+;R26iDTR/+
and (B) WT mice held under ultradian cycle of 4 h light and 4 h darkness are shown. The sessions of darkness are shown by shaded area. Activity
profile is plotted as in Figure 5A. (C) Average percentage (6SEM, n = 3 to 6 mice) of daily activity during the bouts of light sessions (on left) or during
darkness (on right) for five different groups of mice are shown. Groups with percent activity during light phase significantly different (Student’s t test,
p,0.05) from that of the DT-treated WT mice are shown with asterisk. doi:10.1371/journal.pone.0002451.g006 Figure 6. Lack of negative masking in mRGC ablated mice. Representative wheel running activity profile of DT-treated (A) Opn4Cre/+;R26iDTR/+
and (B) WT mice held under ultradian cycle of 4 h light and 4 h darkness are shown. The sessions of darkness are shown by shaded area. Activity
profile is plotted as in Figure 5A. (C) Average percentage (6SEM, n = 3 to 6 mice) of daily activity during the bouts of light sessions (on left) or during
darkness (on right) for five different groups of mice are shown. Groups with percent activity during light phase significantly different (Student’s t test,
p,0.05) from that of the DT-treated WT mice are shown with asterisk. doi:10.1371/journal.pone.0002451.g006 ganglion cells. Although the tests employed here did not find any
profound loss of image forming function of the retina in acute tests,
we cannot rule out the possibility that mRGCs play a subtle direct
or indirect role in sculpting the image-forming function of the
retina. For instance, in the retina mRGCs may influence function
of other
RGCs
via intercellular
coupling
[11],
they may
participate in relaying luminance information to the image
processing [18], or by playing an indispensible role in the pupil
constriction response, they may reduce direct photodamage to the
retina. in mRGC morphology (Provencio, personal communication),
dendritic arborization [31], light induced calcium responses
[11,32], electrical activity [33] and immunostaining [23] has
already been reported. PLoS ONE | www.plosone.org Discussions However, we found up to
10% of melanopsin expressing cells escaped cell death due to
potential stochastic noise in CRE or DTR expression. It is
interesting to note that these surviving cells are unable to generate
AOPs. This implies that the SCN and OPN require a strong
photic input to reset intrinsic activity. Alternatively, the surviving
cells could represent a distinct mRGC population that does not
play a role in relaying light information to the SCN or OPN. Our finding that mRGCs do not overtly disrupt image-forming
processes implies a divergence of signals at the level of the retinal June 2008 | Volume 3 | Issue 6 | e2451 June 2008 | Volume 3 | Issue 6 | e2451 7 Melanopsin RGC Ablation Melanopsin RGC Ablation integration of Cre leading to loss of melanopsin protein in Opn4Cre/
Cre mice was verified by lack of anti-OPN4 immunostaining (data
not shown). Opn4Cre/+;Z/EG mice were used for assessment of
CRE function and fluorescent tagging of mRGCs in the retina. Littermate Opn4Cre/+;R26iDTR/+ and WT mice were used in all
subsequent experiments for acute ablation of mRGCs. / ablated mice firmly establishes a unique mammalian-specific
cellular network design to integrate light information from these
photopigments to the circadian clock. The mRGCs in this design
serve as the principal site of signal integration and therefore, have
now
emerged
as
a
unique
cellular
target
for
therapeutic
intervention in circadian clock related disorders. The complete
loss of non-image forming photoresponses also highlights the role
of mRGCs as the principal cellular node where photic information
for image-forming and non-image forming responses diverge. The Opn42/2 mice used in this study were generated in 129S
background and characterized earlier [15]. These mice were bred
to a line of C57BL/6 carrying the Pde6brd/rd mutation. Progeny
from this breeding, which were also in a 129S;C57BL/6
background were genotyped and mice of Opn42/2, rd/rd and
Opn42/2;rd/rd genotypes were used for behavioral studies. During the course of this study, another manuscript [37]
describing specific ablation of mRGCs was published. That study
employed direct expression of an attenuated diphtheria toxin from
the melanopsin locus, which caused slow degeneration of the
mRGCs over several months, as assessed by the loss of a Tau:LacZ
transgene expression or melanopsin immunostaining. Both these
techniques detect only a subset of mRGCs ([23] and Figure 2). Therefore, the slow degeneration of non-image forming photo-
sensitivity in the study by Guler et al. DT injection DT (D0564, Sigma-Aldrich, St. Louis, MO) was dissolved in
sterile PBS (1 mg/ml) and stored at 280uC till use. Freshly thawed
DT stock solution was diluted in sterile PBS and injected
intraperitoneally (50 mg/kg body weight) [21] to 8–12 weeks old
Opn4Cre/+;R26iDTR/+ and WT littermate mice. The dose was
repeated once or twice at 3 d interval. Generation of mice All animal care and procedures were approved by the
Institutional Animal Care and Use Committee of the Genomics
Institute of the Novartis Research Foundation and The Salk
Institute for Biological Studies. R26iDTR/+ mouse strain was a kind
gift of Dr. Ari Waisman [20] and Z/EG mouse strain [22] was
purchased from Jackson Laboratory. A schematic diagram of the
targeting construct for generating the R26iDTR/+ mouse is shown in
Figure 1D. The Opn4Cre/+ mouse was generated by replacing the first seven
exons of the mouse melanopsin gene with a gene cassette containing
Cre (Figure 1D). For generation of the targeting construct,
genomic DNA of 129S1SvImJ inbred mouse strain was used as a
template and 3.5 kb of genomic sequence immediately upstream of
the translation start site and 3.4 kb of sequence 39 distal to the 7th
exon of the of Opn4 gene were PCR amplified and cloned into two
multicloning
sites
flanking
the
Cre:IRES:bTau:loxP:Neo:lox-
P:eYFP cassette. The left arm of the targeting vector also carried
a HSV-TK resistance gene. The construct was linearized by NotI
digestion and microinjected into an embryonic stem (ES) cell line
from 129S mice. ES cell clones with integration of the targeting
construct were selected on G418, and 288 antibiotic resistant
clones were screened by PCR for homologous recombination of
the targeting construct to the melanopsin locus. Genomic DNA from
seven PCR positive clones was digested with NheI and subjected to
Southern blot hybridization with a probe that lies within the right
arm. Sequences of the genotyping PCR primers are; wild type
(Primer a = CACTTCAGAGACAGCCAGAAGCAGG, Primer
b = GACTGACACTGAAGCCTGGCAAACG)
and
mutant
(Primer a and Primer c = CCATTTCCGGTTATTCAACTTG-
CACC). One clone with appropriately recombined DNA was
injected into C57BL/6J blastocysts and introduced into C57BL/6J
pseudopregnant females. Chimeric male progeny were bred to
C57BL/6J females and the resulting heterozygous agouti coat-
colored progeny were mated with C57BL/6J. Heterozygous mice
were bred among each other and with Z/EG or Rosa26iDTR/+ mice
which were also in 129S;C57BL/6 mixed background. Targeted Retina staining For flat mount, adult mice were sacrificed, the eyes quickly
removed and placed into aerated Ames medium (Sigma-Aldrich,
St. Louis, MO). After removal of the corneas and lenses, eyecups
were fixed in 4% paraformaldehyde for 15 min. Following three
washes in PBS, retinas were dissected from eyecups, stretched onto
filter paper, and processed in 24-well plates. The retinas were
incubated in a blocking solution (0.3% Triton X-100, 5% normal
donkey serum, and 0.5% glycine in PBS) for 1 h at room
temperature. After three washes in PBS, the retinas were
incubated in a 1:5,000 dilution of rabbit anti-OPN4 antiserum
(against a peptide consisting of the 15 N-terminal amino acids of
mouse melanopsin [38]) or in 1:500 dilution of rabbit anti-GFP
antibody (Cat# A11122, Invitrogen, Carlsbad, CA) in the
blocking solution for overnight at 4uC and rinsed with PBS. Melanopsin immunoreactivity was visualized with Cy3-conjugated
donkey anti-rabbit IgG (1:500, Cat# 711-165-152, Jackson
ImmunoResearch Laboratories, West Grove, PA) or FITC-
conjugated anti-rabbit IgG (1:20, Cat# 401314, Calbiochem,
San Diego, CA) in blocking solution. Finally, retinas were washed
with PBS and mounted with PermaFluor (Cat# IM0752,
Beckman Coulter, Fullerton, CA). Fluorescent images were
captured using an Olympus Fluoview500 confocal microscope or
Leica TCS SP2 AOBS confocal microscope. For Hematoxylin and
Eosin (H&E) staining, eyes were removed and eyecups were fixed
in 4% paraformaldehyde for 2 h, washed three times with PBS,
and embedded in paraffin. Five micron thick paraffin sections
were used for H&E staining. Stained slides were visualized under a
Leica microscope. Discussions [37] may have resulted from
likely heterogeneity in toxin production from the native promoter,
homeostatic synaptic strengthening and/or early developmental
compensation. Despite the differences in the pace of cell death,
and the approach, both manuscripts arrived at similar conclusion
that the mRGCs are the primary node of convergence of
melanopsin and rod/cone initiated photoresponses. Anterograde tracing with cholera toxin g
g
Mice were anesthetized with ketamine (70 mg/kg) and xylazine
(10 mg/kg) and one drop each of 1% tropicamide and 0.5%
proparacaine (Bausch & Lomb, Tampa, FL) were applied to their
eyes. An incision was made with a 30 gauge needle below the
limbus region and 1 ml of 1% Cholera Toxin-B subunit (CTB)
conjugated to Alexa Fluor 488 (Cat# C34775, Invitrogen, CA)
was injected into the vitreous. After 48 h, the mice were
anesthetized with ketamine (70 mg/kg) and xylazine (10 mg/kg),
perfused with 4% paraformaldehyde in 16PBS. The brain was
extracted, further fixed at 4uC overnight, embedded in OCT and
40 mm thick sections cut in a microtome were imaged using an
Olympus Fluoview500 confocal microscope. PLoS ONE | www.plosone.org June 2008 | Volume 3 | Issue 6 | e2451 PLoS ONE | www.plosone.org 8 Melanopsin RGC Ablation camera to improve the image quality. After collecting images of
dark adapted pupils, the contralateral eye was exposed to light
from the integrating sphere. Light from a 300 Watt Xenon Arc
lamp light source (Sutter Instrument, Novato, CA, USA) was
filtered, collimated and delivered to the integrating sphere through
a liquid light guide. An inline 470 nm filter, a filter wheel with a
series of neutral density filters, and a Lambda 10-3 optical filter
changer with SmartShutterTM were used to control the spectral
quality, intensity and duration of light. Light intensity was
measured with a Melles Griot power meter. Video images of
pupil constriction were analyzed by ViewPoint EyeTrackerTM
software (Arrington Research Inc., Scottsdale, AZ). Dark adapted
pupil area prior to light pulse and pupil area after 30 sec of light
were measured and pupil constriction was defined as 1-(dark
adapted pupil width)ˆ2/(pupil width after 30 sec of light)ˆ2. The
squares come in to account for the calculation of pupil area. Pupil
area constriction was plotted against the log of irradiance. Data
was fitted to a sigmoidal dose response curve with the lower
asymptote fixed to y = 0 using Origin lab 8 software. ERG Mice were dark adapted for 1 h, anesthetized with ketamine
(70 mg/kg) and xylazine (10 mg/kg). The eyes were applied with
1% tropicamide, 0.5% proparacaine and Genteal lubricant eye gel
(CIBA vision, Duluth, GA, USA). Electrodes were connected
under dim red light. ERGs were recorded from both eyes with an
ERG system (Model LE-2000, Tomey, Nagoya, Japan). y
13. Panda S, Provencio I, Tu DC, Pires SS, Rollag MD, et al. (2003) Melanopsin is
required for non-image-forming photic responses in blind mice. Science 301:
525–527. 15. Panda S, Sato TK, Castrucci AM, Rollag MD, DeGrip WJ, et al. (2002)
Melanopsin (Opn4) requirement for normal light-induced circadian phase
shifting. Science 298: 2213–2216. 14. Lucas RJ, Hattar S, Takao M, Berson DM, Foster RG, et al. (2003) Diminished
pupillary light reflex at high irradiances in melanopsin-knockout mice. Science
299: 245–247. Locomotor activity measurement Daily locomotor activity of mice individually housed in wheel
running cages was measured as described in [41]. Typically, 6–
10 week old mice were individually housed in wheel running cages
placed inside light tight boxes with independent illumination. During the light phase, the mice received ,150 lux of white light
from fluorescent light source. Wheel running activity in 1 min bins
was continuously collected and later analyzed by Clocklab
software (Actimetrics, Evanston, IL, USA). All routine animal
husbandry practices during the dark phase were performed under
dim red light. Anterograde tracing with cholera toxin White
LED light embedded in contact lens recording electrodes were
used as light sources. ERG recordings were done following the
standards of the International Society for Clinical Electrophysiol-
ogy of Vision (ISCEV; http://www.iscev.org/standards/erg1999. html). After an additional 5 min of dark adaptation, rod response
was measured by averaging 4 dim light flashes of 80 cd.m2260.12
msec at .2 sec interval. Then mice were further dark adapted for
3 min and maximal combined (rod and cone) response and
oscillatory potential were determined from the average of
responses to four flashes of 6,000 cd.m2260.5 msec at .10 sec
inter-stimulus interval. Finally, after 10min light adaptation (25
cd.m22), average single-flash cone response was determined from
4 flashes of 6,000 cd.m2260.5 msec at .15 sec interval. For tracking the gradual changes in PLR response following DT
injection, pupil area constrictions over 8 days were normalized to
maximum constriction taken as 100%. PLR PLR was assessed as described in [40] with some modification. Mice were dark adapted for at least an hour prior to PLR
assessment. Unanaesthetized mice were hand restrained with the
left eye apposed to one of the outlets of an integrating sphere. The
pupil of right eye was video monitored under infrared light with a
consumer grade digital video camera. An additional +2 diaptor
lens and an infrared filter were fitted to the light path of the Author Contributions Conceived and designed the experiments: SP. Performed the experiments:
CS SP TJ MH HL NT SK. Analyzed the data: SP MH VC. Contributed
reagents/materials/analysis tools: CS TJ. Wrote the paper: SP MH. Conceived and designed the experiments: SP. Performed the experiments:
CS SP TJ MH HL NT SK. Analyzed the data: SP MH VC. Contributed
reagents/materials/analysis tools: CS TJ. Wrote the paper: SP MH. Acknowledgments We would like to thank Dr. Ari Waisman for providing R26iDTR/+ mouse
strain, Van M. Lee, Victoria Piamonte, Kathryn Spencer and Dusko
Trajkovic for technical assistance, and members of the Panda lab for
critical discussion of the results. Visual cliff test The visual cliff test was assessed as described in [39]. Half of a
31 cm length645 cm width650 cm tall clear acrylic box had a black
and white checkerboard pattern on the top (the ‘‘safe’’ side) and the
other side had a clear top (the ‘‘unsafe’’ side/cliff side). The unsafe
side had a checkerboard pattern on the bottom to give an illusion of
added depth. Black paper was placed on the inside of the walls to
reduce reflections. A block of 31 cm length63.5 cm width62.5 cm
height was placed at the center of the box to separate these two sides. The mouse was placed on the block and allowed to make a
spontaneous step down between the safe side and the unsafe side. Data was measured as positive when the mouse chose to step down
onto the horizontal checkered surface (the safe side) and as negative
onto the clear side (the unsafe side). Each mouse was tested for 10
consecutive trials. Fifty percent positive indicated that the mouse
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English
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Monitoring of Dual CRISPR/Cas9-Mediated Steroidogenic Acute Regulatory Protein Gene Deletion and Cholesterol Accumulation Using High-Resolution Fluorescence In Situ Hybridization in a Single Cell
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Frontiers in endocrinology
| 2,017
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cc-by
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Original Research
published: 25 October 2017
doi: 10.3389/fendo.2017.00289 Monitoring of Dual crisPr/cas9-
Mediated steroidogenic acute
regulatory Protein gene Deletion
and cholesterol accumulation Using
high-resolution Fluorescence In Situ
hybridization in a single cell Jinwoo Lee1,2* and Colin Jefcoate1,2,3,4* 1 Department of Cell and Regenerative Biology, University of Wisconsin, Madison, WI, United States, 2 Endocrinology and
Reproductive Physiology Program, University of Wisconsin, Madison, WI, United States, 3 Molecular and Environmental
Toxicology Center, University of Wisconsin, Madison, WI, United States, 4 Molecular and Cellular Pharmacology, University of
Wisconsin, Madison, WI, United States Keywords: steroidogenic acute regulatory protein, CRISPR, fluorescence in situ hybridization, cholesterol, lipid
droplets Edited by: Edited by:
Michael Schumacher,
Institut national de la santé et de la
recherche médicale, France Institut national de la santé et de la
recherche médicale, France
Reviewed by:
Vassilios Papadopoulos,
University of Southern California,
United States
Emilie Lesport,
Institut Professeur Baulieu, France
*Correspondence:
Jinwoo Lee
jwlee7@wisc.edu;
Colin Jefcoate
jefcoate@wisc.edu Reviewed by:
Vassilios Papadopoulos,
University of Southern California,
United States
Emilie Lesport,
Institut Professeur Baulieu, France Reviewed by:
Vassilios Papadopoulos,
University of Southern California,
United States
Emilie Lesport,
Institut Professeur Baulieu, France Specialty section:
This article was submitted to
Cellular Endocrinology,
a section of the journal
Frontiers in Endocrinology Received: 25 June 2017
Accepted: 11 October 2017
Published: 25 October 2017 Recent advances in fluorescence microscopy, coupled with CRISPR/Cas9 gene editing
technology, provide opportunities for understanding gene regulation at the single-cell
level. The application of direct imaging shown here provides an in situ side-by-side
comparison of CRISPR/Cas9-edited cells and adjacent unedited cells. We apply this
methodology to the steroidogenic acute regulatory protein (StAR) gene in Y-1 adrenal
cells and MA-10 testis cells. StAR is a gatekeeper protein that controls the access of
cholesterol from the cytoplasm to the inner mitochondria. The loss of this mitochon-
drial cholesterol transfer mediator rapidly increases lipid droplets in cells, as seen in
StAR−/− mice. Here, we describe a dual CRISPR/Cas9 strategy marked by GFP/mCherry
expression that deletes StAR activity within 12 h. We used single-molecule fluorescence
in situ hybridization (sm-FISH) imaging to directly monitor the time course of gene editing
in single cells. We achieved StAR gene deletion at high efficiency dual gRNA targeting to
the proximal promoter and exon 2. Seventy percent of transfected cells showed a slow
DNA deletion as measured by PCR, and loss of Br-cAMP stimulated transcription. This
DNA deletion was seen by sm-FISH in both loci of individual cells relative to non-target
Cyp11a1 and StAR exon 7. sm-FISH also distinguishes two effects on stimulated StAR
expression without this deletion. Br-cAMP stimulation of primary and spliced StAR RNA
at the gene loci were removed within 4 h in this dual CRISPR/Cas9 strategy before any
effect on cytoplasmic mRNA and protein occurred. StAR mRNA disappeared between
12 and 24 h in parallel with this deletion, while cholesterol ester droplets increased four-
fold. These alternative changes match distinct StAR expression processes. This dual
gRNA and sm-FISH approach to CRISPR/Cas9 editing facilitates rapid testing of editing
strategies and immediate assessment of single-cell adaptation responses without the
perturbation of clonal expansion procedures. INTRODUCTION course of deletion by PCR amplification of the targeted StAR gene
segment and by probing the edited StAR DNA segment with sm-
FISH after RNase removal of all RNA. We compared this targeted
StAR deletion to a non-targeted region of the StAR locus (exon 7)
or to another similarly expressed gene (Cyp11a1 loci). We used
this Cas9 procedure to examine the immediate consequences of
StAR deletion. The StAR transfer of cholesterol from LDs to the
cleavage enzyme, Cyp11a1, located on the inner face of the inner
membrane, is integrated with the cleavage of cholesterol esters
(CEs) by hormone-sensitive lipase (HSL), under control of protein
kinase A (PKA) (18). In StAR−/− mice and human deficiency, the
loss of steroidogenesis in the adrenal glands, testes, and ovaries is
matched by large accumulations of CE (3, 19). The capacity to resolve individual RNA species in single cells by
single-molecule Fluorescence in Situ Hybridization (sm-FISH)
(1, 2) now provides the means to examine the CRISPR/Cas9
gene editing in single cells. Here, we describe a dual CRISPR/
Cas9 cleavage of steroidogenic acute regulatory protein (StAR),
the prime regulator of cholesterol metabolism, in Y-1 adrenal
cells and MA-10 testis cells. We used direct sm-FISH to compare
StAR expression in dual-transfected CRISPR (+) cells to non-
transfected (NT) adjacent cells. The goal was to separate the tim-
ing, respectively, of the transfection, editing, and gene expression
processes. We also measured the subsequent adaptation resulting
from the loss of StAR function. We demonstrated dramatic
increases of lipid droplets (LDs) that mimic the human adrenal
deficiency condition (3).h In this report, we show that editing was extensive within 12 h
and that cholesterol trafficking was then rapidly redirected from
intra-mitochondrial oxidation to the formation of cytoplasmic
CE accumulations. The cholesterol captured as esters in these
accumulations was much greater than the small amounts diverted
from StAR-dependent steroid synthesis. StAR has been linked
to several cell types to cholesterol export (20), to transcription
controlled by hydroxy-metabolites through liver X receptors
(LXR) (21, 22), and to other mitochondrial processes (23).h i
This single-cell detection depends on sm-FISH, which uses
multiple fluorescent 20-base oligomers (4) to detect primary
transcripts (p-RNA) and spliced transcripts (sp-RNA) at gene
loci and, then, to detect mRNA as single molecules in the cyto-
plasm (1, 2). INTRODUCTION cAMP analogs extensively induce these StAR RNA
species in the Y-1 adrenal and MA-10 testis cells that we used
here (5, 6). The Y-1 cells are distinguished by basal StAR mRNA
expression, which was sufficient for maximum stimulation by
cAMP within 10 min of steroid synthesis (7). sm-FISH imaging of
StAR expression showed that the loci responded asymmetrically
to cAMP stimulation within asynchronous cell populations. Stimulation of StAR transcripts at the gene loci not only increased
the levels of different types of RNA but also decreased inter-cell
differences. These direct sm-FISH analyses of editing can be completed
within 24 h of transfection. The speed this procedure provides the
opportunity for a relatively quick optimization of the CRISPR/
Cas9 editing strategy, notably for gRNA selection and validation. We have designed gRNA sequences with an online CRISPR design
tool and used the fast algorithm Cas-OFFinder, to searches for
potential off-target sites of Cas9 (24). Various PCR-based meth-
odologies are available for single-guide RNA (sgRNA) validation,
including the following: mismatch cleavage assays (25), indel
detection by amplicon analysis (IDAA) (26), and digital PCR (27). However, a complete workflow of gRNA validation in single-cell
colony expansion is time intensive. This direct combined sm-FISH
and CRISPR/Cas9 strategy provided an intermediate step in the
isolation of appropriately targeted clonal lines. f
Understanding the effects of CRISPR/Cas9 on StAR expres-
sion requires an appreciation of the editing process. The CRISPR/
Cas9 technology was developed from bacterial adaptive immune
systems (8–10). Cas9 is an RNA-guided DNA endonuclease that
fuses with a guide RNA (gRNA). The gRNA includes a four-base
endonuclease cleavage sequence and a Cas9 recognition site
[protospacer adjacent motif (PAM)] at the 3′end (11–13). The
association of Cas9 and gRNA directs specific localization to
complementary DNA sequences selected for gene editing (14, 15). Here, we used a dual Cas9 vector strategy in which mCherry and
GFP expression marked the respective deliveries of the 5′- and 3′-
gRNA sequences. The guided Cas9 creates a double-stranded break
(DSB) 3 bp upstream of the PAM sites, within the gRNA hybrid-
ized sequence (13, 16, 17). The dual cleavages this design provided
lead to an excision and re-ligation to produce an edited StAR gene
lacking the early proximal promoter, exon 1, and intron 1. This
deletion removed the possibility of functional mRNA expression. We directly assessed the deletion by measuring the deletion time Citation: Lee J and Jefcoate C (2017)
Monitoring of Dual CRISPR/
Cas9-Mediated Steroidogenic Acute
Regulatory Protein Gene Deletion and
Cholesterol Accumulation Using
High-Resolution Fluorescence In Situ
Hybridization in a Single Cell. Front. Endocrinol. 8:289. doi: 10.3389/fendo.2017.00289 October 2017 | Volume 8 | Article 289 Frontiers in Endocrinology | www.frontiersin.org 1 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate Abbreviations: 3′EU, oligomer set targeting extended 3′UTR; Cas9, CRISPR-
associated protein 9; CE, cholesterol ester; CRISPR, the clustered regularly inter-
spaced short palindromic repeat; DSB, double-strand breaks; FISH, fluorescence
in situ hybridization; gRNA, guide RNA; HSL, hormone-sensitive lipase; LD, lipid
droplets; N-SIM, Nikon’s Structured Illumination Microscope; PAM, protospacer
adjacent motif; PKA, protein kinase A; p-RNA, primary RNA; Single Molecule
Fluorescence in Situ Hybridization (sm-FISH); sp-RNA, spliced primary RNA;
StAR, steroidogenic acute regulatory protein. Cell Linesh The MA-10 mouse Leydig tumor cell line (a gift of Dr. Mario
Ascoli) was derived from the Leydig tumor, M5480P (28). The
Y-1 cell line (a gift of Dr. Bernard Schimmer) was derived from
a mouse adrenocortical tumor (29). General procedures for
cell culture were as described previously (1). A day before the
experiment, approximately 3–6 × 105 cells were seeded onto the
coverslip (Corning) coated with ploy-Lysine (Sigma) in wells of a
six-well plate (Corning). Real-time RT-PCR and Genomic
DNA-PCR for Analysis of StAR
Primer design and cDNA synthesis were performed as previ-
ously described (1, 30). qPCR was performed using BioRad
CFX96™ Real-Time PCR Detection System. The qPCR
protocolfor StAR expression time courses was done as follows:
initial denaturation at 95°C for 3 min, followed by 40 cycles of Plasmid Preparation p
Digested plasmids [plasmid (1 µg), FastDigest Bbsl (1 µl),
FastAP (1 µl), 10× FastDigest Buffer (2 µl), ddH2O (total 20 µl),
30 min, 37°C] were purified using QIAquick Gel extraction kit. The oligo pairs encoding the guide sequences were annealed and
ligated into the plasmids. We prepared the mixture [sgRNA left
(100 µM, 1 μ), sgRNA right (100 µM, 1 µl), 10× T4 ligation buffer
l (1 µl), T4 PNK (1 µl), ddH2O (total 10 µl)] for phosphorylating,
and annealing the sgRNA oligos in a thermocycler (37°C for
30 min; 95°C for 5 min; ramp down to 25°C at 5°C min−1). We
set up a ligation reaction (Bbsl), and the mixture were incubated
for 10 min at RT. We also prepared a no-insert pSpCas9 (BB)-
only negative control for ligation. DH5 alpha cells were used for
transformation. The product (2 µl) was added to ice-cold DH5
alpha cells (50 µl), incubated the mixture on ice for 30 min,
heat-shocked the cell mixture at 42°C for 45 s, and returned it
immediately to ice for 2 min. Then, we added 500 µl of SOC
medium and incubated for 1 h before plating the cells onto an
LB plate containing 100 µg ml−1 ampicillin. After an overnight
incubation at 37°C, we picked 10 colonies to check for the cor-
rect insertion of the sgRNA. We ran the colony PCR reaction
with the guide sequence (forward) and a primer on the plasmid
backbone (reverse: CAG_enhancer region). The sm-FISH method has been shown to be effective at
visualizing well-expressed mRNA transcripts in most cells. Glass coverslips with plated cells were washed with sterile PBS
and placed in six-well plates. Cells were plated on glass cover-
slips, fixed with 0.4% paraformaldehyde, stored at 4°C in 70%
ethanol overnight, and hybridization was performed the next
day. For single mRNA detection, sm-FISH probe sets consist of
multiple singly labeled oligonucleotides (generally 30–48 bases
long) designed to hybridize along targeted RNA transcripts. Hybridization was performed in a dark humidified condi-
tion. Samples were re-suspended in 2× SSC and added GLOX
buffer without enzymes for equilibration, incubated, and then
re-suspended in GLOX buffer with enzymes (glucose oxidase
and catalase). Again, antifade reagent was used prior to Z-stack
imaging. Transfection DNA was transfected with 7.5 µl of Lipofectamine 3000 reagent,
5 µl P3000, and 2.5 µg of plasmid DNA cocktail following the
manufacturer’s instructions. CRISPR (+) cells under conditions
used here are present in 25–30% of the cells 24 h post-transfection. We analyzed all NT cells in randomly selected fields. The smaller
proportion of CRISPR (+) cells necessitates analysis of all GFP
(+) cells in wider fields. Design of sgRNA Sequence The term sm-FISH has been used to distinguish the use of a
probe set consisting of 40 fluorescent 20mers. The RNA probe
sets for StAR were generated by using the Stellaris probe design-
er.4 Samples were prepared according to a previously described
method (1). Freshly prepared StAR p-RNA (Quasar-570,
Biosearch Technology), StAR sp-RNA (Quasar-670, Biosearch
Technology), StAR 3′ EU (Quasar-570, Biosearch Technology),
and Cyp11a1 p-RNA (Quasar-670, Biosearch Technology)
probe sets and antibody for StAR were used. A clean coverslip
was placed over the sample to prevent drying of the RNA
hybridization solution [10% dextran sulfate (Sigma), 10%
deionized formamide (Ambion), 2× SSC] during the incuba-
tion. Samples were incubated in a dark humidified chamber at
37°C overnight. After a 30-min wash in wash buffer, samples
were incubated in DAPI nuclear stain (wash buffer with 5 ng/
ml DAPI) to counterstain the nuclei for 30 min. In the case of
the combined FISH, an extra washing step is needed with the
secondary antibody. Samples were processed according to as
previously described (1).hf g
g
q
pSpCas9 (BB)-2A-GFP (Addgene plasmid # 48138) and pU6-
(BbsI)_CBh-Cas9-T2A-mCherry (Addgene plasmid # 64324)
were used for the sgRNA expression plasmids (31). pSpCas9
(BB)-2A-GFP express a chimeric gRNA plus EGFP and human
codon-optimized Cas9. EGFP was replaced into Cherry in pU6-
(BbsI)_CBh-Cas9-T2A-mCherry. The components of type II
CRISPR/Cas9 system crRNA and tracrRNA were fused to gener-
ate a sgRNA. For the S. pyogenes system, the target sequence leads
a 5′-NGG PAM and the guide sequence pairs with the opposite
strand at ~3 bp upstream of the PAM. The targeted sequences are
as follows: StAR exon 2 GGTGGATGGGTCAAGTTCGACGT
CGG and StAR promoter AGTCATCAGTCATTGTGCAG AGG. To clone the guide sequence into the sgRNA scaffold, we designed
guide sequences with online CRISPR design tools.1,2 To search for
potential off-target sites of Cas9, we used Cas-OFFinder.3 Real-time RT-PCR and Genomic
DNA-PCR for Analysis of StAR Primer design and cDNA synthesis were performed as previ-
ously described (1, 30). qPCR was performed using BioRad
CFX96™ Real-Time PCR Detection System. The qPCR
protocolfor StAR expression time courses was done as follows:
initial denaturation at 95°C for 3 min, followed by 40 cycles of October 2017 | Volume 8 | Article 289 Frontiers in Endocrinology | www.frontiersin.org 2 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate 10 s at 95°C and 30 s at 60°C. Primer sequences are as follows:
Star E1/I1, 5′-AGACATATGCGGAATATGAAAGGTG-3′ and
5′-CCCAAGAGCTTTCCCACAAA-3′; E5/E6, 5′-GAGTGGTG
TCATCAGAGCTGAAC-3′ and 5′-TGAGTTTAGTCTTGGAG
GGACTTCC-3′. Genomic DNA extraction was done with TRIzol
following the manufacturer’s protocol. The genomic DNA PCR
protocol was done as follows: initial denaturation at 95°C for 3 min,
followed by 35 cycles of 15 s at 95°C, 30 s at 58°C, and 150 s at
72°C. Primer sequences are as follows: 5′-CCTCTGCACAATG
ACTGATGACT-3′ and 5′-GGATGGGTCAAGTTCGACGTC
GG-3′. PCR products were separated on a 0.5% agarose gel. 4 http://www.biosearchtech.com/products/rna-fish 1 http://tools.genome-engineering.org.
2 http://chopchop.cbu.uib.no.
3 http://www.rgenome.net/cas-offinder/. Image Acquisition and Analysis To detect and visualize p-RNA, sp-RNA, and mRNA, we used
the Olympus wide-field fluorescence microscope (Model IX81)
and the Nikon’s Structured Illumination Microscope (N-SIM)
for higher resolution images according to a previously described
method (1, 33). We used two microscope settings representing
high and low sensitivity to compare p-RNA and sp-RNA at loci
under, respectively, basal conditions and after 1 and 3 h of stim-
ulation by Br-cAMP (Figure S1B in Supplementary Material). The high sensitivity setting is also used to image mRNA, which
appears as single molecules. The N-SIM uses different opti-
cal processing methods but provided results that were fully
consistent with the Olympus IX81. Five fluorescence imaging
filter set (DAPI, GFP, Orange, mCherry, and Red) was used
for IX81 in this study. GFP (488ext/507emi nm) and mCherry
(587/610 nm) channels were used for the detection of Cas9-
GFP and Cas9-mCherry. Orange (538/559 nm, pseudocolored
green) and red (618/637 nm, pseudocolored red) channels were
used for the detection of StAR p-RNA and sp-RNA/mRNA. Yellow color indicates colocalization of p-RNA and sp-RNA. We used the “StAR Search” software developed by the Arjun
Raj lab at University of Pennsylvania.5 Statistical significance
was determined by Student’s t-test or ANOVA; p < 0.05 was
considered statistically significant where *p < 0.05, **p < 0.01,
and ***p < 0.001. Data were analyzed by using the PRISM
software (San Diego, CA, USA). We have previously reported a set of sm-FISH oligomers (1, 2)
that image specific segments of the StAR gene (intron 1 and the
3′end of exon 7) and equivalent sequences in RNA transcription
products. We have resolved p-RNA and sp-RNA of StAR gene
loci and single mRNA molecules in the cytoplasm (Figure 1C). Although mRNA expression in individual cells was variable,
image analyses of sets of adjacent cells provided a mean expres-
sion as copy numbers per cell that matched equivalent qPCR
analyses based on several million cells in a culture well (1, 2). Here, we have applied these sm-FISH probes to in situ single-cell
analysis of CRISPR/Cas9 editing.h y
g
The StAR sm-FISH images obtained from Y-1 adrenal and
MA-10 testis cells differ by over 50-fold ranging. We have pre-
viously reported single hybridizations for StAR gene DNA, for
each resolved mRNA molecule and minimum p-RNA expres-
sion in basal Y-1 loci (2, 33). Plasmid Preparation For DNA sm-FISH, cells were treated with RNase A at 37°C
for 1 h, washed and equilibrated with PBS for 5 min before dehy-
dration by consecutive 5 min incubations in 70, 85, and 100%
ethanol. After air-drying, cells were heated at 80°C for 5 min in
hybridization mixture (50% formamide, 10% dextran, 0.5 µM
EDTA, and 4× SSC), and then washed using an ethanol series
(ice old 70, 80, and 95%). After air-drying, cells were placed in
RNA hybridyzation with probes for overnight. October 2017 | Volume 8 | Article 289 Frontiers in Endocrinology | www.frontiersin.org 3 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate targeted by CRISPR/Cas9 and the associated impact on StAR
expression. We tested both basal, and Br-cAMP-stimulation
of StAR expressed either as RNA at gene loci or as mRNA
and protein in the cytoplasm. The final adaptation phase was
indicated by fluorescence imaging of significant increases in CEs
that form the major constituents in cytoplasmic LDs and also
accumulate in late endosomes (34). These accumulations were
readily detected in single cells by ORO, which delivered a low-
intensity fluorescence (32). ORO (+) droplets were increased
extensively in CRISPR (+) cells. We measured the time course
of this response in individual cells in relation to the StAR RNA
editing responses. Oil Red O (ORO) (Lipid Stain) Staining
Oil Red O stock solution was prepared by dissolving 0.3 g of ORO
(MP Biomedicals, OH, USA) in 100 ml of isopropanol. ORO
working solution was prepared by mixing three parts (30 ml) of
ORO stock solution with two parts (20 ml) of water and filtered
through 0.2 µm filters. Cells were washed with water and incu-
bated with 60% isopropanol for 5 min. After 5 min, the wells were
completely dry. ORO working solution was added to the cells, and
the cells were incubated for 10 min in the solution. Stained cells
were washed three times with water. Again, the antifade reagent
was used prior to Z-stack imaging. ORO is a fat-soluble dye and
can be used for fluorescence microscopy wherein a lipid environ-
ment has enough fluorescence (32). g
p
CRISPR/Cas9 editing produces sequence deletions that
remove expression or activity when two gRNA sequences target
double-strand DNA cleavage sites that span a functionally
essential part of the gene locus. Plasmid Preparation The StAR gene sequence between
these two gRNAs is lost, and ligation between the 5′- and 3′-ends
restores the integrity of the gene. We have selected a 5′ target
that overlaps C/EBPβ-binding site (−90 ~ −80 bp) (35) in the
StAR promoter and a site on the reverse strand at the end of the
exon 2 region. A deletion of key promoter elements then ensues,
including the transcription StAR site, exon 1, intron 1, and most
of intron 2 (Figure 1B). To test the efficiency of gRNA and to
visualize the impact of gene deletion, we have combined this dual
CRISPR/Cas9 strategy with sm-FISH analyses of the various steps
in gene expression. 5 http://www.rajlab.seas.upenn.edu/StarSearch/launch.html. Image Acquisition and Analysis Peak unresolved fluorescence for,
respectively, p-RNA and sp-RNA is seen in these loci after 1 h
stimulation by Br-cAMP. StAR protein expression determined by
immunohistochemistry has a similar range of expression, which
corresponds to accumulation in mitochondria. The comparison
of image intensities required the adjustment of microscope
settings (Figures S1A–C in Supplementary Material). We also
showed that the 3D distribution of loci and mRNA in these cells
was appreciably different. Fortunately, the active StAR loci were
found close to the nuclear midline thus facilitating detection (Z
axis). Most of the mRNAs were found closer to the adherent sur-
face in the widest part of the cell (Figure S1D in Supplementary
Material). Optimum sensitivity for single DNA/RNA molecules
was obtained with single probe sets where pixels can be directly
counted with the aid of an imaging program (star search) (Figure
S1E in Supplementary Material). Experimental Design for CRISPR/Cas9
Deletion from the StAR Genehi CRISPR (+) cells
expressed both gRNAs and Cas9 conjugated with mCherry (PX330) or GFP (PX458), which indicates gRNA expression. (C) Characterization of editing: high-
resolution fluorescence in situ hybridization probes were used for identification of altered StAR p-RNA and sp-RNA at loci during CRISPR/Cas9 editing. Targeted
StAR DNA at gene locus was also visualized by the p-RNA probe for intron 1 after the RNase treatment. Single molecules of mRNA were detected by sp-RNA
probes. StAR antibody was also added for immunofluorescence (IF) images. Before any stimulation, Y-1 cells showed a consistent basal
expression of p-RNA and sp-RNA/mRNA, which were unre-
solved when measured by q-PCR. Sm-FISH resolved this expres-
sion to highly variable levels of p-RNA and sp-RNA at the loci
in the individual cells (Figure 2B). About one-third of the cells
showed mRNA in the cytoplasm, and about one-third show none. There is little relationship between expression of sp-RNA at the
loci and the mRNA in the cytoplasm. However, the expression
at the loci turns over much more quickly than the cytoplasmic
mRNA. In MA-10 cells, this basal level p-RNA and sp-RNA was
undetectable (2, 36). Experimental Design for CRISPR/Cas9
Deletion from the StAR Genehi This study has two general goals; first, to separate the timing of
CRISPR/Cas9 editing of StAR through the temporal resolution
in individual cells of transfection, editing, and gene expression;
second, to measure the direct adaptation of the cells due to
loss of StAR function (Figure 1A). The progress in editing was
measured in whole cultures by PCR analysis of the targeted seg-
ment of StAR DNA and then in single cells by direct sm-FISH
imaging. We examined both the StAR DNA sequence that was October 2017 | Volume 8 | Article 289 Frontiers in Endocrinology | www.frontiersin.org 4 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate Figure 1 | Design for CRISPR/Cas9 editing of steroidogenic acute regulatory protein (StAR). (A) The sequence of steps used for dual CRISPR/Cas9 deletion of the
StAR DNA from Y-1 and MA-10 cells. Transfection and DNA editing by Cas9 were assessed by impacts on StAR RNA at loci, on StAR mRNA and protein in the
cytoplasm, plus evaluation of effects on StAR activity based on Oil Red O (ORO) staining of cholesterol in lipid droplets (LDs). Side-by-side comparison of NT and
CRISPR (+) cells was performed. (B) Dual CRISPR/Cas9 design for StAR. Two gRNAs were used to generate a large deletion of the gene. CRISPR (+) cells
expressed both gRNAs and Cas9 conjugated with mCherry (PX330) or GFP (PX458), which indicates gRNA expression. (C) Characterization of editing: high-
resolution fluorescence in situ hybridization probes were used for identification of altered StAR p-RNA and sp-RNA at loci during CRISPR/Cas9 editing. Targeted
StAR DNA at gene locus was also visualized by the p-RNA probe for intron 1 after the RNase treatment. Single molecules of mRNA were detected by sp-RNA
probes. StAR antibody was also added for immunofluorescence (IF) images. Figure 1 | Design for CRISPR/Cas9 editing of steroidogenic acute regulatory protein (StAR). (A) The sequence of steps used for dual CRISPR/Cas9 deletion of the
StAR DNA from Y-1 and MA-10 cells. Transfection and DNA editing by Cas9 were assessed by impacts on StAR RNA at loci, on StAR mRNA and protein in the
cytoplasm, plus evaluation of effects on StAR activity based on Oil Red O (ORO) staining of cholesterol in lipid droplets (LDs). Side-by-side comparison of NT and
CRISPR (+) cells was performed. (B) Dual CRISPR/Cas9 design for StAR. Two gRNAs were used to generate a large deletion of the gene. Frontiers in Endocrinology | www.frontiersin.org Characterization of StAR Expression in
Untransfected Y-1 Cells To assess the impact of CRISPR/Cas9 editing in Y-1 cells, we
first used q-PCR to characterize the normal expression of
StAR p-RNA and sp-RNA after Br-cAMP stimulation. p-RNA
increased 10-fold over 1 h to a steady state, while sp-RNA
showed a delay of about 20 min [Figure 2A (a,b)]. We previously
characterized the delay in sp-RNA as a pause in elongation at the
end of the terminal exon that is coupled with splicing (1, 33). This increase is biphasic, corresponding first to the generation of
sp-RNA from the splicing of p-RNA at the gene locus, and then
to a slower process that corresponds to export of the processed
mRNA from the loci to the cytoplasm. The primary p-RNA StAR
transcripts were only found in the nucleus at the gene loci (1, 2). p-RNA and sp-RNA were expressed at the same loci in rep-
resentative Y-1 cells at low sensitivity. Such robust expression
was only visible in a few select basal cells. Two loci exhibited this
robust expression in most Y-1 cells after of stimulation. There was October 2017 | Volume 8 | Article 289 Frontiers in Endocrinology | www.frontiersin.org 5 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate no further increase at 3 h [Figure 2C (a)]. The dot blots indicated
the range of expression at Y-1 loci under basal conditions, and
with stimulations for 1 and 3 h [Figure 2C (b)]. MA-10 cells where cytoplasmic mRNA is scarcely detectable at 1 h
even as StAR protein exhibits peak translation. A major factor in
this discrepancy is that StAR protein accumulates in mitochondria
with slow turnover. By contrast, low levels of StAR mRNA, which
can sustain peak StAR mitochondrial activity (7), may turnover
more rapidly than the excess that induced by high concentrations
of Br-cAMP. We compared sm-FISH images of cytoplasmic mRNA under
basal conditions and after 1 and 3 h of stimulation by Br-cAMP
[Figure 2D (a)]. There was no net increase in cytoplasmic mRNA
in the initial 1 h, despite substantial increases of sp-RNA in the
loci. The cytoplasmic mRNA rose extensively at 3 h. We have
previously shown (1, 33) that cytoplasmic mRNA increased to
this steady-state level after 2 h. The sm-FISH image showed a
significant increase in StAR protein at 1 and 3 h, which evidently
preceded the increase in mRNA. This anomaly is even clearer in CRISPR Cas9 Editing of Basal and
Stimulated StAR We examined NT and CRISPR (+) cells under basal conditions
or after 1 h stimulation by Br-cAMP. This stimulation provides
optimal responses of p-RNA and sp-RNA at the loci. mCherry
and GFP fluorescence mark the expression of 5′ gRNA and 3′
gRNA, respectively. Their dual fluorescence is therefore indicative
of CRISPR (+) cells. We first examined p-RNA and sp-RNA in
Y-1 cells under basal conditions. We examined NT and CRISPR (+) cells under basal conditions
or after 1 h stimulation by Br-cAMP. This stimulation provides
optimal responses of p-RNA and sp-RNA at the loci. mCherry
and GFP fluorescence mark the expression of 5′ gRNA and 3′
gRNA, respectively. Their dual fluorescence is therefore indicative
of CRISPR (+) cells. We first examined p-RNA and sp-RNA in
Y-1 cells under basal conditions. to be processed to provide a statistically valid assessment of the
CRISPR impact on basal Y-1 expression. To improve the detection of StAR transcripts, we took advan-
tage of the rapid stimulation of StAR p-RNA and sp-RNA at
loci in Y-1 cells. After 1 h of Br-cAMP stimulation, p-RNA and
sp-RNA were visible even at low sensitivity settings. In five rep-
resentative NT cells, all loci expressed both p-RNA and sp-RNA
with 3/5 exhibiting two active loci and 2/5 with a single active
locus. No CRISPR (+) cell showed an active locus [Figure 3B (a)]. However, a lower proportion of CRISPR (+) cells retained active
loci as shown [Figure 3B (b)]. Examination of all CRISPR (+)
cells from several microscope fields showed that 33/149 (22%)
retained some p-RNA expression, compared to 315/394 (80%)
in NT cells [Figure 3B (c)]. Similar proportions were shown for
sp-RNA [CRISPR (+) 14/55 (25%); NT 175/222 (78%)]. Thus, the
ratio of primary to spliced StAR transcripts retained in the active
CRISPR (+) loci appears similar to the NT loci, consistent with
no effect of dual CRISPR/Cas9 on StAR splicing. High-sensitivity microscope settings were needed to
detect the expression at these minimally active loci (Figure
S2A in Supplementary Material). The transfected cells, which
were identified by the expression of both mCherry and GFP,
did not show expression at the level reached by the subset of
higher-expressing basal cells (3/7 Figure 3A; inserts a–c). Only
one CRISPR (+) cell, out of 10 examined cells, retained active
StAR loci [Figure 3A (b)]. CRISPR Cas9 Editing of Basal and
Stimulated StAR We applied this sm-FISH analysis to basal and induced Y-1 cells
24 h after application of the dual CRISPR/Cas9 transfection. Figure 2 | Continued Figure 2 | Continued October 2017 | Volume 8 | Article 289 Frontiers in Endocrinology | www.frontiersin.org 6 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate Figure 2 | Characterization of steroidogenic acute regulatory protein (StAR) expression in Y-1 cells. (A) qPCR analysis of StAR transcription in Y-1 adrenal cells
after stimulation by Br-cAMP. (a) Location of PCR primers to detect the primary transcripts [pRNA(E1/I1)] and spliced transcripts [spRNA(E5/E6)]. (b) Time course for
the stimulation of StAR RNA as determined by q-PCR. (B) High sensitivity sm-FISH detection of basal expression of sp-RNA at loci and mRNA in cytoplasm. (C) (a)
Low sensitivity paired sm-FISH images of the time-dependent appearance of p-RNA and sp-RNA at loci after stimulation by Br-cAMP (1 mM). (b) Relative integral
density (IntDen) was measured based on the integrated density subtracted with background readings. (D) High sensitivity sm-FISH of StAR mRNAs (a) and IF of
protein (b) after 1 and 3 h Br-cAMP (1 mM) stimulation. The “Star search” program was used for counting fluorescence of mRNA and protein immunohistochemistry
in individual cells. Error bars show SEM. **p < 0.01, ***p < 0.001; ns, not significant by ANOVA with post hoc Tukey. Scale bar represents 10 µm. Figure 2 | Characterization of steroidogenic acute regulatory protein (StAR) expression in Y-1 cells. (A) qPCR analysis of StAR transcription in Y-1 adrenal cells
after stimulation by Br-cAMP. (a) Location of PCR primers to detect the primary transcripts [pRNA(E1/I1)] and spliced transcripts [spRNA(E5/E6)]. (b) Time course for
the stimulation of StAR RNA as determined by q-PCR. (B) High sensitivity sm-FISH detection of basal expression of sp-RNA at loci and mRNA in cytoplasm. (C) (a)
Low sensitivity paired sm-FISH images of the time-dependent appearance of p-RNA and sp-RNA at loci after stimulation by Br-cAMP (1 mM). (b) Relative integral
density (IntDen) was measured based on the integrated density subtracted with background readings. (D) High sensitivity sm-FISH of StAR mRNAs (a) and IF of
protein (b) after 1 and 3 h Br-cAMP (1 mM) stimulation. The “Star search” program was used for counting fluorescence of mRNA and protein immunohistochemistry
in individual cells. Error bars show SEM. **p < 0.01, ***p < 0.001; ns, not significant by ANOVA with post hoc Tukey. Scale bar represents 10 µm. Frontiers in Endocrinology | www.frontiersin.org CRISPR Cas9 Editing of Basal and
Stimulated StAR Overall, in the basal NT cells, 27/40
expressed p-RNA, and 23/47 expressed sp-RNA with most
expressing both in varying ratios [Figure 3A (c)]. To confirm
the specificity of these low-level CRISPR responses as well as
the sensitivity of p-RNA and sp-RNA detection in the presence
of a background, we examined expression in Y-1 cells with
CRISPR/Cas9 transfection carried out in the absence of gRNAs
(Figure S2B in Supplementary Material). Without gRNAs, the
basal expression of p-RNA and sp-RNA was not hindered in the
mCherry-positive cells. Nevertheless, the variability and low
detection mean that still larger numbers of CRISPR (+) need f
To establish dual sgRNA-directed gene deletion, we designed
PCR primer sets that amplifies the StAR genomic DNA which
we expect to be deleted by the dual CRISPR editing [Figure 3C
(a)]. The full StAR gene product (WT, 1.8 kb) and deleted gene
product [CRISPR (+), 0.65 kb] were separated on a 0.5% agarose
gel. In the experiment, the deletion shown by a 0.65 kb band was
about 25% at 12 h and at least 50% at 24 h [Figure 3C (b)]. October 2017 | Volume 8 | Article 289 Frontiers in Endocrinology | www.frontiersin.org 7 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate Figure 3 | CRISPR Cas9 editing of basal and stimulated steroidogenic acute regulatory protein (StAR). (A) (a) CRISPR/Cas9 StAR deletion under the basal
condition at the locus. The boxes indicate the active StAR loci (p-RNA, sp-RNA), and the dashed line (circle) shows the CRISPR/Cas9-positive cell. GFP and
mCherry were used as a marker for gRNA expression. (b) a CRISPR (+) cell that retained an active StAR loci. (c) Percent of NT basal cells expressing p-RNA and
sp-RNA at loci (the segment is shown in blue). Only 1/10 CRISPR (+) cells had detectable RNA at the locus. (B) (a) CRISPR/Cas9 StAR deletion was measured at
the loci after 1 h stimulation with Br-cAMP (1 mM). (b) An example of a CRISPR (+) cell that retained active StAR loci. (c) Percent of non-transfected cells (NT, left)
and CRISPR (+) cells (right) that expressed active StAR loci (p-RNA and sp-RNA) (segment shown in blue). (C) (a) PCR strategy to validate StAR gene deletion. (b)
PCR amplification in wild type (WT) and transfected (24 h) cells with various CRISPR/Cas9 plasmids. (D) (a) Detection of p-RNA (StAR, cyp11a1) after 1 h
stimulation with Br-cAMP in Y-1 cells. Application of gRNA to Manipulation of
StAR Editing A similar probing with the
StAR exon 7 DNA probe (3′EU) showed that StAR DNA outside
the targeted segment was retained in the CRISPR (+) cells to the
same extent as in the adjacent NT cells (Figure 5D; Figure S5B in
Supplementary Material). Again the 3′EU sequence was detected
at one locus in each of the CRISPR (+) cells. Thus, both StAR 3′EU
DNA and Cyp11a1 shared similar detection efficiencies. The locus
DNA hybridization that requires denaturation at a much higher
temperature than the locus-associated RNA is likely to be easier for
regions of the chromatin that are actively transcribed. A distal StAR
promoter sequence with similar base composition requires still a
harsher denaturation (Figure S5C in Supplementary Material). The 3D spatial distribution of StAR loci, mRNA, and protein
which is central to the sm-FISH assessment of gene expression
was best seen in MA-10 cells. Thus, StAR protein and mRNA
after 3 h of stimulation showed very characteristic 3D organiza-
tions (38) (Figure 4B). The StAR protein in these mitochondria
is located in the matrix and is inactive in promoting cholesterol
transfer to Cyp11a1. StAR is only active immediately after the
translation of mRNA at the mitochondrial surface (7). We next assessed whether this dual CRISPR/Cas9 system is
needed for this removal of StAR sp-RNA/mRNA. MA-10 cells
were transfected with the dual gRNAs, with each gRNA singly or
without gRNAs and then assessed for mRNA expression. Without
the gRNA, the three Cas9/GFP (+) cells retained the same StAR
mRNA distribution as surrounding NT cells [Figure 4C (a)],
with no statistical difference between NT and GFP (+) cells in
the extent of expression [Figure 4C (b)]. Consistent with the Y-1
experiments, StAR mRNA was completely removed in the dual
gRNA CRISPR (+) MA-10 cells [Figure 4D (a,b)]. Single 5′-gRNA/Cas9 or 3′-gRNA/Cas9 transfections of
MA-10 cells for 24 h were marked by, respectively, mCherry or
GFP. 5′gRNA CRISPR transfections produced similar effects
after 24 h to the dual protocol. PCR analyses showed that neither
single CRISPR/Cas9 produced any of the DNA deletion seen
for the dual editing [Figure 3C (c)]. Out of eight mCherry (+)
cells, six cells showed complete loss of transcription while two
retained active StAR loci [Figure S4A (a,b) in Supplementary
Material]. One of these cells with active loci also showed loss of
mRNA, while one retained the mRNA. Application of gRNA to Manipulation of
StAR Editing To verify whether excision of the targeted sequence had occurred
in individual loci of StAR, DNA sm-FISH was performed with
the p-RNA probe, which hybridizes to intron 1 which is targeted
by the dual gRNACRISPR/Cas9. We compared this hybridiza-
tion to non-target DNA either within the StAR locus (distal
promoter-3′EU/exon 7) or in Cyp11a1, another expressed gene
(intron 3/Cyp11a1) (Figure 5A). The key steps in distinguishing
DNA sm-FISH from RNA sm-FISH are the RNase removal of
all RNA and the stronger denaturation conditions needed to
open the chromatin structure for hybridization (Figure 5B). The
gene hybridization targets a single DNA sequence at each locus
whereas the RNA expression also detected by p-RNA may vary
from 1 to >50 transcripts. Single mRNA particles are harder to image than the accumulated
RNA at loci, particularly in Y-1 cells, which slowly round up when
stimulated by Br-cAMP. This cell rounding which is larger in Y-1
cells than in MA-10 cells arises from activation of PTPases that
affect adhesion complexes (37). Overall, the stimulation of StAR
p-RNA and sp-RNA in MA-10 cells by Br-cAMP was similar to
that seen in Y-1 cells (Figure 2A) except that MA-10 cells were
devoid of basal expression measured either by qPCR (Figure 4A)
or sm-FISH (Figure S3 in Supplementary Material). Again,
q-PCR showed that Br-cAMP stimulation was characterized by a
steady state for p-RNA from 1 to 3 h. The formation of sp-RNA,
which included increases first at the locus and then as mRNA in
the cytoplasm, showed a 25-min delay.h We compared StAR and Cyp11a1 DNA in two CRISPR (+) cells
with four adjacent NT-cells. CRISPR (+) cells (#1 and #2) each
showed a pair of Cyp11a1 loci but no StAR intron 1 (Figure 5C). Overall, these CRISPR (+) cells showed that about 20% retained
at least 1 StAR locus that retained intron 1. Of the four adjacent
NT cells, Cyp11a1 loci are seen in three with an average of one locus
detected per cell. The NT cells each show at least the intron 1 DNA
segment from StAR loci (Figure S5A in Supplementary Material). A similar heterogeneity in sm-FISH hybridization was seen in the
Cyp11a1 transcription (Figure 3D). CRISPR Cas9 Editing of Basal and
Stimulated StAR (b) Quantitation of active loci per cell for StAR and Cyp11a1 in NT cells and CRISPR (+) cells after 1 h Br-cAMP stimulation. Error bars show SEM. **p < 0.01, ***p < 0.001; ns, not significant by ANOVA with post hoc Tukey. Scale bar represents 10 µm. Figure 3 | CRISPR Cas9 editing of basal and stimulated steroidogenic acute regulatory protein (StAR). (A) (a) CRISPR/Cas9 StAR deletion under the basal
condition at the locus. The boxes indicate the active StAR loci (p-RNA, sp-RNA), and the dashed line (circle) shows the CRISPR/Cas9-positive cell. GFP and
mCherry were used as a marker for gRNA expression. (b) a CRISPR (+) cell that retained an active StAR loci. (c) Percent of NT basal cells expressing p-RNA and
sp-RNA at loci (the segment is shown in blue). Only 1/10 CRISPR (+) cells had detectable RNA at the locus. (B) (a) CRISPR/Cas9 StAR deletion was measured at
the loci after 1 h stimulation with Br-cAMP (1 mM). (b) An example of a CRISPR (+) cell that retained active StAR loci. (c) Percent of non-transfected cells (NT, left)
and CRISPR (+) cells (right) that expressed active StAR loci (p-RNA and sp-RNA) (segment shown in blue). (C) (a) PCR strategy to validate StAR gene deletion. (b)
PCR amplification in wild type (WT) and transfected (24 h) cells with various CRISPR/Cas9 plasmids. (D) (a) Detection of p-RNA (StAR, cyp11a1) after 1 h
stimulation with Br-cAMP in Y-1 cells. (b) Quantitation of active loci per cell for StAR and Cyp11a1 in NT cells and CRISPR (+) cells after 1 h Br-cAMP stimulation. Error bars show SEM. **p < 0.01, ***p < 0.001; ns, not significant by ANOVA with post hoc Tukey. Scale bar represents 10 µm. To test the specificity of genome editing introduced by CRISPR/
Cas9, we examined whether CRISPR (+) and NT cells maintained
normal expression of Cyp11a1 p-RNA. StAR p-RNA was not sup-
pressed in CRISPR (+) cells [Figure 3D (a)]. The four NT cells expressed Cyp11a1 in 3/4 cells, with two showing both loci and
one only a single locus. Two CRISPR (+) cells showed one cell
with two active Cyp11a1 loci and one without Cyp11a1 expres-
sion. Neither CRISPR (+) cell showed StAR expression. Overall, October 2017 | Volume 8 | Article 289 Frontiers in Endocrinology | www.frontiersin.org 8 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate in exon 2. Application of gRNA to Manipulation of
StAR Editing By contrast, the pattern
of responses for 3′gRNA indicated much lower locus disruption
[Figure S4B (a,b) in Supplementary Material]. Ten GFP (+)
single transfections showed six that retained locus transcription,
five of which expressed mRNA. Thus, half of the 3′gRNA-Cas9
transfections remain similar to the surrounding NT cells. Clearly,
DSB introduced around the 5′ PAM site, located in the proximal
promoter, more readily introduce mutations that remove StAR
expression than DSB introduced around the 3′PAM site located An analysis of the StAR gene in 277 cells showed recognition of
at least 1 StAR intron 1 sequence in 157/226 UT cells (70%) but in
only 11/51 CRISPR (+) cells (20%) (Figure 5E). The CRISPR (+)
cells that retain StAR intron 1 closely parallel the 25% that retain
p-RNA and sp-RNA transcription without mRNA and protein. CRISPR Cas9 Editing of Basal and
Stimulated StAR The only difference between the 5′gRNA CRISPR (+)
cells and the dual edit is that 1/8 of these cells retain both locus
transcription and the full processing to mRNA. in 39 NT cells, StAR was expressed in an average of 1.4 loci per
cell, with Cyp11a1 in 1.3 loci per cell. In CRISPR (+) cells, the
Cyp11a1 expression decreased to 0.85 loci per cell, whereas StAR
loci were undetected in these 13 CRISPR (+) cells [Figure 3D (b)]. Frontiers in Endocrinology | www.frontiersin.org Time Course of CRISPR Initiation and
StAR Editing in Y-1 Cells We have addressed the time dependence of several key steps: the
entry of editing CRISPR vectors into the cells, their expression as
marked by GFP and mCherry, the deletion of the targeted StAR
DNA, and the several changes in StAR expression. We examined
each at 4, 12, and 24 h after transfection. The editing responses
were compared after a 2 h Br-cAMP stimulation [Figure 6A (a)]. Sm-FISH provides an assessment of the impact of increasing October 2017 | Volume 8 | Article 289 Frontiers in Endocrinology | www.frontiersin.org 9 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate Figure 4 | Role of gRNA in steroidogenic acute regulatory protein (StAR) editing; Br-cAMP stimulation of MA-10 Cells and suppression of mRNA. (A) qPCR
analysis of StAR transcription in MA-10 testis cells. (B) XY and XZ projection of StAR mRNA and protein in MA-10 cells after 3 h of stimulation by Br-cAMP (1 mM). (C) CRISPR/Cas9 system without gRNA. (a) The high sensitivity of FISH image to StAR mRNA with sp-RNA probes. The dotted circles indicate the dual-transfected
cells (left). GFP and mCherry’s dual fluorescence (right). (b) Quantitation of mRNA copy numbers in NT and GFP/mCherry (+) cells after 3 h of stimulation by
Br-cAMP (1 mM). Expression differences were not significant in the t-test (ns). (D). Dual CRISPR/Cas9 system with gRNAs. (a) High sensitivity to sp-RNA detection
of mRNA (left) with GFP and mCherry detection of the transfected cells (right). Expression difference was significant by Student’s t-test ***p < 0.001. Scale bar
represents 10 µm. Figure 4 | Role of gRNA in steroidogenic acute regulatory protein (StAR) editing; Br-cAMP stimulation of MA-10 Cells and suppression of mRNA. (A) qPCR
analysis of StAR transcription in MA-10 testis cells. (B) XY and XZ projection of StAR mRNA and protein in MA-10 cells after 3 h of stimulation by Br-cAMP (1 mM). (C) CRISPR/Cas9 system without gRNA. (a) The high sensitivity of FISH image to StAR mRNA with sp-RNA probes. The dotted circles indicate the dual-transfected
cells (left). GFP and mCherry’s dual fluorescence (right). (b) Quantitation of mRNA copy numbers in NT and GFP/mCherry (+) cells after 3 h of stimulation by
Br-cAMP (1 mM). Expression differences were not significant in the t-test (ns). (D). Dual CRISPR/Cas9 system with gRNAs. (a) High sensitivity to sp-RNA detection
of mRNA (left) with GFP and mCherry detection of the transfected cells (right). Time Course of CRISPR Initiation and
StAR Editing in Y-1 Cells The dashed lines (circle) indicate the nucleus. Blue
loci/dots are Cyp11a1, and red loci/dots are StAR intron 1. Orange channel (pseudocolored red) was used for the detection of StAR int1 and StAR oligomer set
targeting extended 3′UTR (3′EU). Red channel (pseudocolored blue) was used for Cyp11a1 int3. (D) Probes hybridizing CRISPR non-target sequences in StAR
(3′EU) and cyp11a1 were applied in CRISPR (+) and NT cells. Red loci/dots are StAR exon 7. (E) Quantitation of StAR intron 1. The blue segment in the bar denotes
the percentage of cells with the active locus (StAR p-RNA). Scale bar represents 10 µm. Figure 5 | Detection of DNA deletion from the steroidogenic acute regulatory protein (STAR) loci of Y-1 cells by Cas9. (A) Probe design of DNA FISH. The probe
hybridizes the StAR intron 1 (538/559 nm, pseudocolored red) in the CRISPR targeted sequence compared to non-target exon 7 [UTR/oligomer set targeting
extended 3′UTR (3′EU), 538/559 nm, pseudocolored red] and Cyp11a1 int3 (618/637 nm, pseudocolored blue). (B) Procedure for DNA FISH. After RNase
treatment, the slide was heated to denature DNA. Probes for CRISPR target StAR (int1) and non-target Cyp11a1 show the difference between NT and CRISPR (+)
cells (top). Non-target StAR (3′EU) and Cyp11a1 were used as a control (bottom). (C) StAR intron1 was deleted in CRISPR (+) cells. StAR intron 1 compared to
Cyp11a1 locus DNA retained in NT (a–d) cells. GFP and mCherry were used as markers for gRNA expression. The dashed lines (circle) indicate the nucleus. Blue
loci/dots are Cyp11a1, and red loci/dots are StAR intron 1. Orange channel (pseudocolored red) was used for the detection of StAR int1 and StAR oligomer set
targeting extended 3′UTR (3′EU). Red channel (pseudocolored blue) was used for Cyp11a1 int3. (D) Probes hybridizing CRISPR non-target sequences in StAR
(3′EU) and cyp11a1 were applied in CRISPR (+) and NT cells. Red loci/dots are StAR exon 7. (E) Quantitation of StAR intron 1. The blue segment in the bar denotes
the percentage of cells with the active locus (StAR p-RNA). Scale bar represents 10 µm. expression time profile. In Figure 5, we show that DNA hybridiza-
tion in intron 1 remains heterogeneous in ways that may relate
to the transcription access. The intensity of GFP fluorescence in
each cell has been assessed for all cells in the fields of two CRISPR-
transfected slide cultures at 4, 12, and 24 h [Figure 6A (b)]. Time Course of CRISPR Initiation and
StAR Editing in Y-1 Cells Expression difference was significant by Student’s t-test ***p < 0.001. Scale bar
represents 10 µm. Figure 4 | Role of gRNA in steroidogenic acute regulatory protein (StAR) editing; Br-cAMP stimulation of MA-10 Cells and suppression of mRNA. (A) qPCR
analysis of StAR transcription in MA-10 testis cells. (B) XY and XZ projection of StAR mRNA and protein in MA-10 cells after 3 h of stimulation by Br-cAMP (1 mM). (C) CRISPR/Cas9 system without gRNA. (a) The high sensitivity of FISH image to StAR mRNA with sp-RNA probes. The dotted circles indicate the dual-transfected
cells (left). GFP and mCherry’s dual fluorescence (right). (b) Quantitation of mRNA copy numbers in NT and GFP/mCherry (+) cells after 3 h of stimulation by
Br-cAMP (1 mM). Expression differences were not significant in the t-test (ns). (D). Dual CRISPR/Cas9 system with gRNAs. (a) High sensitivity to sp-RNA detection
of mRNA (left) with GFP and mCherry detection of the transfected cells (right). Expression difference was significant by Student’s t-test ***p < 0.001. Scale bar
represents 10 µm. DSB within a few minutes of their expression, which is marked by
synthesis of GFP and mCherry proteins. In this event, the Cas9/
StAR editing is likely to occur on the leading edge of the GFP/RFP periods of CRISPR/Cas9 activity on StAR expression (locus sp-
RNA versus cytoplasmic mRNA and protein). The gRNA/Cas9’s
complex interacts with the StAR gene and generates the initial October 2017 | Volume 8 | Article 289 Frontiers in Endocrinology | www.frontiersin.org 10 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate Figure 5 | Detection of DNA deletion from the steroidogenic acute regulatory protein (STAR) loci of Y-1 cells by Cas9. (A) Probe design of DNA FISH. The probe
hybridizes the StAR intron 1 (538/559 nm, pseudocolored red) in the CRISPR targeted sequence compared to non-target exon 7 [UTR/oligomer set targeting
extended 3′UTR (3′EU), 538/559 nm, pseudocolored red] and Cyp11a1 int3 (618/637 nm, pseudocolored blue). (B) Procedure for DNA FISH. After RNase
treatment, the slide was heated to denature DNA. Probes for CRISPR target StAR (int1) and non-target Cyp11a1 show the difference between NT and CRISPR (+)
cells (top). Non-target StAR (3′EU) and Cyp11a1 were used as a control (bottom). (C) StAR intron1 was deleted in CRISPR (+) cells. StAR intron 1 compared to
Cyp11a1 locus DNA retained in NT (a–d) cells. GFP and mCherry were used as markers for gRNA expression. Time Course of CRISPR Initiation and
StAR Editing in Y-1 Cells We
have used GFP fluorescence as the marker since mCherry expres-
sion overlapped in all cases. The GFP expression was robust in
some cells after 4 h and approximately doubled in both numbers
and intensities after 12 h. Between 12 and 24 h the number of cells
expressing GFP remained at about 30%, but the mean expression has doubled. We presented three GFP expression ranges: low/
background in which transfection is minimal, medium, which is
sufficient to generate DSB and a higher level for which the editing
impact is unknown [Figure 6A (c)]. We have used the PCR amplification of StAR DNA to ana-
lyze the effects of the single and dual CRISPR/Cas9 processes
on the StAR locus (Figure 3C). We examined the excision as a
time course in relation to the CRISPR/Cas9 GFP transfection
[Figure 6A (d)]. After 12 h, there was about a 15–20% excision
compared to the 50% excision after 24 h. However, after 4 h, the October 2017 | Volume 8 | Article 289 Frontiers in Endocrinology | www.frontiersin.org 11 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate Figure 6 | (A) Time course of steroidogenic acute regulatory protein (StAR) editing in relation to Cas9 transfection. (a) Design for varying post-transfection time for
CRISPR editing: after the transfection period, Br-cAMP (1 mM) was treated for 2 h. p-RNA, sp-RNA/mRNA, and protein in CRISPR (+) and NT cells were analyzed
by high-resolution fluorescence in situ hybridization. (b) Duplicate transfections (#1 and #2) were used to show GFP expression in cells as a function of post-
transfection time (4, 12, and 24 h). (c) Relative GFP intensity in individual cells was shown. Two dotted lines separate three levels of expression: lower—background;
middle—effective CRISPR (+); upper—effective but in excess of the amount needed for CRISPR. (d) PCR strategy to validate StAR gene deletion. Time course
experiment was performed (2–24 h). (B) Impact of CRISPR on StAR editing at various transfection times: (a) StAR locus (sp-RNA), 4 versus 12 h; (b,c) loss of mRNA
and protein, 4, 12, and 24 h. (d) The impact of CRISPR/Cas9 on StAR mRNA and protein. Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not
significant by ANOVA with post hoc Tukey Scale bar represents 10 µm Figure 6 | (A) Time course of steroidogenic acute regulatory protein (StAR) editing in relation to Cas9 transfection. Time Course of CRISPR Initiation and
StAR Editing in Y-1 Cells (a) Design for varying post-transfection time for
CRISPR editing: after the transfection period, Br-cAMP (1 mM) was treated for 2 h. p-RNA, sp-RNA/mRNA, and protein in CRISPR (+) and NT cells were analyzed
by high-resolution fluorescence in situ hybridization. (b) Duplicate transfections (#1 and #2) were used to show GFP expression in cells as a function of post-
transfection time (4, 12, and 24 h). (c) Relative GFP intensity in individual cells was shown. Two dotted lines separate three levels of expression: lower—background;
middle—effective CRISPR (+); upper—effective but in excess of the amount needed for CRISPR. (d) PCR strategy to validate StAR gene deletion. Time course
experiment was performed (2–24 h). (B) Impact of CRISPR on StAR editing at various transfection times: (a) StAR locus (sp-RNA), 4 versus 12 h; (b,c) loss of mRNA
and protein, 4, 12, and 24 h. (d) The impact of CRISPR/Cas9 on StAR mRNA and protein. Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not
significant by ANOVA with post hoc Tukey. Scale bar represents 10 µm. October 2017 | Volume 8 | Article 289 12 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate transfection (Figure 7C). To assess this adaptation to the Cas9
editing of StAR, we examined ORO staining in Y-1 cells at 12,
24, and 48 h after the CRISPR transfection. Each time, Br-cAMP
was added for the final 1 h to match the conditions used for the
assessment of StAR editing. A comparison of ORO staining in
CRISPR (+) and adjacent NT cells shows a large increase in CE/
ORO in the CRISPR (+) cells that lack StAR activity (Figure 7D). excision fragment was not detectable despite CRISPR/Cas9 trans-
fection at about half the 12 h level. The 0.65 kb product should
be readily detectable at 10% of the 24 h level representing 5%
excision. Overall, the excision process matched the intensity of
CRISPR/Cas9 transfection. We examined the effects of editing in CRISPR (+) cells at the
three times after CRISPR transfection followed by a 2 h Br-cAMP
stimulation. Threefold increases in sp-RNA/mRNA in Y-1 cells
produced by Br-cAMP after 2 h (Figure 2C) matched the increase
in cytoplasmic mRNA (Figure 2D) and previous assessments of
this response (1, 33). Time Course of CRISPR Initiation and
StAR Editing in Y-1 Cells After 12 h, the suppression of locus
sp-RNA continued, but there was now a modest decline in StAR
mRNA and protein [Figure 6B (b–d)]. However, 24 h after transfec-
tion, there was the complete removal of StAR mRNA and protein
in CRISPR (+) cells. The decline in StAR protein follows the loss of
mRNA. The NT cells again showed modestly progressive increases. The more rapid effect on the expression at the loci compared
to the mRNA and protein indicated an editing mechanism that
discriminates between locus expression and mRNA generation. The complete losses of locus RNA, mRNA, and protein at 24 h
match the measured DNA deletion shown in Figure 6A (d). The
diminished effects on mRNA and protein at 4 and 12 h correspond
well with the diminished DNA deletion at these earlier times. The
extensive suppression of sp-RNA at 4 and 12 h far exceed the
DNA deletion at these times and therefore arise from another
effect of the CRISPR/Cas9. As noted earlier, the single 5′gRNA
CRISPR/Cas9 can impact StAR without any deletion. h
The more rapid effect on the expression at the loci compared
to the mRNA and protein indicated an editing mechanism that
discriminates between locus expression and mRNA generation. The complete losses of locus RNA, mRNA, and protein at 24 h
match the measured DNA deletion shown in Figure 6A (d). The
diminished effects on mRNA and protein at 4 and 12 h correspond
well with the diminished DNA deletion at these earlier times. The
extensive suppression of sp-RNA at 4 and 12 h far exceed the
DNA deletion at these times and therefore arise from another
effect of the CRISPR/Cas9. As noted earlier, the single 5′gRNA
CRISPR/Cas9 can impact StAR without any deletion. f
g
p
The relationship between ORO and StAR editing remains simi-
lar to the plot for GFP accumulation. The accumulation of GFP
in vacuoles raised a possibility of interference with CE trafficking. To check this possibility, we followed the conditions shown in
Figure 4C and omitted both the gRNAs that are necessary for the
Cas9 cleavage of StAR without affecting GFP expression. We meas-
ured the ORO changes in NT and GFP (+) cells. Without gRNA
to initiate StAR editing, the GFP (+) cells showed no stimulation
of ORO staining to match the high cytoplasmic GFP expression
[Figure S7A in Supplementary Material]. Time Course of CRISPR Initiation and
StAR Editing in Y-1 Cells This control was con-
firmed for multiple gRNA (−) cells [Figure S7B in Supplementary
Material]. The significant increase in CE accumulations, therefore,
directly derives within a few hours from the redirection of cyto-
plasmic cholesterol dynamics resulting from the removal of StAR. Time Course of CRISPR Initiation and
StAR Editing in Y-1 Cells Thus, at least 75% of the RNA at the end of
each 2 h Br-cAMP treatment was produced within this period
and after the CRISPR–Cas9 transfection.t A high-resolution N-SIM image of ORO in adjacent NT and
CRISPR (+) cells showed CE/ORO lining the inside of vacuoles
[Figure 7E (a)]. We further examined the NT/CRISPR (+) pair
[Figure 7E (a)] with a 3D compilation [Figure 7E (b)]. For the
most part, the ORO accumulations are separate from the GFP
accumulations and are positioned above them. There are also
many vacuoles lined with GFP. The second set of three CRISPR
(+) cells [Figure 7E (c)] showed appreciable local heterogeneity
in the stimulation of ORO (+) CE accumulations. There was little
difference in the accumulation of CE regardless of the presence
or absence of Br-cAMP, which activates both StAR and HSL. The regions of high ORO staining in NT cells again avoided
regions of StAR expression in these cells [Figure 7F (a)]. We
also tested the effect of a 1 h addition of the Cyp11a1 inhibitor
aminoglutethimide (AMG), which fully inhibits mitochondrial
cholesterol metabolism (39). AMG also increased ORO staining
in NT cells in regions devoid of StAR. After the AMG treatment,
60% of the basal cells showed increased ORO with enlarged
LDs or CE-laden endosomes, much like what was seen in StAR
deficiency in CRISPR (+) cells. The AMG treatment of CRISPR
(+) cells produced additive increases in ORO [Figure 7F (b)]. The effect of AMG is remarkably rapid, raising the question of
whether AMG is affecting CE distribution at an additional point.h When the stimulation was initiated after 4 h, we saw the complete
loss of locus sp-RNA expression from CRISPR (+) cells [Figure 6B
(a)]. Surprisingly, mRNA expression was retained (Figure S6 in
Supplementary Material). After 12 h, the suppression of locus
sp-RNA continued, but there was now a modest decline in StAR
mRNA and protein [Figure 6B (b–d)]. However, 24 h after transfec-
tion, there was the complete removal of StAR mRNA and protein
in CRISPR (+) cells. The decline in StAR protein follows the loss of
mRNA. The NT cells again showed modestly progressive increases.hf When the stimulation was initiated after 4 h, we saw the complete
loss of locus sp-RNA expression from CRISPR (+) cells [Figure 6B
(a)]. Surprisingly, mRNA expression was retained (Figure S6 in
Supplementary Material). LDs Accumulate in CRISPR (+) Y-1 Cells
but Only after StAR mRNA Deletionf but Only after StAR mRNA Deletion
We cannot directly image the effect of StAR on steroid synthesis,
but LDs, which can be readily imaged by ORO, may provide an
alternative target (19). In steroidogenic cells, these droplets mostly
comprise CE. The CE content of LDs reflects the complex choles-
terol dynamics of the cytoplasm, which involves transport through
endosomes, biosynthesis from acetyl-CoA, and acylation by cho-
lesterol acyltransferase (34). LDs include some endosomes that are
filled with CE derived from low-density lipoprotein (LDL) but in
rodent cells mostly comprise CE droplets that derive from high-
density lipoprotein (HDL) taken up by Srb1. These droplets are
surrounded by organizing proteins that also control interactions
with processing enzymes such as HSL and StAR (18). A change of
CE in LDs in CRISPR (+) cells visualized by ORO may, therefore,
represent an adaptation to StAR deletion CRISPR (+) (Figure 7A). There appears to be an inverse relationship between the levels
and location of ORO (+) droplets and the mitochondria that
express StAR protein. Y-1 cells with high StAR have low ORO stain-
ing, and this inverse relationship extends to the cells (Figure 7B). We have shown that CRISPR (+) cells that are marked by GFP
show substantial increases in ORO, which start 12 h after CRISPR We cannot directly image the effect of StAR on steroid synthesis,
but LDs, which can be readily imaged by ORO, may provide an
alternative target (19). In steroidogenic cells, these droplets mostly
comprise CE. The CE content of LDs reflects the complex choles-
terol dynamics of the cytoplasm, which involves transport through
endosomes, biosynthesis from acetyl-CoA, and acylation by cho-
lesterol acyltransferase (34). LDs include some endosomes that are
filled with CE derived from low-density lipoprotein (LDL) but in
rodent cells mostly comprise CE droplets that derive from high-
density lipoprotein (HDL) taken up by Srb1. These droplets are
surrounded by organizing proteins that also control interactions
with processing enzymes such as HSL and StAR (18). A change of
CE in LDs in CRISPR (+) cells visualized by ORO may, therefore,
represent an adaptation to StAR deletion CRISPR (+) (Figure 7A).h Frontiers in Endocrinology | www.frontiersin.org DISCUSSION This report describes the direct imaging with sm-FISH of the
dual-gRNA-directed CRISPR/Cas9 inactivation of the key
steroidogenic gatekeeper gene StAR. PCR analyses showed the
excision of a central segment of proximal promoter/exon 1/intron
1 from the StAR genomic DNA. In addition, sm-FISH provided
comprehensive single-cell imaging of CRISPR editing at the
level of primary and spliced transcripts at gene loci and single
molecules of cytoplasmic mRNA in relation to expressed protein
and gene function. The sm-FISH method has also been extended
to provide novel direct single-cell imaging of the DNA deletion in There appears to be an inverse relationship between the levels
and location of ORO (+) droplets and the mitochondria that
express StAR protein. Y-1 cells with high StAR have low ORO stain-
ing, and this inverse relationship extends to the cells (Figure 7B). We have shown that CRISPR (+) cells that are marked by GFP
show substantial increases in ORO, which start 12 h after CRISPR October 2017 | Volume 8 | Article 289 Frontiers in Endocrinology | www.frontiersin.org 13 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate Figure 7 | Cholesterol esters (CEs) in lipid droplets (LDs) appear selectively and rapidly in cells with CRISPR steroidogenic acute regulatory protein (StAR) deletion. (A) Model for StAR cytoplasmic activity. Protein kinase A activates CE conversion to cholesterol (CH) mediated by the hormone-sensitive lipase; transfer into the
mitochondria-mediated by StAR leading to CYP-mediated metabolism (Cyp11a1 > Cyp27a1). StAR may also impact cholesterol uptake into the cell mediated by
the high-density lipoprotein (HDL) receptor Srb1; internal transfer in and out of LDs and exit from the cell due to an ATP-dependent pump activity (Abca1, Abcg1). StAR deletion increases CE-containing LDs visualized and quantified through Oil Red O (ORO) staining. (B) CE in LDs measured by ORO in Y-1 cells after 24 h of
CRISPR transfection separated from StAR protein expression in NT cells. CRISPR (+) cells are circled. (C) ORO staining in Y-1 cells at 12 and 24 h after the CRISPR
transfection. (D) Quantitation of ORO intensity in individual cells at 12–48 h after transfection in Y-1 cells. (E) N-SIM images of GFP expression and ORO droplets in
Y-1 cells. (a) XY section of CRISPR (+) cells expressing GFP and showing high ORO and an adjacent NT cell with typical low ORO. DISCUSSION We also observed this accumulation in
humans with StAR deficiency and in StAR−/− mice (40). i
However, in addition to excision, 30% of the CRISPR (+) cells
retained this StAR sequence and normal p-RNA and sp-RNA tran-
scripts at the loci, but remarkably, lost StAR mRNA and protein in
the cytoplasm (Figure 3). We detected similar losses when using
a single 5′gRNA (but not a 3′gRNA) (Figure S5 in Supplementary
Material), thus establishing that alternative mutation-based
mechanisms can operate in parallel with DNA excision.fi g
y
The 4-h CRISPR effect was consistent with disruption of the
uncoupled process, which provided locus RNA but contributed
little to mRNA. The slower 12–24 h alternative editing without
excision targeted the rapid coupled process, which did not con-
tribute to locus p-RNA or sp-RNA but generated most of the StAR
mRNA and protein. This alternative to CRISPR-induced excision
implies a distinct slower Cas9 intervention in the StAR promoter
that may arise as an error in the excision ligation step (Figure S8B
in Supplementary Material). The equivalent effects of the single
5′gRNA CRISPR/Cas9 strategy established that a single promoter
intervention proximal to the 5′PAM site can produce such changes,
presumably from mutations derived from errors in the initial
NHEJ-type repair process (42). Interestingly, the endonuclease
cleavage site overlapped the C/EBPβ-binding sequence, which
likely mediated of the delayed coupled StAR transcription (41).h p
p
Y-1 cells exhibit modest basal StAR expression, which is suffi-
cient to sustain maximum cholesterol metabolism when activated
by Br-cAMP (7). This expression is diversely distributed among
the individual cells and their loci [Figure 2 (b)]. The impact on
basal expression in CRISPR (+) cells is consistent with deletion
but currently, lacks a sufficient number of images from more active
basal cells to achieve a statistical assessment. We overcame this dif-
ficulty by applying CRISPR/Cas9 before the appreciable induction
by Br-cAMP, when most cells exhibit strong locus and cytoplasmic
responses (Figures 2 and 3). The precision of the sm-FISH in
resolving transcription and splicing at the locus from processing
to mRNA established distinct early (4 h) and delayed (12–24 h)
alternative disruptions of the StAR locus without deletion that
nevertheless retained transcription. Sm-FISH analyses of MA-10
cells differ in that they lack measurable basal locus and cytoplasm
expression. This distinction facilitated our analysis of the complex
StAR response to Br-cAMP stimulation (Figure 4; Figure S4 in
Supplementary Material). DISCUSSION We applied this dual gRNA
Cas9 targeting method to the StAR gene in Y-1 adrenal cells and
MA-10 interstitial testis cells, each of which showed an acute PKA
stimulation, thus providing extra insight into CRISPR’s impact. This strategy, which is applicable to any responsive gene, provides
novel direct assessment of the immediate impact of CRISPR edit-
ing on both the gene expression mechanism and the function.l These two alternative CRISPR editing effects were consistent
with selective interventions in two StAR multistep expression
processes that functioned in parallel after 2 h of Br-cAMP
stimulation of both Y-1 or MA-10 cells (1, 33) (Figure S8 in
Supplementary Material). StAR loci are initially activated with
substantial restraints over splicing and possibly polyadenylation
such that p-RNA and sp-RNA build up at the loci with the only
slow transfer of mRNA to the cytoplasm. Protein translation,
however, was robust despite this low cytoplasmic mRNA. This
early stimulus corresponded to maximum cholesterol metabo-
lism. This activity depends on the translation of a labile 3.5 kb
mRNA that locates to mitochondria. This location depends
on the extended 3′UTR of StAR, which delivers cleavage and
polyadenylation at specific sites (30). After about 1 h, a much
faster transcription process began in which formation of p-RNA
at loci is bypassed through a coupling of splicing to transcription. We suspect that this surge derived from Br-cAMP induction of
additional StAR transcription factors, notably c/EBPβ and
NR4a1 (6, 41). The rapid sp-RNA processing meant that sp-RNA
remained low in the loci while mRNA accumulated extensively
in the cytoplasm, particularly at the mitochondria. At the end of
a 2-h Br-cAMP stimulus, both transcription mechanisms were
functioning equally. We marked CRISPR (+) cells using mCherry and GFP fluo-
rescence (Figures 2, 4 and 6), which additionally provides a time
course for effective transfection (Figure 6). PCR shows that half
of the StAR DNA exhibited the predicted 1.1 kb deletion 24 h
after transfection (Figure 6), while sm-FISH shows a complete
loss of StAR expression of mRNA and protein from CRISPR (+)
cells (Figure 3). This loss was matched by excisions of the dual
CRISPR target sequence as shown by sm-FISH assessment of
intron 1 (Figure 5). This loss of StAR expression was matched by a
large increase in CE accumulation in LDs and endosomes, which
we imaged using ORO. DISCUSSION (b) The 3D assemblage of the
same cell (c) different CRISPR cell with more extensive ORO staining but heterogeneous distribution. (F) Distribution of ORO and StAR in NT cells under basal
conditions and after 1 h stimulation by Br-cAMP or aminoglutethimide. Left and right panels show same cells marked by, respectively, GFP and ORO (left) and StAR
protein. In each case, ORO several large LDs locate together and displace StAR/mitochondria. Figure 7 | Cholesterol esters (CEs) in lipid droplets (LDs) appear selectively and rapidly in cells with CRISPR steroidogenic acute regulatory protein (StAR) deletion. (A) Model for StAR cytoplasmic activity. Protein kinase A activates CE conversion to cholesterol (CH) mediated by the hormone-sensitive lipase; transfer into the
mitochondria-mediated by StAR leading to CYP-mediated metabolism (Cyp11a1 > Cyp27a1). StAR may also impact cholesterol uptake into the cell mediated by
the high-density lipoprotein (HDL) receptor Srb1; internal transfer in and out of LDs and exit from the cell due to an ATP-dependent pump activity (Abca1, Abcg1). StAR deletion increases CE-containing LDs visualized and quantified through Oil Red O (ORO) staining. (B) CE in LDs measured by ORO in Y-1 cells after 24 h of
CRISPR transfection separated from StAR protein expression in NT cells. CRISPR (+) cells are circled. (C) ORO staining in Y-1 cells at 12 and 24 h after the CRISPR
transfection. (D) Quantitation of ORO intensity in individual cells at 12–48 h after transfection in Y-1 cells. (E) N-SIM images of GFP expression and ORO droplets in
Y-1 cells. (a) XY section of CRISPR (+) cells expressing GFP and showing high ORO and an adjacent NT cell with typical low ORO. (b) The 3D assemblage of the
same cell (c) different CRISPR cell with more extensive ORO staining but heterogeneous distribution. (F) Distribution of ORO and StAR in NT cells under basal
conditions and after 1 h stimulation by Br-cAMP or aminoglutethimide. Left and right panels show same cells marked by, respectively, GFP and ORO (left) and StAR
protein. In each case, ORO several large LDs locate together and displace StAR/mitochondria. October 2017 | Volume 8 | Article 289 Frontiers in Endocrinology | www.frontiersin.org 14 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate CRISPR (+) cells and adjacent NT cells. The extent of this StAR
deletion is compared to retentions of, respectively, a non-targeted
segment of the StAR gene and the Cyp11a1 gene, the immediate
partner in the steroidogenic pathway. Frontiers in Endocrinology | www.frontiersin.org DISCUSSION The time course for CRISPR/Cas9
effects showed that editing was effective within 4 h in removing
StAR expression at the locus, even though mRNA and protein
were retained. Examination of single 5′end targeting was selec-
tively effective within 24 h in suppressing StAR expression without
DNA deletion suggesting that this 4 h disruption may arise from
an error in the early DSB repair of the 5′gRNA Cas9 process in the
proximal promoter. Alternatively, direct inhibition of StAR tran-
scription factors such as CREB or C/EBPβ may be produced by
this high affinity Cas9 complex. Removal of this complex through
the slow mutation will remove this direct disruption. The CRISPR editing machinery appears to function more rap-
idly than expected based on the in vitro modeling (43). However,
a novel recent approach to the measurement of the dissociation of
gRNA/Cas9 complexes in cells has indicated much faster rates (44). These cell dissociation rates vary appreciably, probably because of
different competing binding effects from transcription factors. Here, the 5′gRNA interaction in the StAR proximal promoter
region overlapped several functional transcription factor sites.h The role of StAR as a regulator of cholesterol metabolism in
mitochondria has been extensively documented (7, 45). Here, the
CRISPR/Cas9 removal of the StAR activity between 12 and 24 h
has been linked to a large increase in CE in LDs (Figure 7), which
StAR affects through interactions with a HSL and activation of
alternative transcription processes mediated by LXRa (22, 46). This
accumulation in single cells imaged by ORO staining provides a
measure of the cell adaptation to StAR loss (Figure 7A). Increases
in CE in LD and late endosomes in these cells were enriched in
regions where StAR protein and associated mitochondria were low
(Figure 7). The Cyp11a1/cholesterol metabolism inhibitor AMG October 2017 | Volume 8 | Article 289 Frontiers in Endocrinology | www.frontiersin.org 15 Dual CRISPR/Cas9-Mediated Gene Deletion Lee and Jefcoate Figure 8 | Schematic diagrams of selective CRISPR/Cas9 impact on synthesis of steroidogenic acute regulatory protein (StAR) mRNA and cholesterol transfer
activity in the cytoplasm. CRISPR/Cas9 editing of StAR increases accumulation of cholesterol esters (CEs) detected by Oil Red O (ORO). Newly translated p37 StAR
becomes phosphorylated by protein kinase A and then imported into mitochondria concomitant with cholesterol (CH) transfer and metabolism by Cyp11a1 (52). FUNDING This study was financially supported by NIH (RO1 DK074819,
RO1 DK090249). This research was also supported by funding
from the University of Wisconsin School of Medicine and Public
Health Department of Cell and Regenerative Biology. AUTHOR CONTRIBUTIONS JL and CJ designed and analyzed the experiments; wrote the
manuscript. JL carried out all experiments. DISCUSSION StAR deletion appears
to affect enhance the complex cytoplasmic cholesterol exchange
dynamics involving cholesterol movement between multiple orga-
nelles. Interestingly, NPC/STARD3 mediates removal from late
endosomes/lysosomes (LE/Ly), which are inhibited by lipophilic
amines (48) that may potentially include AMG. NPC1/Stard3
mediates contacts between LE/Ly and endoplasmic reticulum
(ER), which mediate cholesterol transfer between these organelles
(49, 50). ER contacts with mitochondria facilitate their fusion,
which is now recognized as a prime site of StAR activity (51). This rapid response to dual CRISPR Cas9 editing of StAR closely
matched the cytoplasmic large CE accumulations in the adrenals,
testes, and ovaries of StAR−/− mice, as well as in humans, who carry
the loss of function StAR mutations (40).f the means to identify alternative variations, for example,
changing the gRNA sites while also providing insight into the
optimum conditions for more detailed single-cell cloning or
sequencing. ACKNOWLEDGMENTS We thank Dr. Mario Ascoli for the MA-10 mouse Leydig tumor
cell, and Dr. Bernard Schimmer for the Y-1 cell line. DISCUSSION Without phosphorylation, StAR may promote cytoplasmic steps that promote exit of excess CH from the cells; for example by stimulation of liver X receptor (LXR)
induction of ATP-dependent cholesterol export pumps: AbcA1 to low-density lipoprotein (LDL) and AbcG1 to high-density lipoprotein (HDL) (21). Entry from LDL
deposits cholesterol in the late endosomes/lysosomes. Re-distribution, including endoplasmic reticulum or mitochondria, is directed by NPC1 and 2 together with
StARD3/MLN64 (48). Entry from HDL deposits CE in LDs. CE is converted to CH by hormone-sensitive lipase (HSL) and in LE by acid-sensitive esterase (18, 53). Acylation by cholesterol acyltransferase (ACAT) produces the reverse formation of CE. StAR inactivation by Cas9 increases CE. This can arise through decreased
removal from LE and LD and decreased export from the cell via AbcA1 and AbcG1. Aminoglutethimide (AMG) and StAR/Cas9 share similar effects on ORO/CE. Decreased CH metabolism cannot produce AMG effect (1 h). Other points of overlap include effects on mitochondrial cholesterol that reduce ATP, or decreased
distribution of CE from LE. Lipophilic amines like AMG, notably imipramine, disrupt the release of CH from LE (54–56). StAR may also mediate the activation of LXR,
notably to maintain AbcA1-mediated CH transport. Figure 8 | Schematic diagrams of selective CRISPR/Cas9 impact on synthesis of steroidogenic acute regulatory protein (StAR) mRNA and cholesterol transfer
activity in the cytoplasm. CRISPR/Cas9 editing of StAR increases accumulation of cholesterol esters (CEs) detected by Oil Red O (ORO). Newly translated p37 StAR
becomes phosphorylated by protein kinase A and then imported into mitochondria concomitant with cholesterol (CH) transfer and metabolism by Cyp11a1 (52). Without phosphorylation, StAR may promote cytoplasmic steps that promote exit of excess CH from the cells; for example by stimulation of liver X receptor (LXR)
induction of ATP-dependent cholesterol export pumps: AbcA1 to low-density lipoprotein (LDL) and AbcG1 to high-density lipoprotein (HDL) (21). Entry from LDL
deposits cholesterol in the late endosomes/lysosomes. Re-distribution, including endoplasmic reticulum or mitochondria, is directed by NPC1 and 2 together with
StARD3/MLN64 (48). Entry from HDL deposits CE in LDs. CE is converted to CH by hormone-sensitive lipase (HSL) and in LE by acid-sensitive esterase (18, 53). Acylation by cholesterol acyltransferase (ACAT) produces the reverse formation of CE. StAR inactivation by Cas9 increases CE. This can arise through decreased
removal from LE and LD and decreased export from the cell via AbcA1 and AbcG1. Aminoglutethimide (AMG) and StAR/Cas9 share similar effects on ORO/CE. DISCUSSION Decreased CH metabolism cannot produce AMG effect (1 h). Other points of overlap include effects on mitochondrial cholesterol that reduce ATP, or decreased
distribution of CE from LE. Lipophilic amines like AMG, notably imipramine, disrupt the release of CH from LE (54–56). StAR may also mediate the activation of LXR,
notably to maintain AbcA1-mediated CH transport. (39) rapidly increased these effects in NT cells independent of
StAR, making the involvement of cholesterol metabolism unlikely. Each ORO image following StAR deletion in CRISPR (+) cells
and AMG stimulation in NT cells showed similar accumulations
in cytoplasmic endosome-like vacuoles (Figure 7E). CE in LDs
is supplied by HDL via SRB1 at the plasma membrane but in
late endosomes by LDL through LDL receptors. Each import is
balanced by ATP-dependent export pumps (Abcg1 and Abca1). Potential mechanisms include stimulation of ATP-dependent cho-
lesterol export (47), mitochondrial ATP and apoptotic processes
(46), and the effects of hydroxyl-cholesterol metabolites on LXR
receptors (21). These cytoplasmic disruptions are represented
in relation to CRISPR editing (Figure 8). StAR deletion appears
to affect enhance the complex cytoplasmic cholesterol exchange
dynamics involving cholesterol movement between multiple orga-
nelles. Interestingly, NPC/STARD3 mediates removal from late
endosomes/lysosomes (LE/Ly), which are inhibited by lipophilic
amines (48) that may potentially include AMG. NPC1/Stard3
mediates contacts between LE/Ly and endoplasmic reticulum
(ER), which mediate cholesterol transfer between these organelles
(49, 50). ER contacts with mitochondria facilitate their fusion,
which is now recognized as a prime site of StAR activity (51). This rapid response to dual CRISPR Cas9 editing of StAR closely
matched the cytoplasmic large CE accumulations in the adrenals,
testes, and ovaries of StAR−/− mice, as well as in humans, who carry
the loss of function StAR mutations (40).f (39) rapidly increased these effects in NT cells independent of
StAR, making the involvement of cholesterol metabolism unlikely. Each ORO image following StAR deletion in CRISPR (+) cells
and AMG stimulation in NT cells showed similar accumulations
in cytoplasmic endosome-like vacuoles (Figure 7E). CE in LDs
is supplied by HDL via SRB1 at the plasma membrane but in
late endosomes by LDL through LDL receptors. Each import is
balanced by ATP-dependent export pumps (Abcg1 and Abca1). Potential mechanisms include stimulation of ATP-dependent cho-
lesterol export (47), mitochondrial ATP and apoptotic processes
(46), and the effects of hydroxyl-cholesterol metabolites on LXR
receptors (21). These cytoplasmic disruptions are represented
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embj.201591481 Copyright © 2017 Lee and Jefcoate. This is an open-access article distributed under
the terms of the Creative Commons Attribution License (CC BY). The use, distribu-
tion or reproduction in other forums is permitted, provided the original author(s)
or licensor are credited and that the original publication in this journal is cited, in
accordance with accepted academic practice. No use, distribution or reproduction is
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embj.201695917 October 2017 | Volume 8 | Article 289 Frontiers in Endocrinology | www.frontiersin.org 18
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Mining tissue specificity, gene connectivity and disease association to reveal a set of genes that modify the action of disease causing genes
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BioMed Central BioMed Central g
Antonio Reverter*, Aaron Ingham and Brian P Dalrymple Address: Computational and Systems Biology, CSIRO Livestock Industries, Queensland Bioscience Precinct, 306 Carmody Road, St. Lucia,
B i b
Q
l
d
A
li Address: Computational and Systems Biology, CSIRO Livestock Industries, Queensland Bioscience Precinct, 306 Carmody Road, St. Lucia,
Brisbane, Queensland 4067, Australia Email: Antonio Reverter* - Tony.Reverter-Gomez@csiro.au; Aaron Ingham - Aaron.Ingham@csiro.au;
Brian P Dalrymple - Brian.Dalrymple@csiro.au
* Corresponding author Email: Antonio Reverter* - Tony.Reverter-Gomez@csiro.au; Aaron Ingham - Aaron.Ingham@csiro.au;
Brian P Dalrymple - Brian.Dalrymple@csiro.au Received: 7 January 2008
Accepted: 19 September 2008 Received: 7 January 2008
Accepted: 19 September 2008 BioData Mining 2008, 1:8
doi:10.1186/1756 0381 1 8
This article is available from: http://www.biodatamining.org/content/1/1/8 This article is available from: http://www.biodatamining.org/content/1/1/8 © 2008 Reverter et al; licensee BioMed Central Ltd. © 2008 Reverter 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. ticle distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2. d use, distribution, and reproduction in any medium, provided the original work is properly cited. Open Ac
Research
Mining tissue specificity, gene connectivity and disease association
to reveal a set of genes that modify the action of disease causing
genes
Antonio Reverter*, Aaron Ingham and Brian P Dalrymple Open Access Data resources, edits and nomenclature We merged three large datasets as follows: Firstly, we
accessed expression data drawn from massively parallel
signature sequencing (MPSS) covering 182,719 tag signa-
tures across 32 tissues [2]. Tissues represented on the
MPSS data included nine different central nervous system
(CNS) areas (amygdale, caudate nucleus, cerebellum, cor-
pus callosum, fetal brain, hypothalamus, thalamus, spinal
cord, and pituitary gland) and 23 non-CNS organs (adre-
nal gland, bladder, bone marrow, heart, kidney, lung,
mammary gland, pancreas, placenta, prostate, retina, sali-
vary gland, small intestine, spleen, stomach, testis, thy-
mus, thyroid, trachea, uterus, colon, monocytes and
peripheral blood lymphocytes). A total of 18,677 unique
genes were represented on the MPSS data and the number
of expressed genes per tissue averaged 8,943 and ranged
from 5,845 in pancreas to 12,267 in testis. Among the high-throughput technologies, gene expres-
sion profiling has led to the identification of genes that
perform in a coordinated manner allowing researchers to
reasonably predict the role of genes for which no biologi-
cal function was attributed, based on the known perform-
ance of other group members. These predictions rely on
the guilt-by-association heuristic, widely invoked in
genomics and with proven applicability [1]. At the same time, a comprehensive atlas of transcribed
genes in humans has revealed that genes may be split into
two broad categories based on the number of tissues they
are expressed in [2]. Genes that are expressed in many tis-
sues are designated as housekeeping (HK) while those
that are expressed in few tissues are termed tissue-specific
(TS). Secondly, we downloaded a set of 55,606 true positive
interactions among 7,197 genes that were defined from
functional studies [15]. This interactions dataset was built
including 2,788 confirmed, direct, physical protein-pro-
tein interactions derived from the Biomolecular Interac-
tion Network Database (BIND; http://binddb.org) [16],
18,176 confirmed human protein interactions from the
Human Protein Reference Database (HPRD; http://
www.hprd.org/) [17], 22,012 direct functional interac-
tions from the Kyoto Encyclopedia of Genes and
Genomes (KEGG; http://www.genome.jp/kegg) [18], and
16,295 interactions derived from Reactome http://
www.reactome.org[19]. Tissue specificity has subsequently been linked to a
number of significant outcomes including level of expres-
sion [3], ability to detect cis-acting and trans-acting
expression- quantitative trait loci [4], and differential rates
of polymorphism [5], evolution [6] and disease-associa-
tion [7]. Abstract Background: The tissue specificity of gene expression has been linked to a number of significant
outcomes including level of expression, and differential rates of polymorphism, evolution and
disease association. Recent studies have also shown the importance of exploring differential gene
connectivity and sequence conservation in the identification of disease-associated genes. However,
no study relates gene interactions with tissue specificity and disease association. Methods: We adopted an a priori approach making as few assumptions as possible to analyse the
interplay among gene-gene interactions with tissue specificity and its subsequent likelihood of
association with disease. We mined three large datasets comprising expression data drawn from
massively parallel signature sequencing across 32 tissues, describing a set of 55,606 true positive
interactions for 7,197 genes, and microarray expression results generated during the profiling of
systemic inflammation, from which 126,543 interactions among 7,090 genes were reported. Results: Amongst the myriad of complex relationships identified between expression, disease,
connectivity and tissue specificity, some interesting patterns emerged. These include elevated rates
of expression and network connectivity in housekeeping and disease-associated tissue-specific
genes. We found that disease-associated genes are more likely to show tissue specific expression
and most frequently interact with other disease genes. Using the thresholds defined in these
observations, we develop a guilt-by-association algorithm and discover a group of 112 non-disease
annotated genes that predominantly interact with disease-associated genes, impacting on disease
outcomes. Conclusion: We conclude that parameters such as tissue specificity and network connectivity can
be used in combination to identify a group of genes, not previously confirmed as disease causing,
that are involved in interactions with disease causing genes. Our guilt-by-association algorithm
should be useful for the discovery of additional modifiers of genetic diseases, and more generally,
for the ability to associate genes of unknown function to clusters of genes with defined functions
allowing for novel biological inference that can be subsequently validated. Page 1 of 11
(page number not for citation purposes) http://www.biodatamining.org/content/1/1/8 BioData Mining 2008, 1:8 Background disease, may impact the development of diseases, includ-
ing cancers, and hypothesize that many other members of
this list will ultimately be confirmed as modifiers of vari-
ous genetic diseases. g
The understanding of the biology underlying phenotype
is still a limiting factor in delivering the promise of high
throughput genomics. However, as new datasets are avail-
able, new data mining methods are developed and the
goal appears ever more achievable. The impact of tissue-specificity Among the myriad of complex relationships, some inter-
esting patterns emerged. Consistent with previous find-
ings [3], we observed a strong relationship between the
number of tissues in which a gene was expressed and its
level of expression (Table 1). Importantly, this relation-
ship was unaffected by disease or interaction status. Results and discussion
Initial gene groupings and unknown biological processes Initial gene groupings and unknown biological processes
Figure 1 illustrates the way in which the 15,050 genes
were simultaneously annotated as either disease-associ-
ated or included in the true positive interactions and the
inflammation datasets. These genes were further classified
as either TS, NS or HK, and the number of disease-associ-
ated and/or interacting genes contained within each of the
resulting 12 categories was determined. The proportion of
genes with unknown biological process was also regis-
tered. These criteria resulted in 15,050 genes [see Additional file
1] of which 5,198 and 4,950 were included in the true
positive interactions and the inflammation datasets,
respectively, and with 2,499 genes in common. In addi-
tion, a total of 6,151 (41%) of the genes were associated
with disease according to OMIM database [22] as of Sep-
tember 19, 2007; and with 1,445 of them defined as dis-
ease-causing (i.e., associated with either known disease
phenotype or polymorphic sequence known). As expected, the discovery of interactions as well as dis-
ease-association for a given gene provides additional bio-
logical knowledge, allowing inferences as to its genomic
functionality. Nevertheless, the biological process of
about 10% of these presumably well-characterized genes
remains to be elucidated. On the other extreme, and high-
lighting the extent to which further research is needed, as
many as 85% of NDIS, NINT genes and across the three
expression categories (TS, NS and HK) belong to an
unknown biological process. Hereafter, we refer to DIS to indicate the 6,151 genes from
our resulting dataset that are disease-associated according
to OMIM, and to NDIS to indicate the remaining 8,899
non-disease-associated genes also according to OMIM. Similarly, we refer to INT (and NINT) to indicate genes in
our dataset for which interactions have (and have not)
been reported. Data mining approaches In order to further characterize the relationship existing
between tissue specificity, gene connectivity and disease
association, the 15,050 genes were classified as either TS
or HK. To ensure that these two categories together repre-
sented the majority of the genes, we searched for category
limits from either extreme of the distribution of the
number of genes expressed in one, two, and up to 32 tis-
sues, until equivalent categories were defined, cumula-
tively representing > 50% of the total number of genes. In
doing so, there were 4,232 (28%) TS genes expressed in 1
to 4 tissues, and 4,006 (27%) HK genes expressed in more
than 25 tissues. The remaining 6,812 (45%) genes were
classified as non-specific (NS). Overall, the distribution of the expression of genes among
tissues was grossly bimodal. However, this bimodality
vanished when the distribution was examined separately
for INT and NINT genes (Figure 2). INT genes are over-
represented among HK genes, while NINT genes are pre-
dominantly TS. We conclude that the more tissues a gene
is expressed in, the higher its chances of interacting with
at least one other gene, irrespective of the tissue-specificity
of this second gene. Figure 3 presents the relationship between tissue specifi-
city and proportion of disease-associated genes. The over-
all Pearson correlation coefficient (PCC) was moderate
(0.53) yet significant (P = 0.0019) indicating an increase
in the number of DIS genes among broadly expressed
genes. Computing the PCC conditional on interaction sta-
tus results in a non-significant PCC of -0.26 (P = 0.1459)
for NINT genes, and a strong negative PCC of -0.73 (P <
0.0001) for INT genes. This counterintuitive pattern of
correlation is representative of the Simpson's Paradox
[23] with the paradox being that, although INT genes tend
to be expressed in many tissues, those that are expressed
in a tissue specific manner are more likely to be DIS. This
is likely due to the increased number of relationships an Finally, and in order to identify novel candidate genes
impacting disease, we developed a guilt-by-association
algorithm. Selection thresholds based on the average
number of known interactions combined with the average
proportion of DIS genes among their interactors were
determined from DIS genes. These thresholds were then
applied to genes in the NDIS category. Genes exceeding
both thresholds were identified as likely disease-associ-
ated candidates. Data resources, edits and nomenclature In addition, we [8] and others [9,10] have dem-
onstrated the importance of exploring differential gene
connectivity in the identification of disease-associated
genes using microarray gene expression data. More
recently, the combination of text mining with gene inter-
action network analysis has been proposed to infer
unknown gene-disease associations [11]. Furthermore, genes with a high degree of connectivity
(network hubs) have been shown to be conserved across
species [12] and their knockout phenotype more likely to
be lethal [13]. Finally, based on sequence conservation
across species, a computational algorithm has been devel-
oped to identify genes associated with disease [14]. How-
ever, no study relates gene interactions with tissue
specificity and its subsequent likelihood of association
with disease. Finally, we used the microarray expression results gener-
ated during the profiling of systemic inflammation across
44,924 probe sets [20] and from which 126,543 interac-
tions among 7,090 genes were reported [8]. The microar-
ray
experiment
used
92
Affymetrix
GeneChips
(Affymetrix, Santa Clara, CA) to examine gene expression
profiles in whole blood leukocytes immediately before
and at 2, 4, 6, 9 and 24 h after intravenous administration
of bacterial lipopolysaccharide (LPS) endotoxin to four
healthy human subjects. For the control (placebo) data,
four additional subjects were studied under identical con-
ditions but without LPS administration. To address this situation, we mined three large independ-
ent datasets and classified transcribed human genes based
on transcript abundance, tissue specificity, gene connec-
tivity and disease association. We discuss how these fac-
tors relate to each other and, based on this new
knowledge, implement a simple yet powerful guilt-by-
association algorithm that allows us to identify several
candidate genes that, while not previously associated with For the present study, and to enable the merging of the
three datasets, a number of edits were performed as fol-
lows: For the MPSS data, tags not expressed at more than
5 transcripts per million (tpm), in at least one tissue, were Page 2 of 11
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(page number not for citation purposes) Results and discussion
I i i l
i
d disregarded. The threshold of 5 tpm corresponds to the
sensitivity of MPSS technology as claimed by the manu-
facturers and independently assessed in our laboratory
[21]. Also, when the same gene was represented by more
than one MPSS tag, the reading from the most abundant
tag, summed across all tissues, was assigned to that gene. Finally, for the true positive interactions and the inflam-
mation datasets, interactions involving genes not sur-
veyed in the MPSS data were also discarded. Identification of candidate disease genes via guilt-by-
association Given our measurement confirming that like associates
with like, we developed a guilt-by-association algorithm
with the aim of identifying candidate genes among the
previously classified non-disease subset. Our guilt-by-
association algorithm starts by examining the connectivity
properties of the DIS genes. In this context, DIS genes
were found to be involved, on average, in 12 interactions
(ranging from 0 to 176). Also on average, their interactors
were themselves DIS genes in 75% of instances. Impor-
tantly, while only 1,132 (or 18.4%) of DIS genes had > 12
interactions (revealing the skewedness in the number of
interactions), 651 (or 57.5%) of them interacted with DIS
genes > 75% of the time. When these same thresholds
(i.e., > 12 interactions and > 75% of DIS genes among
interactors) were applied to NDIS genes, we revealed the
presence of 112 genes [see Additional file 2], including 26
TS, 50 NS and 36 HK, that while not being associated with
disease, have higher than average connectivity degree (> Data mining approaches Page 3 of 11
(page number not for citation purposes) Page 3 of 11
(page number not for citation purposes) http://www.biodatamining.org/content/1/1/8 BioData Mining 2008, 1:8 Gene groupings
Figure 1
Gene groupings. Genes were classified as tissue-specific (TS), non-specific (NS) or housekeeping (HK). Among each class,
the number of interacting and disease-associated genes is noted, and for each of the resulting 12 categories, the percentage of
genes with unknown biological process ontology is given. HK Y Y
HK N Y
HK Y N
HK N N
NS Y Y
NS N Y
NS Y N
NS N N
TS Y Y
TS N Y
TS Y N
TS N N
0 10 20 30 40 50 60 70 80 90 100
Interaction
Disease
# Genes
% Unknown Biological Process
1,581
219
1,288
918
2,034
775
1,325
2,678
954
588
467
2,223 Gene grou
Figure 1 Gene groupings
Figure 1
Gene groupings. Genes were classified as tissue-specific (TS), non-specific (NS) or housekeeping (HK). Among each class,
the number of interacting and disease-associated genes is noted, and for each of the resulting 12 categories, the percentage of
genes with unknown biological process ontology is given. genes (Figure 4). Consistent with our results, genes associ-
ated with similar disorders have been shown to have
higher likelihood of physical interactions between their
products and a higher expression profiling similarity for
their transcripts [24]. interacting HK gene would have compared to a TS equiv-
alent, thereby increasing the likelihood of a mutation
leading to a detrimental and potentially lethal outcome,
as previously determined [6]. We conclude that it is not so
much that TS genes are more likely to be associated with
disease, but rather that HK genes associated with disease
are rarely observed. Page 4 of 11
(page number not for citation purposes) Gene interactions in the context of tissue-specificity and
disease association Our analyses revealed that interacting HK genes are more
likely to interact with genes that are also HK (PCC = 0.89;
P < 0.0001) and vice-versa (i.e., TS genes are more likely
to interact among themselves). Importantly, this correla-
tion remained strong when conditioning on disease status
(Table 1). Also, interactions between two HK genes were
12.8 times more frequent (P < 0.0001) and 3.3 times
more cohesive (P < 0.0001) as measured by the clustering
coefficient, than interactions between two TS genes. The
clustering coefficient is a measure of network cohesive-
ness and captures how many neighbours of a given gene
are connected to each other. Similarly, interactions between two DIS genes were 3.1
times more frequent (P < 0.0001) and 1.6 times more
cohesive (P < 0.001) than interactions between two NDIS Page 4 of 11
(page number not for citation purposes) Page 4 of 11
(page number not for citation purposes) http://www.biodatamining.org/content/1/1/8 BioData Mining 2008, 1:8 Table 1: Relationship between the number of tissues in which a gene is expressed and a series of variables. Variable
Correlation
Regression
Expression:
Overall genes
0.706
2.034
Non-Interacting (NI) genes only
0.709
1.802
Interacting genes only
0.707
2.107
Non-Disease (ND)
0.707
1.769
Disease (D)
0.709
2.382
Non-Interacting and Non-Disease
0.691
1.759
Non-Interacting and Disease
0.764
2.039
Interacting and Non-Disease
0.719
1.803
Interacting and Disease
0.702
2.438
Proportion of interacting genes:
Overall genes
0.949
0.012
Non-Disease genes only
0.942
0.013
Disease genes only
0.917
0.008
Proportion of disease genes:
Overall
0.527
0.002
Non-Interacting genes only
-0.263
-0.001
Interacting genes only
-0.733
-0.004
Tissue specificity of interactors:
Overall genes
0.887
0.112
Non-Disease genes only
0.736
0.062
Disease genes only
0.872
0.151
Proportion of disease genes among interactors:
Overall genes
0.229
0.000
Non-Disease genes only
-0.048
0.000
Disease genes only
0.575
0.001 Table 1: Relationship between the number of tissues in which a gene is expressed and a series of variables. Table 1: Relationship between the number of tissues in which a gene is expressed and a series of variable In order to further ascertain the optimality of various loca-
tion parameters to be used as thresholds in the guilt-by-
association algorithm, we explored the proportion of truly
disease associated genes from the total number of cap-
tured genes and the results are presented in Table 3. While
the median performs slightly better (i.e. Gene interactions in the context of tissue-specificity and
disease association by up to 1.03
times better, or 78.9 over 76.3) than the mean when used
as a threshold for the proportion of disease genes among
interactors, this improvement is at the expense of generat-
ing substantially larger lists of candidate genes. When
exploring the number of connections, the mean is very
close to the 75th percentile, indicating the skewness in the
connectivity distribution with most genes having few con-
nections and few genes having many connections. Also, as
a threshold for the number of connections, the mean per-
forms favourably against either inter-quartile. 12 connections) and higher than average proportion (>
75%) of genes in OMIM among their connectors. Table 2
presents the number of genes in the contingency table
underlying our guilt-by-association algorithm. To assess the optimality of our approach, we repeated the
analyses using only the 1,445 DIS genes (out of the initial
6,151) with known disease phenotype and either
sequence mutation or molecular basis known as those
declared as truly disease-associated. The new thresholds
for connectivity and proportion of DIS genes among inter-
actors were 12 and 35%, respectively. The new list of can-
didate genes included 127 genes of which 107 were
assessed as DIS in the initial list of 6,151. Assuming the
remaining 20 genes are indeed false positives, this implies
a precision of at least 84%. However, the infeasibility of directly computing perform-
ance measures associated with a given algorithm in the
absence of negative examples should be acknowledged. That is, although one can be relatively sure that certain It should be noted that precision alone is not enough to
assess the goodness of a classifier, as it is only concerned
with the ratio of identified genes that are positive, but not
with the total number of discovered genes. Page 5 of 11
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http://www.biodatamining.org/content/1/1/8
Frequency histogram of gene expression
Figure 2
Frequency histogram of gene expression. For each gene, the tissues showing expression at more than 5 transcript per
million were counted and the histogram explored separately for non-interacting (green) and interacting genes (red). The two
distributions are statistically different (Kolmogorov-Smirnov test P-value < 0.001). Gene interactions in the context of tissue-specificity and
disease association Non-Interacting genes
Interacting genes http://www.biodatamining.org/content/1/1/8 BioData Mining 2008, 1:8 Non-Interacting genes
Interacting genes Non-Interacting genes
Interacting genes Frequency
Figure 2 q
y
g
g
p
g
Frequency histogram of gene expression. For each gene, the tissues showing expression at more than 5 transcript per
million were counted and the histogram explored separately for non-interacting (green) and interacting genes (red). The two
distributions are statistically different (Kolmogorov-Smirnov test P-value < 0.001). (hypergeometric P = 7.5 × 10-16) than the 14% predicted
by OMIM across the genome, with 2,549 genes defined as
the basis of heritable disease out of the 18,091 total. genes are associated with a disease, it is not possible to
ensure that a set of genes is not involved in any disease. In
other words: Absence of evidence is not evidence of
absence. On the other extreme, some of the genes anno-
tated as disease associated by OMIM could also be false
positives. In these situations, partially supervised learning
algorithms have been proposed to address this issue and
in the context of identifying disease genes [14]. Clusters of disease among candidate genes f
g
g
In order to determine what diseases these genes might
impact, we explored the gene networks spanned by the
members of our guilt-by-association list, alone and in
combination with their interactors. Based on the disease
associations shown [see Additional file 2], each cluster
was examined for a common disease. In this fashion, we
identified two clusters of genes that impact on either
breast or gastric cancer. Figure 5 depicts the Cytoscape
[25] representation of the breast cancer cluster where
seven of our guilt-by-association genes (APBA2BP,
CCNA2, COBRA1, PCAF, RAD51, SMARCA4 and
STAT5A) were linked to the well characterized human Nevertheless, a literature survey revealed that 44 of the
112 candidate genes [see Additional file 2] have been pre-
viously associated with polymorphisms or differential
gene expression leading to a modified risk of disease. A
further 10 genes exist within chromosomal regions associ-
ated with disease. The remaining 58 genes have no obvi-
ous association to disease in any system. The 39% rate of
disease association determined here is much higher Page 6 of 11
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http://www.biodatamining.org/content/1/1/8
Disease association and tissue specificity
Figure 3
Disease association and tissue specificity. Relationship between tissue specificity (x-axis) and proportion of disease-asso-
ciated genes (y-axis) computed using all genes (blue pattern), and separate for non-interacting (green pattern) and interacting
genes (red pattern). Number of tissues
Proportion of disease genes
1
7
13
19
25
31
0.1
0.3
0.5
0.7 http://www.biodatamining.org/content/1/1/8 BioData Mining 2008, 1:8 Disease as
Figure 3 Disease association and tissue specificity
Figure 3
Disease association and tissue specificity. Relationship between tissue specificity (x-axis) and proportion of disease-asso-
ciated genes (y-axis) computed using all genes (blue pattern), and separate for non-interacting (green pattern) and interacting
genes (red pattern). Conclusion 3.1
1.0
2.6
2.6
7.4
9.7
7.4
2.6
2.6
6.7
0.2
0.5
0.7
2.7
3.8
2.7
0.7
0.5
1.9
0.4
1.0
1.2
3.9
5.7
4.2
3.1
1.0
1.2
1.2
1.0
0.4
1.0
1.2
4.2
3.9
5.7
Disease……
Non-Disease
Non-Disease
Non-Disease
Disease……
Disease……
TS
NS
HK
TS
NS
HK 3.1
1.0
2.6
2.6
7.4
9.7
7.4
2.6
2.6
6.7
0.2
0.5
0.7
2.7
3.8
2.7
0.7
0.5
1.9
0.4
1.0
1.2
3.9
5.7
4.2
3.1
1.0
1.2
1.2
1.0
0.4
1.0
1.2
4.2
3.9
5.7
Disease……
Non-Disease
Non-Disease
Non-Disease
Disease……
Disease……
TS
NS
HK
TS
NS
HK Relating ge
Figure 4 Relating gene connectivity with disease association and tissue specificity
Figure 4
Relating gene connectivity with disease association and tissue specificity. Percentage of gene-gene interactions that
exists between two groups of genes depending on their tissue specificity (TS: tissue-specific, NS: non-specific, and HK: house-
keeping) and disease association. Colours indicate interactions between two disease-associated genes (red), between a disease-
associated and a non-disease-associated gene (yellow), and between two non-disease-associated genes (green). The size of the
rectangles indicates the relative number of interacting genes in each group. parameter in our guilt-by-association algorithm obviates
the need to also include tissue specificity. members of this list will ultimately be confirmed as mod-
ifiers of various genetic diseases. However, it should also be acknowledged that probability
values associated with testing the null hypothesis of a
given PCC not being statistically different from zero were
computed assuming asymptotic normality and as such are
prone to inaccuracies. With this in mind, we focussed on
combining discrete parameters such as number of connec-
tions and the association to disease-associated genes to
identify a group of genes, not previously confirmed as dis-
ease causing, that are involved in interactions with disease
causing genes. The nature of these newly identified inter-
actions could range from epistatic interactions (i.e., the
action of one gene is suppressed by another such as the
case of RAD51 and BRCA1) to physical gene-gene interac-
tions to correlated co-expression. Based on bibliographi-
cal validation and network re-construction we have
identified several candidate genes that may impact the
development of cancer and hypothesize that many other Finally, it should be noted that while new algorithms are
being proposed in the literature on a rather frantic pace,
the task of comprehensively comparing algorithms could
be unattainable if not futile. Conclusion breast cancer susceptibility genes, BRCA1 and BRCA2. Although none of these genes are annotated as disease
causing in OMIM, five have been previously associated
with the development of breast cancer, for example, alle-
les of RAD51 are epistatic with alleles of BRCA2. However,
CCNA2 is only mentioned in a very small number of
reports on breast cancer and APBA2BP is not a well stud-
ied gene. Data mining approaches have allowed us to gain an
insight into the complex relationships existing between
gene expression, disease association, network connectivity
and tissue specificity. We have identified elevated rates of
expression and network connectivity among broadly
expressed genes, and among disease-associated tissue-spe-
cific genes. In particular, when exploring the relationship between tis-
sue specificity and disease association, we found this rela-
tionship most interesting. While there is a moderate
positive relationship between the number of tissues in
which a gene is expressed and the proportion of disease
genes, we show that this relationship is reversed when
only considering genes for which interactions have been
reported. We present this phenomenon as an example of
the well-reported Simpson's Paradox. To a great extent,
the inclusion of number of interactions as a threshold For the case of gastric cancer, another cluster of seven
genes (AKT3, KRAS, MAP2K4, PIK3CB, PLCB1, PIK3R5
and PPP3R2) was identified. Four of these genes have
been previously associated with gastrointestinal disease
while AKT3, PIK3CB and PIK3R5 have not, although the
differential expression of AKT3 in gastric cancer is well
defined [see Additional file 2]. We suggest these previ-
ously non-associated genes are strong candidates for fur-
ther study into the basis of these diseases and are potential
prognostic markers. Page 7 of 11
(page number not for citation purposes) Page 7 of 11
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Figure 4
Relating gene connectivity with disease association and tissue specificity. Percentage of gene-gene interactions that
exists between two groups of genes depending on their tissue specificity (TS: tissue-specific, NS: non-specific, and HK: house-
keeping) and disease association. Colours indicate interactions between two disease-associated genes (red), between a disease-
associated and a non-disease-associated gene (yellow), and between two non-disease-associated genes (green). The size of the
rectangles indicates the relative number of interacting genes in each group. Conclusion Instead, we claim that our
conservative thresholds for predicting disease association
is justified because using thresholds of known disease
genes increases our likelihood of success given any estima-
tion process is going to have a degree of false positives. We
acknowledge the list does not exhaust all possible disease
genes but merely gives researchers the best short list for
further study. Page 8 of 11
(page number not for citation purposes) Competing interests The authors declare that they have no competing interests. Abbreviations HK: housekeeping; MPSS: massively parallel signature
sequencing; NS: non-specific; PCC: Pearson correlation
coefficient; TS: tissue-specific; DIS: genes in our dataset
that are disease-associated according to OMIM as of Sep-
tember 19, 2007; NDIS: genes in our dataset that are non- Page 8 of 11
(page number not for citation purposes) Page 8 of 11
(page number not for citation purposes) BioData Mining 2008, 1:8 Table 2: Contingency table underlying the guilt-by-association algorithm
Disease Associated? Number of Connections
% Disease-associated genes among interactors
≤ 75
> 75
Yes
≤ 12
3,112
1,907
> 12
481
651
No
≤ 12
7,853
705
> 12
229
112
Number of disease- and non-disease-associated genes by thresholds on number of connections and percentage of disease-associated genes among
interactors. The thresholds are obtained from exploring disease-associated genes and correspond to the average number of connections (12)
among disease-associated genes and the average proportion of disease-associated genes (75%) among their interactors. The 112 non-disease-
associated genes (bottom right cell) form the basis of the newly reported disease-associated genes [see Additional file 2]. Table 2: Contingency table underlying the guilt-by-association algorithm Number of disease- and non-disease-associated genes by thresholds on number of connections and percentage of disease-associated genes among
interactors. The thresholds are obtained from exploring disease-associated genes and correspond to the average number of connections (12)
among disease-associated genes and the average proportion of disease-associated genes (75%) among their interactors. The 112 non-disease-
associated genes (bottom right cell) form the basis of the newly reported disease-associated genes [see Additional file 2]. disease-associated genes also according to OMIM; INT:
genes in our dataset for which interactions have been
reported; NINT: genes in our dataset for which interac-
tions have not been reported. Authors' contributions AR conceived the study, carried out the data mining
approaches and drafted the manuscript. AI directed the
design and coordination of the biological/immunological
relevance of the results and drafted the manuscript. BD
participated in the coordination of the whole study and
drafted the manuscript. All authors read and approved the
final manuscript. Table 3: Precision analysis of the guilt-by-association algorithm
Threshold for number of connections (TC)
Threshold for % disease genes among interactors (TD)
Q1
TC = 1
Q2
TC = 4
Q3
TC = 13
Mean
TC = 12
Q1
TD = 12.8
N Captured
1,943
1,391
638
683
% Known
73.3
75.0
76.5
76.4
Q2
TD = 28.6
N Captured
1,024
563
195
219
% Known
74.8
78.9
85.1
84.9
Q3
TD = 50.0
N Captured
251
118
16
19
% Known
70.5
67.8
75.0
78.9
Mean
TD = 35.0
N Captured
748
409
109
127
% Known
73.4
76.3
84.4
84.2
The optimality of various location parameters to be used as thresholds in the guilt-by-association algorithm was explored by computing the
proportion of known (% Known) disease associated genes from the total number of captured genes (N Captured). The analysis was performed
using only the 1,445 genes (out of the initial 6,151) with known disease phenotype as the set of truly disease causing, and with the remaining 4,706
declared as disease associated. The three inter-quartiles (Q1: 25th percentile; Q2: 50th percentile or median; and Q3: 75th percentile) plus the mean
were used as thresholds. Table 3: Precision analysis of the guilt-by-association algorithm Threshold for number of connections (TC) The optimality of various location parameters to be used as thresholds in the guilt-by-association algorithm was explored by computing the
proportion of known (% Known) disease associated genes from the total number of captured genes (N Captured). The analysis was performed
using only the 1,445 genes (out of the initial 6,151) with known disease phenotype as the set of truly disease causing, and with the remaining 4,706
declared as disease associated. The three inter-quartiles (Q1: 25th percentile; Q2: 50th percentile or median; and Q3: 75th percentile) plus the mean
were used as thresholds. The optimality of various location parameters to be used as thresholds in the guilt-by-association algorithm was explored by computing the
proportion of known (% Known) disease associated genes from the total number of captured genes (N Captured). Page 9 of 11
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English
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New insights gained from museum collections: Deep-sea barnacles (Crustacea, Cirripedia, Thoracica) in the Muséum National d’Histoire Naturelle, Paris, collected during the Karubar expedition in 1991
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Zoosystematics and evolution/Mitteilungen aus dem Museum für Naturkunde in Berlin. Zoologische Reihe
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Abstract An examination of the deep-sea barnacles (Cirripedia, Thoracica) collected by the Karubar expedition to Indonesia (1991) and
deposited in the Muséum National d’Histoire Naturelle, Paris, identified 40 species contained in three families of stalked and five
families of acorn barnacles. Information on these species is presented, including descriptions, updated distributions and images to aid
species identification. Thirty of the species, treated herein, are new records for the Indonesian Kei Islands and Tanimbar Island, which
increases the total number of species recorded from Kei Islands, Aru Island and Tanimbar Island to 40. This study demonstrates the
value of museum collections as a resource in biodiversity science. New insights gained from museum collections: Deep-sea barnacles
(Crustacea, Cirripedia, Thoracica) in the Muséum National d’Histoire
Naturelle, Paris, collected during the Karubar expedition in 1991 Pipit Pitriana1,2,5, Diana S. Jones3, Laure Corbari4, Kristina von Rintelen1 1 Museum für Naturkunde – Leibniz Institute for Evolution and Biodiversity Science, Invalidenstrasse 43, 10115, Berlin, Germany
2 Research Centre for Deep-sea, Indonesian Institute of Science (LIPI), Jl. Y. Syaranamual, Poka, Tlk. Ambon, Kota Ambon, Maluku, Indonesia
3 The Western Australian Museum, 49 Kew Street, Welshpool WA 6106, Locked Bag 49, Welshpool DC WA 6986, Australia
4 Muséum national d’Histoire naturelle, Institut de Systématique, Évolution, Biodiversité ISYEB – UMR 7205 – CNRS, MNHN, UPMC, EPHE,
57 rue Cuvier, CP 26, 75005, Paris, France 5 Institute of Geological Sciences, Freie Universität Berlin, Malteserstrasse 74–100 Building C and D, 12249, Berlin, Germany 5 Institute of Geological Sciences, Freie Universität Berlin, Malteserstrasse 74–100 Building C and D, 12249, Berlin, Germany
http://zoobank.org/DF25E94F-EDEC-4FD4-BA1D-E4AC288282AD
Corresponding author: Pipit Pitriana (pipit.pitriana@mfn.berlin)
Academic editor: Martin Husemann ♦ Received 23 June 2020 ♦ Accepted 13 August 2020 ♦ Published 28 September 2020 Academic editor: Martin Husemann ♦ Received 23 June 2020 ♦ Accepted 13 August 2020 ♦ Published 28 Sep Key Words acorn barnacles, Indonesian biodiversity, new record, stalked barnacles, taxonomy Copyright Pipit Pitriana et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Zoosyst. Evol. 96 (2) 2020, 649–698 | DOI 10.3897/zse.96.55733 Zoosyst. Evol. 96 (2) 2020, 649–698 | DOI 10.3897/zse.96.55733 New insights gained from museum collections: Deep-sea barnacles
(Crustacea, Cirripedia, Thoracica) in the Muséum National d’Histoire
Naturelle, Paris, collected during the Karubar expedition in 1991 Introduction present). This programme was a collaboration between
the Muséum National d’Histoire Naturelle (MNHN), Par
is and the Institut de Recherche pour le Développement
(IRD) (formerly ORSTOM), to explore the deep-sea fau
na of the tropical Indo-Pacific. As the programme was
inspired and guided by carcinologists, it is not surprising
that ~ 33% of the papers resulting from these cruises con
cern crustaceans, especially crabs, lobsters and shrimps
(Richer de Forges et al. 2013). In 1991, scientists from France and Indonesia conducted
collaborative research through the Karubar expedition in
Indonesia. The acronym for this expedition, which collect
ed the material reported on herein, is a contraction of the
names of the Kei, Aru and Tanimbar Islands. These Islands
attracted attention after Professor Th. Mortensen’s Danish
expedition to the Kei Islands (1914–16). Mortensen sug
gested that the Islands were an ideal place for a marine
laboratory to study deep-sea fauna, as he had found stalked
crinoids, elasipods and other abyssal creatures at depths of
200–400 m around the Kei Islands (Crosnier et al. 1997). Publications on the thoracic barnacles collected by
the Karubar expedition are those of Buckeridge (1994,
1997) who focused on barnacles from the suborder Verru
comorpha. He reported six species of verrucomorph bar
nacles: Newmaniverruca albatrossiana (Pilsbry, 1912),
Altiverruca navicula (Hoek, 1913), Brochiverruca dens The Karubar expedition was part of the MUSOR
STOM-Tropical Deep-Sea Benthos programme (1976– Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 650 (Broch, 1932), Metaverruca recta (Aurivillius, 1898),
Rostratoverruca intexta (Pilsbry, 1912) and R. kruegeri
(Broch, 1922). Here, we present a study of the remaining
barnacle material collected by the Karubar expedition, to
cover the entire taxonomic range of deep-sea barnacles
collected by this expedition. two genera and species; Pachylasmatidae: one genus and
species; Archaeobalanidae: three genera, six species; Pyr
gomatidae: one genus and species; Balanidae: one genus
and species). A list of the species of Cirripedia collected during
the Karubar expedition and deposited in the Muséum
National d’Histoire Naturelle (MNHN) Paris is present
ed, including additional information on substrate, as
well as the depth where the sample was found (Suppl. material 1: Table S1). Material and methods In total, 459 specimens were studied at the Muséum Na
tional d’Histoire Naturelle (MNHN), of which 89 speci
mens were studied in detail at the Museum für Naturkunde
(MfN), Berlin, Germany. Species were determined on ex
ternal shell morphology and mouthpart and arthropodal
characters, as described in Darwin (1852, 1854), Hoek
(1883, 1907, 1913), Pilsbry (1890, 1907, 1916), Annan
dale (1905, 1906, 1908, 1909, 1910, 1913, 1916), Broch
(1916, 1922, 1931), Foster (1974, 1978, 1980, 1981),
Newman and Ross (1971, 1976), Rosell (1981, 1989,
1991), Calman (1918, 1919), Ren (1989), Chan et al. (2009, 2010) and Shalaeva and Boxshall (2014). Genus Heteralepas Pilsbry, 1907 Alepas Darwin, 1852: 156, pl. III, figs 5, 6. Heteralepas (Heteralepas) Pilsbry, 1907a: 100. Heteralepas s. str. – Newman, 1960: 109. Alepas Darwin, 1852: 156, pl. III, figs 5, 6. Heteralepas (Heteralepas) Pilsbry, 1907a: 100. Heteralepas s. str. – Newman, 1960: 109. Type genus. Heteralepas Pilsbry, 1907a. Type genus. Heteralepas Pilsbry, 1907a. Remarks. The family consists of two genera: Heterale
pas Pilsbry, 1907a and Paralepas Pilsbry, 1907a. Suborder HETERALEPADOMORPHA New
man, 1987 Diagnosis. Capitulum and peduncle without calcareous
armament. According to Buhl-Mortensen and Mifsud
(2017), Heteralepadomorpha consists of seven families
of poorlyknown Pedunculata. In the “Taxonomic account” section, the diagnosis is
followed by description, type species and localities and
known distributions for each species are given. Each
species is listed under its current valid binomen and all
known synonyms are also provided. Photographs of each
species are provided to facilitate their recognition. Family HETERALEPADIDAE Nilsson-Cantell, 1921 Heteralepadidae Nilsson-Cantell, 1921: 245
Lepadidae Darwin, 1852: 8 (part.) In the “Material examined” section, there are four station
codes dependent on the type of dredge used and are listed as
follows: DW for dredge Warén, ED (drague épibenthique)
for epibenthic dredge, CP (chalut à perche) for beam trawl
and CC (chalut à crevettes) for otter trawl (shrimp). Diagnosis. Capitulum naked, thick-walled, globular, sup
ported by stout peduncle; filamentary appendage at base
of cirrus I; caudal appendage multi-segmented; cirri cten
opod or acanthopod, rarely lasiopod. Measurements were made using digital callipers (ac
curate to 0.1 mm) as follows: for acorn barnacles, bas
al length of shell (LB), basal width (WB), orifice length
(LO), orifice width (WO) and carinal height (H); for
stalked barnacles, total height (TH), capitular height
(CH), diameter of base of capitulum (DBC), distance be
tween carina and scutum (CS), scutal length (LS), scutal
width (WS), tergal length (LT) and tergal width (WT). Taxonomic account Class HEXANAUPLIA Oakley, Wolfe, Lind
gren & Zaharoff, 2013
Subclass THECOSTRACA Gruvel, 1905
Infraclass CIRRIPEDIA Burmeister, 1834
(= Cirrhipèdes Lamarck, 1806)
Superorder THORACICA Darwin, 1854 zse.pensoft.net Order LEPADIFORMES Buckeridge & New
man, 2006 Hard body parts, such as the shell (parietes) and the
opercular plates, were studied using a Leica M125 stereo
microscope. Soft body parts, such as mouthparts and cir
ri, were studied using an Axioskop 20 light microscope. Mouthparts and cirri were mounted on glass slides and
examined under a light microscope. Images were taken
using a stereomicroscope with a digital camera (Leica
Microsystems M205C and Leica Z16 APo-A). Suborder HETERALEPADOMORPHA New
man, 1987 Heteralepas japonica (Aurivillius, 1892)
Figure 1 Alepas japonica Aurivillius, 1892: 125. – 1894: 28, pl. II figs 14, 15,
pl. VIII, figs 3, 7, pl. IX, fig. 3. Alepas indica Gruvel, 1901: 259. – 1905a: 162, fig. 179. Heteralepas (Heteralepas) japonica. – Pilsbry, 1907a: 101. Heteralepas (Heteralepas) japonica var. alba Krüger, 1911: 34, pl. 1,
fig. 2b. Heteralepas (Heteralepas) dubia Broch, 1922: 288, fig. 38. Heteralepas japonica. – Pilsbry 1911: 71, fig. 4. – Zevina et al. 1992:
31, fig. 19. – Chan et al. 2009a: 61. – Chan et al. 2009b: 88–91,
figs 2A–D, 3A–D, 4, 5. – Pitriana et al. 2020: 12, fig. 3. Type locality. Hirado Strait, Japan, 146 m depth (Auriv
illius 1892). Genus Paralepas Pilsbry, 1907 Material examined. Tanimbar Island: 1 specimen,
MNHN-IU-2019-4877, stn. CP 46, 08°01'S, 132°51'E,
271–273 m depth, 29 October 1991. Alepas Darwin, 1852: 156 (part.). Heteralepas (Paralepas) Pilsbry, 1907a: 100, fig. 34A. Paralepas s. str. – Newman, 1960: 108. Diagnosis. Capitulum rounded, plates absent; wall of ca
pitulum thick; orifice crenulated with no more than two
crests on carinal region; cirrus VI with caudal appendage;
maxillule strongly notched. Diagnosis. Maxillule slightly notched; filamentary ap
pendages well-developed; posterior and anterior rami of
cirri V and VI similar in length; segments of cirri V and
VI square-shaped, each armed with semi-circular brush
of many setae on anterior face. Description. Capitulum and peduncle yellowish. Orifice
slightly protuberant, crenulated, occupying one half to
one third capitular length, parallel to or at oblique angle
to capitulum; integument thick, chitinous. Cirrus I with
anterior rami (20-segmented) shorter than posterior rami
(25-segmented); cirrus VI with long caudal appendage,
24-segmented, 2/3 length of anterior ramus. Mandibles
with four large teeth excluding inferior angle; maxillule
strongly notched, two large setae at upper angle, blade-
shaped setae on cutting margin; labrum concave with nu
merous teeth. Measurements of specimen: height of ca Type species. Alepas percarinata Pilsbry, 1907c: 185,
fig. 2, pl. IV, fig. 8; type locality: Molokai, Hawaiian waters. Type species. Alepas percarinata Pilsbry, 1907c: 185,
fig. 2, pl. IV, fig. 8; type locality: Molokai, Hawaiian waters. Results Morphological analyses of all the samples revealed 40
species from three families of stalked barnacles (Heter
alepadidae: two genera, three species; Poecilasmatidae:
four genera, five species; Scalpellidae: twelve genera, 21
species) and five families of acorn barnacles (Verrucidae: Diagnosis. Filamentary appendage present at base of cir
rus I, short; posterior rami of cirri V and VI shorter and
more slender than anterior rami. zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 651 pitulum 18.50 mm, width 12.82 mm, thickness 5.97 mm;
length of peduncle 15.26 mm, width 5.55 mm. pitulum 18.50 mm, width 12.82 mm, thickness 5.97 mm;
length of peduncle 15.26 mm, width 5.55 mm. Type species. Alepas rex Pilsbry, 1907c: 186, fig. 3, pl. IV fig. 7; type locality: Kauai Island, Hawaiian waters. Distribution. Singapore; Indo-west Pacific: Indian
Ocean; Australia; Malay Archipelago; Vietnam; Condor
Island; South China Sea; East China Sea; Taiwan, Philip
pines; South Japan; NE New Zealand; fouling hard rock
substrata, crabs, gorgonians, antipatharians, deep-sea ca
bles; 48–500 m depth (Jones and Hosie 2016). Recently,
the species has also been recorded from the Lifamatola
Sea and the Halmahera Sea, Indonesia (Pitriana et al. 2020). In this study, Heteralepas japonica was found at
Tanimbar Island, Indonesia. Type locality. Mediterranean Sea (Chan et al. 2009b). Paralepas minuta. – Stubbings 1967: 240. – Newman 1960: 109. –
Chan et al. 2009b: 66, figs 55–57. Paralepas minuta. – Stubbings 1967: 240. – Newman 1960: 109. –
Chan et al. 2009b: 66, figs 55–57. Remarks. Paralepas minuta (Philippi, 1836) can be dis
tinguished from Paralepas ovalis (Hoek, 1907) by the
presence of triangular scutal areas and from Paralepas
nodulosa Broch, 1922 by the absence of “well defined,
scanty, almost thorn-like warts” (Broch 1922). Material examined. – Kei Islands: 6 specimens, MNHN-
IU-2019-4882, Stn. CP 25, 05°30'S, 132°52'E, 336–346 m
depth, 26 October 1991, attached to dead stem of gorgonian. – Tanimbar Island: 4 specimens, MNHN-IU-2019-4883,
Stn. CP 85, 09°22'S, 131°14'E, 240–245 m depth, 4 No
vember 1991, attached to spine of sea urchin. – Tanimbar Island: 4 specimens, MNHN-IU-2019-4883,
Stn. CP 85, 09°22'S, 131°14'E, 240–245 m depth, 4 No
vember 1991, attached to spine of sea urchin. Paralepas minuta (Philippi, 1836)
Figure 2 Figure 2 Alepas minuta Philippi, 1836: 254, pl. 12, fig. 23. Alepas (Paralepas) minuta. – Weltner, 1897: 239. Figure 1. Heteralepas japonica (Aurivillius, 1892) (MNHN-IU-2019-4877). a. left lateral view showing the capitulum and pedun
cle; b. right lateral view showing the capitulum and peduncle; c. anterior view, orifice; d. posterior view, capitulum and peduncle. Scale bar: 6 mm (a–d). Figure 1. Heteralepas japonica (Aurivillius, 1892) (MNHN-IU-2019-4877). a. left lateral view showing the capitulum and pedun
cle; b. right lateral view showing the capitulum and peduncle; c. anterior view, orifice; d. posterior view, capitulum and peduncle. Scale bar: 6 mm (a–d). zse.pensoft.net 652 Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) Figure 2. Paralepas minuta (Philippi, 1836) (MNHN-IU-2019-4882). а. left lateral view, capitulum and peduncle; b. right lateral
view, capitulum and peduncle; c. anterior view, orifice. Scale bar: 5 mm (a–c). Figure 2. Paralepas minuta (Philippi, 1836) (MNHN-IU-2019-4882). а. left lateral view, capitulum and peduncle; b. right lateral
view, capitulum and peduncle; c. anterior view, orifice. Scale bar: 5 mm (a–c). Heteralepas (Paralepas) minuta. – Broch, 1927b: 18, fig. 4, pl. 1, figs 1, 2. Paralepas minuta nipponica Utinomi, 1970: 342. Paralepas minuta. – Stubbings 1967: 240. – Newman 1960: 109. –
Chan et al. 2009b: 66, figs 55–57. Heteralepas (Paralepas) minuta. – Broch, 1927b: 18, fig. 4, pl. 1, figs 1, 2. Paralepas minuta nipponica Utinomi, 1970: 342. Type locality. Mediterranean Sea (Chan et al. 2009b). Genus Dianajonesia Koçak & Kemal, 2008 Temnaspis Fischer, 1884: 357. – Broch 1931: 30. Poecilasma (part). – Darwin, 1852: 99. – Pilsbry 1907a: 82. – Nils
son-Cantell 1921: 253. Dianajonesia Koçak & Kemal, 2008: 2. Temnaspis Fischer, 1884: 357. – Broch 1931: 30. Paralepas morula (Hoek, 1907)
Figure 3 Alepas morula Hoek, 1907: 35, pl. IV, figs 9–12. Heteralepas (Paralepas) morula. – Broch, 1922: 28, fig. 34. Paralepas morula. – Newman, 1960: 109. Diagnosis. Capitulum yellowish with globular shape; ante
rior and posterior rami of cirrus V and VI similar in length. Description. Capitulum yellowish, globose, small-sized,
externally smooth; orifice crenulated; peduncle rather
short. Segments of rami of cirrus I wide, anterior ramus
(9-segmented) shorter than posterior ramus (13-seg
mented); cirrus VI with short caudal appendage, 8-seg
mented. Penis annulated, setae sparsely distributed over
most of surface, a few longer setae towards tip. Maxillule
notched, two large and one small setae at upper angle;
mandible with four major teeth; labrum concave, with
numerous teeth. Measurements of specimen: height of
capitulum 8.34 mm, width 6.67 mm, thickness 4.53 mm;
length of peduncle 3.84 mm, width 2.60 mm. Material examined. – Tanimbar Island: 45 specimens,
MNHN-IU-2019-4878, Stn. CP 54, 08°21'S, 131°43'E,
836–869 m depth, 30 October 1991, attached to spine of
sea urchin. Material examined. – Tanimbar Island: 45 specimens,
MNHN-IU-2019-4878, Stn. CP 54, 08°21'S, 131°43'E,
836–869 m depth, 30 October 1991, attached to spine of
sea urchin. Diagnosis. Capitulum globular with numerous tubercles
on surface, scuta absent, orifice small, not protuberant,
peduncular length more than half capitular length. Description. Capitulum and peduncle yellowish. Capit
ulum globular with thick, chitinous, large warts all over
surface. Peduncle rather long, narrower than capitulum;
surface bearing tubercles, smaller than those of capitulum. Cirrus I placed rather far from cirrus II; cirri II–VI similar,
length and number of segments slightly increasing from
second to sixth; cirrus VI with 7-segmented caudal append
age. Penis indistinctly segmented, few long hairs towards
tip. Maxillule notched, two large setae on upper notch;
mandibles with three major teeth, inferior angle considered Distribution. Java Sea (Indonesia); Mediterranean Sea;
West Africa; Indo-west Pacific: Indian Ocean; Australia;
Malay Archipelago; Taiwan; Japan; Philippines; North
ern New Zealand; 485–736 m depth; attached to spines
of cidarids (Jones and Hosie 2016). In this study, Para
lepas minuta was found at Kei Islands and Tanimbar Is
land, Indonesia. zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 653 Figure 3. Paralepas morula (Hoek, 1907) (MNHN-IU-2019-4878). а. left lateral view, capitulum and peduncle; b. right lateral
view, capitulum and peduncle. Scale bar: 3 mm (a, b). Figure 3. Paralepas morula (Hoek, 1907) (MNHN-IU-2019-4878). а. left lateral view, capitulum and peduncle; b. right lateral
view, capitulum and peduncle. Suborder LEPADOMORPHA Pilsbry, 1916 Poecilasma (part). – Darwin, 1852: 99. – Pilsbry 1907a: 82. – Nils
son-Cantell 1921: 253. Poecilasma (part). – Darwin, 1852: 99. – Pilsbry 1907a: 82. – Nils
son-Cantell 1921: 253. Lepadomorpha Pilsbry, 1916: 14 (Lepadidae sensu Darwin, 1852). Lepadoidea Darwin, 1852: 8 (nom. trans. Zevina 1978a, b). Dianajonesia Koçak & Kemal, 2008: 2. Diagnosis. Capitulum with five plates, approximate or
variously reduced; scutum divided into two parts; apex of
carina extending to, or slightly overlapping basal end of
terga, basally terminating in disc. Paralepas morula (Hoek, 1907)
Figure 3 Scale bar: 3 mm (a, b). Trilasmatidae Nilsson-Cantell, 1934: 40. – Hiro 1937a: 79. – Krüger
1940: 29. as fourth tooth; labrum with continuous row of numer
ous short, strong, blunt teeth. Measurements of specimen:
height of capitulum 5.14 mm, width 3.69 mm, thickness
1.81 mm; length of peduncle 5.08 mm, width 1.31 mm. Diagnosis. Capitulum with up to five plates, including
tergum and scutum; scutum in some species split into
two (resulting in seven plates); some or all plates may
be degenerate or absent; umbos of terga apical, those of
carina and scuta fundamentally basal; peduncle without
calcareous scales; maxillule not stepped; cirrus I widely
separated and much shorter than posterior cirri; caudal
appendages uniarticulate, spinose. Diagnosis. Capitulum with up to five plates, including
tergum and scutum; scutum in some species split into
two (resulting in seven plates); some or all plates may
be degenerate or absent; umbos of terga apical, those of
carina and scuta fundamentally basal; peduncle without
calcareous scales; maxillule not stepped; cirrus I widely
separated and much shorter than posterior cirri; caudal
appendages uniarticulate, spinose. Distribution. Flores Sea (Indonesia); Philippines; Bass
Strait, N of Tasmania; attached to spines of echinoids,
for example, Histocidaris elegans (Agassiz, 1879); 182–
538 m depth (Jones and Hosie 2016). In this study, Para
lepas morula was found at Tanimbar Island, Indonesia. Type locality. Bali Sea, North of Lombok; 538 m depth
(Hoek 1907). Type genus. Poecilasma Darwin, 1852: 99. Type genus. Poecilasma Darwin, 1852: 99. Type genus. Poecilasma Darwin, 1852: 99. Remarks. Most of the smaller specimens do not show the
warts that are characteristic of this species. Type species. Poecilasma fissa Darwin, 1852: 109, pl. II,
fig. 4, pl. X, fig. 29. Dianajonesia excavatum (Hoek, 1907)
Figure 5 Poecilasma excavatum Hoek, 1907: 10, pl. I, figs 5–10. Poecilasma excavatum Hoek, 1907: 10, pl. I, figs 5–10. Poecilasma excavatum Hoek, 1907: 10, pl. I, figs 5–10. Poecilasma tridens. – Weltner 1922: 80, pl. 4, fig. 19 (non Aurivillius
Poecilasma (Temnaspis) excavatum. – Nilsson-Cantell 1925: 16, fig. pl. 1, fig. 1. Material examined. – Tanimbar Island: 2 specimens,
MNHN-IU-2019-4861, Stn. CP 79, 09°16'S, 131°22'E,
239–250 m depth, 3 November 1991, attached to crab leg. Diagnosis. Capitulum broadly oval, with five smooth
plates; carina not extending to area between terga; pedun
cle with circles of small protuberances; cirri short. Material examined. – Tanimbar Island: 2 specimens,
MNHN-IU-2019-4861, Stn. CP 79, 09°16'S, 131°22'E,
239–250 m depth, 3 November 1991, attached to crab leg. Poecilasma tridens. – Weltner 1922: 80, pl. 4, fig. 19 (non Aurivillius).i Poecilasma (Temnaspis) excavatum. – Nilsson-Cantell 1925: 16, fig. 5,
pl. 1, fig. 1. Trilasmis (Temnaspis) excavatum. – Hiro 1937b: 412. Diagnosis. Capitulum broadly oval, with five smooth
plates; carina not extending to area between terga; pedun
cle with circles of small protuberances; cirri short. Dichelaspis (Dichelaspis) tridens. – Stubbings 1936: 7, fig. 2. (non Au
rivillius). Trilasmis excavatum. – Nilsson-Cantell 1938: 9. Temnaspis excavatum. – Broch 1931: 31, fig. 10. – Dong et al. 1982: 77. Description. Capitulum yellowish, with five tranparent,
smooth plates. Scutum bilobed, occludent segment nar
row, bow-shaped; tergum sub-triangular, nearly half as
broad as long. Carina very narrow, slightly curved down
wards, end blunt. Peduncle yellow, plainly ringed, spines
absent. Cirrus I with anterior ramus wider than posterior
ramus. Maxillule notched, with two strong teeth in upper
part; mandible with four teeth, fourth pectinated, placed
very close to inferior angle; labrum with row of minute
teeth. Measurements of specimen: basal diameter of ca
pitulum 0.94 mm; capitular height 7.03 mm; total height
10.40 mm; scutal width 4.11 mm; scutal length 5.85 mm;
tergal width 1.00 mm; tergal length 3.13 mm. Temnaspis excavatum. – Broch 1931: 31, fig. 10. – Dong et al. 1982: 77. Dianajonesia excavatum. – Koçak & Kemal, 2008: 2. Dianajonesia excavatum. – Koçak & Kemal, 2008: 2. Dianajonesia excavatum. – Koçak & Kemal, 2008: 2. Dianajonesia excavatum. – Koçak & Kemal, 2008: 2. Material examined. – Tanimbar Island: 3 specimens,
MNHN-IU-2019-4859, Stn. CP 78, 09°06'S, 131°24'E,
284–295 m depth, 3 November 1991. Diagnosis. Family POECILASMATIDAE Annandale, 1910 Lepadidae Darwin, 1852: 8 (part.). – Pilsbry 1907a: 72 (part.). Lepadinae. – Gruvel, 1905: 104 (part.). Poecilasmatinae Annandale, 1910a: 84. Poecilasmatidae. – Nilsson-Cantell, 1921: 253. Lepadidae Darwin, 1852: 8 (part.). – Pilsbry 1907a: 72 (part.). Lepadinae. – Gruvel, 1905: 104 (part.). Poecilasmatinae Annandale, 1910a: 84. Poecilasmatidae. – Nilsson-Cantell, 1921: 253. Type species. Poecilasma fissa Darwin, 1852: 109, pl. II,
fig. 4, pl. X, fig. 29. Type species. Poecilasma fissa Darwin, 1852: 109, pl. II,
fig. 4, pl. X, fig. 29. zse.pensoft.net zse.pensoft.net 654 Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) Dianajonesia amygdalum (Aurivillius, 1894)
Figure 4 Philippines; South Japan; tropical West and central Pacific
Ocean to Fiji and Hawaii; attached to decapod crustaceans;
shallow water (Jones and Hosie 2016). In this study, Diana
jonesia amygdalum was found at Tanimbar Island, Indonesia. Philippines; South Japan; tropical West and central Pacific
Ocean to Fiji and Hawaii; attached to decapod crustaceans;
shallow water (Jones and Hosie 2016). In this study, Diana
jonesia amygdalum was found at Tanimbar Island, Indonesia. Poecilasma amygdalum Aurivillius, 1894: 10, pl. 1, figs 4–6, pl. 8,
fig. 4. – Nilsson-Cantell 1921: 262, fig. 46d–g, pl. 3, fig. 6. Poecilasma fissum (non Darwin). – Hoek, 1907: 8 (part.). Trilasmis fissum hawaiense. – Pilsbry, 1928: 306, pl. 24, figs 1–8. Trilasmis (Temnaspis) amygdalum. – Hiro, 1937a: 85, fig. 69. Temnaspis amygdalum amygdalum. – Zevina, 1982: 70, fig. 62. Temnaspis amygdalum. – Utinomi, 1966: 5. – Dong et al. 1982: 77. Dianajonesia amygdalum. – Koçak & Kemal, 2008: 2. Type locality. Nordwachter Island, Thousand Islands,
Java Sea (Aurivillius 1894). Dianajonesia excavatum (Hoek, 1907)
Figure 5 Capitulum with five plates; scutum with two
distinct segments, united at rostral angle; carina generally
extending up between terga, terminating downwards as
imbedded disc (fork- or cup-shaped). Type species. Octolasmis warwicki Gray, 1825: 100. –
1830: pl. VI, fig. 16. Type species. Octolasmis warwicki Gray, 1825: 100. –
1830: pl. VI, fig. 16. Dichelaspis orthogonia Darwin, 1852
Figure 6 – Broch 1922: 279. – 1931: 38, fig. 14. – Nilsson-Cantell
1925: 21, fig. 8. – 1928: 18, fig. 8. – Hiro 1933: 55, fig. 16, pl. 2,
figs 5a, 5. – 1937a: 91, fig. 71. – 1937b: 415, fig. 12. – Stubbings
1963: 327, fig. 1. – Dong et al. 1982: 79. – Zevina et al. 1992: 26,
fig. 15. – Chan 2009: 68, fig. 2 B and fig. 17A–F. Type locality. Siboga station 253; 5°48.2'S, 132°15'E; depth:
304 m; bottom: grey clay, hard and crumbly (Hoek 1907). Material examined. – Kei Islands: 4 specimens,
MNHN-IU-2019-4864, Stn. CP 27, 05°33'S, 132°51'E,
314–304 m depth, 27 October 1991. –
Tanimbar
Island:
6
specimens,
MNHN-
IU-2019-4862, Stn. DW 24, 05°32'S, 132°51'E, 230–
243 m depth, 26 October 1991. Material examined. – Kei Islands: 4 specimens,
MNHN-IU-2019-4864, Stn. CP 27, 05°33'S, 132°51'E,
314–304 m depth, 27 October 1991. Dianajonesia excavatum (Hoek, 1907)
Figure 5 Capitulum with scutum divided into two seg
ments parallel to occludent margin; tergum with excava
tion in scutal margin near occludent margin, receiving tip
of occludent segment of scutum. Description. Capitulum oval, apex pointed, slightly thick,
swollen. Scutum with larger segment strongly bowed, basal
margin short, apex pointed; smaller segment bowed, termi
nating in point at base, tergal margin rounded, fitting exactly Distribution. Indo-west Pacific: Indian Ocean; Madagascar
through Malaysia, Hong Kong, South China Sea; Taiwan; Figure 4. Dianajonesia amygdalum (Aurivillius, 1894) (MNHN-IU-2019-4861). а. left lateral view, capitulum and peduncle;
b. right lateral view, capitulum and peduncle; c. anterior view, orifice; d. posterior view, carina. Scale bar: 2 mm (a–d). Figure 4. Dianajonesia amygdalum (Aurivillius, 1894) (MNHN-IU-2019-4861). а. left lateral view, capitulum and peduncle;
b. right lateral view, capitulum and peduncle; c. anterior view, orifice; d. posterior view, carina. Scale bar: 2 mm (a–d). zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 655 Figure 5. Dianajonesia excavatum (Hoek, 1907) (MNHN-IU-2019-4859). а. left lateral view showing the capitulum and peduncle;
b. right lateral view showing the capitulum and peduncle; c. anterior view, orifice; d. posterior view, carina. Scale bar: 2 mm (a–d). Figure 5. Dianajonesia excavatum (Hoek, 1907) (MNHN-IU-2019-4859). а. left lateral view showing the capitulum and peduncle;
b. right lateral view showing the capitulum and peduncle; c. anterior view, orifice; d. posterior view, carina. Scale bar: 2 mm (a–d). Octolasmis Gray, 1825: 100. – Pilsbry 1907a: 93. Octolasmis Gray, 1825: 100. – Pilsbry 1907a: 93. into excavation of tergum. Tergum triangular, characteristic
excavation at scutal margin near occludent margin. Carina
narrow, terminating in spatula-shaped disc. Cirrus I with
anterior and posterior rami subequal (each five-segment
ed); cirri II–VI longer, more slender; cirrus VI with caudal
appendages. Penis thick, ringed, especially mid-length, ter
minating in narrower, curved part. Maxillule notched, two
large teeth on upper side; mandible with four teeth, largedis
tance between first and second teeth. Labrum convex, with
numerous blunt teeth. Measurements of specimen: basal
diameter of capitulum 1.06 mm; capitular height 7.12 mm;
total height 12.69 mm; scutal width 3.08 mm; scutal length
6.09 mm; tergal width 1.30 mm; tergal length 3.47 mm. Diagnosis. Capitulum with five plates; scutum with two
distinct segments, united at rostral angle; carina generally
extending up between terga, terminating downwards as
imbedded disc (fork- or cup-shaped). Diagnosis. Dichelaspis orthogonia Darwin, 1852
Figure 6 Dichelaspis orthogonia Darwin, 1852: 130, pl. II, fig. 10a, b. Dichelaspis versluysi Hoek, 1907: 28, pl. III, figs 8–13. Octolasmis (Dichelaspis) orthogonia. – Pilsbry, 1907a: 94. Octolasmis orthogonia. – Krüger, 1911: 462. – Pilsbry 1911: 70, pl. 11,
figs 6, 7. – Broch 1922: 279. – 1931: 38, fig. 14. – Nilsson-Cantell
1925: 21, fig. 8. – 1928: 18, fig. 8. – Hiro 1933: 55, fig. 16, pl. 2,
figs 5a, 5. – 1937a: 91, fig. 71. – 1937b: 415, fig. 12. – Stubbings
1963: 327, fig. 1. – Dong et al. 1982: 79. – Zevina et al. 1992: 26,
fig. 15. – Chan 2009: 68, fig. 2 B and fig. 17A–F. Dichelaspis orthogonia Darwin, 1852: 130, pl. II, fig. 10a, b. Dichelaspis orthogonia Darwin, 1852: 130, pl. II, fig. 10a, b. Distribution. Indo-west Pacific: East coast of Africa
(Zanzibar); Gulf of Aden; Indonesia; Malay Archipela
go; South China Sea; East China Sea; Philippines; South
Japan (Goto Island); attached to echinoid spines, crusta
ceans; palinurids and cirripedes; 189–600 m depth (Jones
and Hosie 2016). In this study, Dianajonesia excavatum
was found at Tanimbar Island, Indonesia. Dichelaspis versluysi Hoek, 1907: 28, pl. III, figs 8–13. Dichelaspis versluysi Hoek, 1907: 28, pl. III, figs 8–13. Octolasmis (Dichelaspis) orthogonia. – Pilsbry, 1907a: 94. Octolasmis orthogonia. – Krüger, 1911: 462. – Pilsbry 1911: 70, pl. 11,
figs 6, 7. – Broch 1922: 279. – 1931: 38, fig. 14. – Nilsson-Cantell
1925: 21, fig. 8. – 1928: 18, fig. 8. – Hiro 1933: 55, fig. 16, pl. 2,
figs 5a, 5. – 1937a: 91, fig. 71. – 1937b: 415, fig. 12. – Stubbings
1963: 327, fig. 1. – Dong et al. 1982: 79. – Zevina et al. 1992: 26,
fig. 15. – Chan 2009: 68, fig. 2 B and fig. 17A–F. Octolasmis orthogonia. – Krüger, 1911: 462. – Pilsbry 1911: 70, pl. 11,
figs 6, 7. – Broch 1922: 279. – 1931: 38, fig. 14. – Nilsson-Cantell
1925: 21, fig. 8. – 1928: 18, fig. 8. – Hiro 1933: 55, fig. 16, pl. 2,
figs 5a, 5. – 1937a: 91, fig. 71. – 1937b: 415, fig. 12. – Stubbings
1963: 327, fig. 1. – Dong et al. 1982: 79. – Zevina et al. 1992: 26,
fig. 15. – Chan 2009: 68, fig. 2 B and fig. 17A–F. Octolasmis orthogonia. – Krüger, 1911: 462. – Pilsbry 1911: 70, pl. 11,
figs 6, 7. Type locality. Unknown. Description. Capitulum and peduncle yellowish. Scutum
with basal segment narrow, L-shaped; tergum triangular,
three prominent ridges at scutal margin; base of carina
crescent-formed cup. Cirrus I with anterior ramus (6-seg
mented) shorter than posterior ramus (8-segmented); cir
rus VI with small caudal appendage. Maxillule notched,
three large setae on upper notch; mandible with four
teeth, inferior angle produced into single strong spine;
labrum concave, with numerous teeth. Measurements of
specimen: basal diameter of capitulum 1.89 mm; capitu
lar height 8.69 mm; total height 10.47 mm; scutal width
3.5 mm; scutal length 7.16 mm; tergal width 0.80 mm;
tergal length 6.53 mm. Remarks. The type locality of this species is unknown. The species was re-discovered in the Malay Archipelago
by the Siboga Expedition in 40–112 m depth (Hoek 1907)
and the Albatross Expedition, off Kagoshima Gulf, Japan,
in 87 m depth (Pilsbry 1907d). Genus Dichelaspis Darwin, 1852 –
Tanimbar
Island:
6
specimens,
MNHN-
IU-2019-4862, Stn. DW 24, 05°32'S, 132°51'E, 230–
243 m depth, 26 October 1991. Dichelasmis Darwin, 1852: 115. Heptalasmis. Agassiz, 1842: 178. zse.pensoft.net 656 Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) Figure 6. Dichelaspis orthogonia Darwin, 1852 (MNHN-IU-2019-4864). а. left lateral view capitulum and peduncle; b. right lateral
view, capitulum and peduncle. Scale bar: 2 mm (a–b). Figure 6. Dichelaspis orthogonia Darwin, 1852 (MNHN-IU-2019-4864). а. left lateral view capitulum and peduncle; b. right lateral
view, capitulum and peduncle. Scale bar: 2 mm (a–b). Diagnosis. Capitulum flattened; five plates, separated by
thin membrane; scutum consisting of two distinct seg
ments, with point of junction perfectly calcified; base of
carina oval. sometimes fouling; 14–818 m depth (Jones and Hosie
2016). In this study, Dichelaspis orthogonia was found at
Kei Islands and Tanimbar Island, Indonesia. Type species. Megalasma (Glyptelasma) subcarinatum
Pilsbry, 1907a: 91, pl. VII, figs 1–5; type locality: Atlan
tic Ocean, East of New Jersey, USA. Genus Glyptelasma Pilsbry, 1907 Glyptelasma Pilsbry, 1907a: 87. Glyptelasma gracile (Hoek, 1883)
Figure 7 long hairs towards tip. Maxillule notched, with strong
teeth on upper side; mandible with four teeth; labrum
with row of small teeth on crest. Measurements of spec
imen: basal diameter of capitulum 1.45 mm; capitular
height 10.44 mm; total height 13.99 mm; scutal width
4.79 mm; scutal length 9.01 mm; tergal width 1.87 mm;
tergal length 5.07 mm. Poecilasma gracile Hoek, 1883: 46, pl. II, figs 2–4. Megalasma gracile gracilius. – Pilsbry, 1907a: 88, pl. V, fig. 16,
pl. VII, figs 6–9. Megalasma gracile. – Nilsson-Cantell 1938: 10. Megalasma (Glyptelasma) gracile. – Zevina 1982: 84, fig. 74. Glyptelasma gracile. – Jones et al. 2001: 239. Poecilasma gracile Hoek, 1883: 46, pl. II, figs 2–4. Poecilasma gracile Hoek, 1883: 46, pl. II, figs 2–4. Megalasma gracile gracilius. – Pilsbry, 1907a: 88, pl. V, fig. 16,
pl. VII, figs 6–9. Megalasma gracile. – Nilsson-Cantell 1938: 10. Megalasma (Glyptelasma) gracile. – Zevina 1982: 84, fig. 74. Glyptelasma gracile. – Jones et al. 2001: 239. Megalasma gracile. – Nilsson-Cantell 1938: 10. Megalasma (Glyptelasma) gracile. – Zevina 1982: 84, fig. 74. Glyptelasma gracile. – Jones et al. 2001: 239. Distribution. West-southwest Pacific: from Australia
(Sydney) to Indonesia (Sumbawa) and the Philippines
(northwest Panay Island); East Indian Ocean; attached to
glassy spicule of hexactinellid sponge, fragments of sea
urchin tests, spines of cidarids; 395–935 m depth (Jones
and Hosie 2016). In this study, Glyptelasma gracile was
found at Tanimbar Island, Indonesia. Material examined. – Tanimbar Island: 14 specimens,
MNHN-IU-2019-4873, Stn. CP 54, 08°21'S, 131°43'E,
836–869 m depth, 30 October 1991, attached to spine
of sea urchin; 1 specimen, MNHN-IU-2019-4875, Stn. CC 57, 08°19'S, 31°53'E, 603–620 m depth, 31 Octo
ber 1991; 3 specimens (1 adult, 2 juveniles), MNHN-
IU-2019-4876, Stn. CP 73, 08°29'S, 131°33'E, 840–
855 m depth, 2 November 1991, attached to spines of
sea urchins. Type locality. Challenger expedition Station 164a (off
Sydney, Australia); 34°13'S, 151°38'E; depth 125 m; bot
tom: grey ooze (Hoek 1883). Diagnosis. Capitulum with five plates; carina enlarged,
keel-shaped; tergum with basal point truncated; max
illule notched; caudal appendages with relatively-long
spines at tip. Glyptelasma Pilsbry, 1907a: 87. Diagnosis. Capitulum with five approximate, fully cal
cified plates; scutum quadrangular, umbone subcentral;
carina becoming larger towards base; peduncle short. Distribution. Indo-west Pacific: East and South African
Coast, Indian Ocean, Australia, Indonesia, Malay Ar
chipelago, Vietnam, South China Sea, East China Sea,
Philippines, Taiwan, South Japan; attached to hydroid,
gorgonians, antipatharians, sea urchin spines, nylon cord, zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 657 Type species. Megalasma striatum Hoek, 1883: 51, pl. II,
figs 5–9, pl. VII, figs 8, 9. Genus Megalasma Hoek, 1883 Megalasma Hoek, 1883: 50. – Pilsbry 1907a: 87. Megalasma Hoek, 1883: 50. – Pilsbry 1907a: 87. Figure 8 Figure 8 Megalasma striatum Hoek, 1883: 51, pl. II, figs 5–9, pl. VII, figs 8, 9. –
Broch 1931: 270, figs 29, 30. Megalasma (Megalasma) elegans. – Zullo & Newman, 1964: 355,
fig. 2a–i. Megalasma (Megalasma) striatum. – Zevina 1982: 80, fig. 71. – Chan
2009: 66, figs 2A, 16 A–H. Distribution. West-southwest Pacific, Indo-west Pacific,
East coast of Africa, Indian Ocean, north Australia, Indo
nesia, Malay Archipelago, East China Sea, South China
Sea, Taiwan, Philippines, south Japan to New Zealand;
attached to echinoid spines, antipatharians, gorgonians,
glassy spicule of hexactinellid sponges, corallines; 125–
984 m depth (Jones and Hosie 2016). In this study, Meg
alasma striatum was found at Kei Islands and Tanimbar
Island, Indonesia. Material examined. – Kei Islands: 1 specimen, MNHN-
IU-2019-4868, Stn. CP 09, 05°23'S, 132°29'E, 368–
389 m depth, 23 October 1991; 1 specimen, MNHN-
IU-2019-4869, Stn. CP 16, 05°17'S, 132°50'E, 315–349 m
depth, 24 October 1991, attached to sea urchin spine; 2
specimens, MNHN-IU-2019-4866, Stn. CP 17, 05°15'S,
133°01'E, 439–459 m depth, 24 October 1991. Type locality. Philippine Archipelago (Hoek 1883). – Tanimbar Island: 1 specimen, MNHN-IU-2019-4867,
Stn. CP 46, 08°01'S, 132°51'E, 271–273 m depth, 29 Oc
tober 1991; 4 specimens (3 attached to sea urchin spine),
MNHN-IU-209-4870, Stn. DW 49, 08°00'S, 132°59'E,
206–210 m depth, 29 October 1991; 6 specimens, MNHN-
IU-2019-4871, Stn. CP 85, 09°22'S, 131°14'E, 240–245 m
depth, 4 November 1991, attached to spines of sea urchin. Order SCALPELLIFORMES Buckeridge &
Newman, 2006 Scalpelloidea Pilsbry, 1916: 14 (nom. trans. Zevina, 1978). Scalpelliformes Buckeridge & Newman, 2006: 22. Scalpelloidea Pilsbry, 1916: 14 (nom. trans. Zevina, 1978). Scalpelliformes Buckeridge & Newman, 2006: 22. Diagnosis. Capitulum ovate, occludent margin almost
same length as carinal margin; capitular plates heavily
calcified, markedly sculptured with radial striations be
tween grooves of growth lines; peduncle short, covered
by capitulum. Megalasma Hoek, 1883: 50. – Pilsbry 1907a: 87. Diagnosis. Capitulum with five strong, calcified plates;
scutal umbones subcentral; apex of carina sometimes
slightly overlapping basal end of terga, progressively be
coming broader towards base; peduncle very short. Description. Capitulum whitish, peduncle brownish. Capitulum compressed, striated, about twice as long as
broad. Scutum with apex pointed; tergum basally trun
cated, flat, oblong. Carina flat, very narrow, enlarged,
keel-shaped in basal region. Cirrus I with anterior and
posterior rami equal length; cirrus II–VI long, slender;
cirrus VI with small caudal appendages. Penis with few Type species. Megalasma striatum Hoek, 1883: 51, pl. II,
figs 5–9, pl. VII, figs 8, 9. Type species. Megalasma striatum Hoek, 1883: 51, pl. II,
figs 5–9, pl. VII, figs 8, 9. Figure 7. Glyptelasma gracile (Hoek, 1883) (MNHN-IU-2019-4875). а. left lateral view, capitulum and peduncle; b. right lateral
view, capitulum and peduncle; c. anterior view, orifice; d. posterior view, carina. Scale bar: 2 mm (a–d). Figure 7. Glyptelasma gracile (Hoek, 1883) (MNHN-IU-2019-4875). а. left lateral view, capitulum and peduncle; b. right lateral
view, capitulum and peduncle; c. anterior view, orifice; d. posterior view, carina. Scale bar: 2 mm (a–d). zse.pensoft.net 658 Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) Megalasma striatum Hoek, 1883
Figure 8 Maxillule with three strong setae at upper angle separat
ed by wide notch; mandible with four teeth, lower angle
sharp. Measurements of specimen: basal diameter of ca
pitulum 2.14 mm; capitular height 9.36 mm; total height
9.36 mm; scutal width 3.81 mm; scutal length 7.28 mm;
tergal width 1.58 mm; tergal length 4.29 mm. Family SCALPELLIDAE Pilsbry, 1907 Pollicipedidae Gray, 1825: 100 (part.). – Annandale 1909: 63 (part.). Lepadidae Darwin, 1852: 8 (part.). Pollicipedidae Gray, 1825: 100 (part.). – Annandale 1909: 63 (part.). Pollicipedidae Gray, 1825: 100 (part.). – Annandale 1909: 63 (part.). Lepadidae Darwin, 1852: 8 (part.). Polyaspidae Gruvel, 1905: 8, 16 (part.; rejected by Pilsbry, 1907a, be
cause family group name not derived from generic name). Polyaspidae Gruvel, 1905: 8, 16 (part.; rejected by Pilsbry, 1907a, be
cause family group name not derived from generic name). Description. Capitulum white, with five calcified plates,
surfaces strongly striated. Scutum with basal margin ro
tated; tergum triangular in lateral view; carina with dorsal
roof widening apically on either side of midline groove. Cirrus I with anterior ramus wider than posterior ramus. Scalpellinae Pilsbry, 1907a: 3 (part.). Scalpellidae. – Krüger, 1911: 7. – Pilsbry 1916: 4. – Nilsson-Cantell
1921: 162. – Foster 1978: 38. – Zevina 1978a: 999. – Buckeridge
1983: 27. – Gale 2016: 296. Figure 8. Megalasma striatum Hoek, 1883 (MNHN-IU-2019-4868). а. left lateral view, capitulum; b. right lateral, capitulum;
c. anterior view, capitulum and peduncle; d. posterior view, carina. Scale bar: 2 mm (a–d). Figure 8. Megalasma striatum Hoek, 1883 (MNHN-IU-2019-4868). а. left lateral view, capitulum; b. right lateral, capitulum;
c. anterior view, capitulum and peduncle; d. posterior view, carina. Scale bar: 2 mm (a–d). zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 659 Diagnosis. Capitulum with fully or partially calcified
plates; peducle with calcareous or phosphorus scales. According to Gale (2016), the Scalpellidae have a max
imum of 14 plates i.e. carina, rostrum, paired scuta, ter
ga, upper latera, carinolatera, rostrolatera and inferior
median latera; and rarely 13 plates, due to secondary
loss of the rostrum. Diagnosis. Capitulum with 14 plates, smooth, white and
reddish coloured; carina simply, strongly bowed, roof
slightly convex with umbo at apex; rostrum triangular,
distinct; upper latus quadrangular, large; infra-median la
tus triangular. Description. Capitulum flat, rather broad, not covered
by distinct membrane. Scutum with occludent margin
arched, forming with tergal margin a triangular portion
projecting over tergum. Tergum surpassing scutal area
with occludent margin almost straight. Upper latus quad
rangular, angle at apex between scutal and tergal margins
distinctly projecting over scutum. Rostrum small, trian
gular; rostrolatus very low, quadrangular; infra-median
latus small, triangular, umbo at apex; carinal latus larger
than other latera with carinal margin arched. Diotascalpellum rubrum (Hoek, 1883)
Figure 9 Scalpellum rubrum Hoek, 1883: 91, pl. IV fig. 18. – Pilsbry 1911: 62,
text-fig. 1, pl. VIII, figs 1–4. – Calman 1918b: 122–123. – Broch
1922: 237. – Nilsson-Cantell 1927: 745–747, text-fig. 2. – 1931: 2. Trianguloscalpellum rubrum. – Zevina 1981: 316–317, fig. 240. –
Rosell 1986: 89, text-fig. 1a. – 1991: 22, fig. 1f, g. – Huang 1994:
517. – 2001: 318. – Jones et al. 2001: 254. – Young 2001: 464, fig. 7. – Jones 2007: 292. – Shalaeva and Boxshall 2014: 49, fig. 34. Diotascalpellum rubrum. – Gale 2016: 297, figs 2A, 6U–Y, 11Q–T,
13K–M, 18A–G. Scalpellum rubrum Hoek, 1883: 91, pl. IV fig. 18. – Pilsbry 1911: 62,
text-fig. 1, pl. VIII, figs 1–4. – Calman 1918b: 122–123. – Broch
1922: 237. – Nilsson-Cantell 1927: 745–747, text-fig. 2. – 1931: 2. Trianguloscalpellum rubrum. – Zevina 1981: 316–317, fig. 240. –
Rosell 1986: 89, text-fig. 1a. – 1991: 22, fig. 1f, g. – Huang 1994:
517. – 2001: 318. – Jones et al. 2001: 254. – Young 2001: 464, fig. 7. – Jones 2007: 292. – Shalaeva and Boxshall 2014: 49, fig. 34. Diotascalpellum rubrum. – Gale 2016: 297, figs 2A, 6U–Y, 11Q–T,
13K–M 18A–G Type species. Scalpellum regium Thomson, 1873; type
locality: Stn 61: Atlantic, Western Central (North Western
Atlantic Basin); 5,210 m depth. Type species. Scalpellum regium Thomson, 1873; type
locality: Stn 61: Atlantic, Western Central (North Western
Atlantic Basin); 5,210 m depth. Genus Diotascalpellum Gale, 2016 Diotascalpellum Gale, 2016: 297. Type locality. Philippines, 12°43'N, 122°10'E; depth,
180 m and 207 m; bottom, mud (near Luzon) (Hoek 1883). Diagnosis. Scalpellines without specialised contact be
tween carina and carinolatus, merely a cresentic concavi
ty on interior of plate that forms a rim slightly overlapping
the upper latus; umbo of carinolatus upright, only slightly
incurved, dorsal margin gently convex; inframedian sur
face of carinolatus well demarcated, slightly inset. Family SCALPELLIDAE Pilsbry, 1907 Cirrus I with
anterior and posterior rami almost same length; cirrus VI
with long caudal appendages. Maxillule not notched, with
large spine on upper side, cutting edge almost straight;
mandible with three large teeth excluding inferior an
gle. Measurements of two specimens: height of capitu
lum 12.51–20.57 mm, width 7.39–10.55 mm, thickness
4.09–6.51 mm; length of peduncle 5.10–7.76 mm, width
4.36–6.62 mm. Subfamily SCALPELLINAE Pilsbry, 1907 Scalpellinae Pilsbry, 1907a: 4. – Zevina 1978a: 1002. – Zevina 1981:
93. – Gale 2016: 296. Scalpellinae Pilsbry, 1907a: 4. – Zevina 1978a: 1002. – Zevina 1981:
93. – Gale 2016: 296. Scalpellinae Pilsbry, 1907a: 4. – Zevina 1978a: 1002. – Zevina 1
93. – Gale 2016: 296. Pollicipedidae. – Annandale, 1909: 63 (part.). Pollicipedidae. – Annandale, 1909: 63 (part.). Pollicipedidae. – Annandale, 1909: 63 (part.). Diagnosis. Formerly, the subfamily was characterised
by a subapical carinal umbo, inflexed carina and subapi
cal umbones of the upper and inframedian latus (Zevina
1978a). Gale (2016) characterised the subfamily by the
broad, low, straplike and gently incurved rostrolatus. The
rostrum is broader than high, rectangular, trapezoidal or
triangular and its large, triangular, lateral surfaces contact
the interior of the rostrolatus. The articulation surface be
tween the rostrum and rostrolatus extends over the entire
height of both plates. Distribution. Java Sea, Indonesia. Pacific: western cen
tral and northwest; Philippines; Kagoshima Sea and Sag
ami Bay, Japan; 133–551 m depth (Shalaeva and Box
shall 2014). In this study, Diotascalpellum rubrum was
found at Kei Islands, Indonesia. Type genus. Scalpellum Leach, 1817: 68. Type genus. Scalpellum Leach, 1817: 68. Type genus. Scalpellum Leach, 1817: 68. Regioscalpellum Gale, 2016: 298. Regioscalpellum Gale, 2016: 298. Diagnosis. Concave scalpellines; internal surface upper
latus covered by epidermis, short carinal margin present
on carinolatus; carinolatus with incurved umbones, later
al margin strongly convex. Trianguloscalpellum moluccanum. – Jones, 2012: 371, table 2. – Sha
laeva and Boxshall 2014: 45, fig. 31. Regioscalpellum moluccanum (Hoek, 1883)
Figure 10 Diotascalpellum rubrum. – Gale 2016: 297, figs 2A, 6U–Y, 11Q–T,
13K–M, 18A–G. Scalpellum moluccanum Hoek, 1883: 104, pl. V figs 3, 4; 1907: 56, 85,
pl. VII, fig. 13. – Gruvel 1905: 76, fig. 85. – Nilsson-Cantell 1927:
747, fig. 3. Material examined. – Kei Islands: 2 specimens,
MNHN-IU-2019-4925, Stn. CP 09, 05°23'S, 132°29'E,
368–389 m depth, 23 October 1991; 1 specimen, MNHN-
IU-2019-4926, Stn. CP 16, 05°17'S, 132°50'E, 330–
350 m depth, 24 October 1991, attached to gorgonian. Arcoscalpellum moluccanum. – Newman & Ross, 1971: 60. – Jones et
al. 1990: 6, 27. – Jones 1992: 172–175, figs 18, 19. Trianguloscalpellum moluccanum. – Jones, 2012: 371, table 2. – Sha
laeva and Boxshall 2014: 45, fig. 31. Trianguloscalpellum moluccanum. – Jones, 2012: 371, table 2. – Sha
laeva and Boxshall 2014: 45, fig. 31. zse.pensoft.net 660 Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) Figure 9. Diotascalpellum rubrum (Hoek, 1883) (MNHN-IU-2019-4925). а. left lateral view showing the capitulum and peduncle;
b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale
bar: 5 mm (a–d). Figure 9. Diotascalpellum rubrum (Hoek, 1883) (MNHN-IU-2019-4925). а. left lateral view showing the capitulum and peduncle;
b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale
bar: 5 mm (a–d). Regioscalpellum moluccanum. – Gale, 2016: 298. mandible with three large teeth excluding inferior an
gle. Measurements of five specimens: height of capitu
lum 7.88–14.89 mm, width 4.81–10.65 mm, thickness
2.15–5.24 mm; length of peduncle 3.38–9.13 mm, width
2.09–6.04 mm. Material examined. – Tanimbar Island: 3 specimens,
MNHN-IU-2019-4909, Stn. CP 38, 07°40'S, 132°27'E,
620–666 m depth, 28 October 1991; 1 specimen, MNHN-
IU-2019-4910, Stn. DW 49, 08°00'S, 132°59'E, 206–210 m
depth, 29 October 1991; 1 specimen, MNHN-IU-2019–4911,
Stn. CC 57, 08°19'S, 131°53'E, 603–620 m depth, 31 Octo
ber 1991; 1 specimen, MNHN-IU-2019-4912, Stn. CP 52,
08°03'S, 131°48'E, 1244–1266 m depth, 30 October 1991. Distribution. Pacific, Central and Southwest; Tasman
Sea, north-eastern Australia; 788–2,745 m depth (Jones
2012; Shalaeva and Boxshall 2014). In this study, Re
gioscalpellum moluccanum was found at Tanimbar Is
land, Indonesia. Distribution. Pacific, Central and Southwest; Tasman
Sea, north-eastern Australia; 788–2,745 m depth (Jones
2012; Shalaeva and Boxshall 2014). In this study, Re
gioscalpellum moluccanum was found at Tanimbar Is
land, Indonesia. Diagnosis. Regioscalpellum moluccanum (Hoek, 1883)
Figure 10 Capitulum with 14 plates, surface covered
by chitinous, almost smooth membrane; carina simply,
not strongly bowed, apex projecting freely, roof not flat;
umbo of the carina apical; upper latus almost triangular. Type locality. Banda Sea (4°21'S, 129°7'E); Challenger
stn 195; 2,606 m depth: bottom temp. 3 °C; substrate grey
ooze (Hoek 1883). Description. Capitulum inflated, subtriangular, apices of
plates projecting freely through membrane, growth lines
fairly distinct. Scutum trapezioid, umbo apical. Tergum
large, oval, truncated, very pointed, extending between
upper latus and carina. Carina simply, slightly bowed,
roof slightly convex, apex projecting freely. Upper latus
quadrangular; rostrum very small, carinate; rostral latus
trapeziform; infra-median latus triangular, moderately
small; carinal margins of carinal latera convex, touching
each other at base and below middle of carina. Peduncle
stout, slightly thinner below apex. Cirrus I unequal, an
terior ramus oval, posterior ramus more slender; cirrus
VI with long caudal appendages. Maxillule not notched,
large spine on upper side, cutting edge almost straight; zse.pensoft.net Genus Scalpellum Leach, 1817 Scalpellum Leach, 1817: 68. – Darwin 1852: 21. – Hoek 1883: 59. –
Gruvel 1905: 23. – Pilsbry 1907d: 181. – Tarasov and Zevina 1957:
126. – Zevina 1978a: 1002. – 1981: 94. – Gale 2016: 297. Strictoscalpellum Broch, 1924: 14. Diagnosis. Scalpellines with carinal and scutal umbones
subapical; carinolatus with horn-like projection, often
recurved, extending beyond carinal margin; inframedian
latus rectangular to slightly trapezoidal, with low umbo;
upper latus rhomboidal with subapical umbo; rostrum
rectangular, pyramidal, with large sub-umbonal surface. Zoosyst. Evol. 96 (2) 2020, 649–698 661 Figure 10. Regioscalpellum moluccanum (Hoek, 1883) (MNHN-IU-2019-4909). а. left lateral view showing the capitulum and
peduncle; b. right lateral view showing the capitulum and peduncle; c. carinal view showing the capitulum and peduncle; d. rostral
view showing the capitulum and peduncle. Scale bar: 4 mm (a–d). Figure 10. Regioscalpellum moluccanum (Hoek, 1883) (MNHN-IU-2019-4909). а. left lateral view showing the capitulum and
peduncle; b. right lateral view showing the capitulum and peduncle; c. carinal view showing the capitulum and peduncle; d. rostral
view showing the capitulum and peduncle. Scale bar: 4 mm (a–d). Type species. Lepas scalpellum Linnaeus, 1767: 1109. Type species. Lepas scalpellum Linnaeus, 1767: 1109. pitulum 15.90 mm, width 10.29 mm, thickness 4.58 mm;
length of peduncle 6.97 mm, width 5.67 mm. pitulum 15.90 mm, width 10.29 mm, thickness 4.58 mm;
length of peduncle 6.97 mm, width 5.67 mm. Type locality. Unknown. Distribution. In this study, Scalpellum sp. was found in
Tanimbar Island, Indonesia. Scalpellum stearnsi Pilsbry, 1890
Figure 12 left lateral view, capitulum and peduncle; b. right lateral view, capitulum and
peduncle; c. rostral view, capitulum and peduncle; d. carinal view. Scale bar: 7 mm (a–d). Figure 11. Scalpellum sp. (MNHN-UI-2019-4929). а. left lateral view, capitulum and peduncle; b. right lateral view, capitulum and
peduncle; c. rostral view, capitulum and peduncle; d. carinal view. Scale bar: 7 mm (a–d). MNHN-IU-2019-4891, Stn. CP 69, 08°42'S, 131°53'E,
356–368 m depth, 2 November 1991; 5 specimens,
MNHN-IU-2019-4892, Stn. CC 41, 07°45'S, 132°42'E,
393–401 m depth, 28 October 1991; 5 specimens,
MNHN-IU-2019-4893, Stn. CP 59, 08°20'S, 132°11'E,
399–405 m depth, 31 October 1991, 2 specimens attached
to glass rope sponge. MNHN-IU-2019-4891, Stn. CP 69, 08°42'S, 131°53'E,
356–368 m depth, 2 November 1991; 5 specimens,
MNHN-IU-2019-4892, Stn. CC 41, 07°45'S, 132°42'E,
393–401 m depth, 28 October 1991; 5 specimens,
MNHN-IU-2019-4893, Stn. CP 59, 08°20'S, 132°11'E,
399–405 m depth, 31 October 1991, 2 specimens attached
to glass rope sponge. north east Australia; Malay Archipelago; Celebes; Sulu
Arch.; Sulu Sea; South China Sea; East China Sea; Phil
ippines; Taiwan; east coast of south Japan, Sagami Bay,
off Hondo; attached to mollusc shells, anchor filaments
of hexactinellid sponges, telegraph cables, carapaces of
crabs, stones; 146–2117 m depth (Jones and Hosie 2016). In this study, Scalpellum stearnsi was found at Tanimbar
Island and Kei Islands, Indonesia. – Kei Islands: 8 specimens, MNHN-IU-2019-4894,
Stn. CP 06, 05°49'S, 132°21'E, 287–298 m depth, 22 Oc
tober 1991. Type locality. East coast of Japan, between the Bay of
Tokyo and the Inland Sea (Jones 1992). Diagnosis. Capitulum rhomboidal, compressed; carina
strongly angled at umbo; scutum large, umbo at tergo-oc
cludent angle; tergum triangular; inframedian latus pen
tagonal; carinolateral horn-shaped. Remarks. For the first time, Scalpellum stearnsi was
found in Japan and described by Pilsbry (1890). During
the Siboga expedition (1899), S. stearnsi was collected
from different locations in the Malay Archipelago with
the depths varying from 204 m to 450 m. Hoek (1907)
found intraspecific variations of the shell plate mor
phology. He then divided S. stearnsi into two groups,
i.e. variety robusta and var. gemina, which differed in
the shape of the tergum. The species S. stearnsi in this
study belongs to the group of var. gemina because of the
V-shaped tergum. Description. Capitulum with 14 fully calcified plates. Scutum large, longer than broad, apex slightly projecting
over tergum; tergum large, triangular, apex erect, slightly
recurved. Scalpellum stearnsi Pilsbry, 1890
Figure 12 Material examined. – Tanimbar Island: 1 specimen,
MNHN-UI-2019-4929, Stn. DW 49, 08°00'S, 132°59'E,
206–210 m depth, 29 October 1991. Figure 12 Figure 12 Scalpellum magnum Darwin, 1852: 18, pl. I, fig. 1. Scalpellum magnum Darwin, 1852: 18, pl. I, fig. 1. Scalpellum calcariferum Fischer, 1891: 116. Scalpellum calcariferum Fischer, 1891: 116. Scalpellum stearnsii var. gemina and var. robusta Hoek, 1907: 69,
pl. VI, figs 2, 3, 8–12. Diagnosis. Capitulum with 14 fully calcified plates; ter
gum triangular; inframedian latus broad, quadrilateral
with umbo near to basal; upper latus rhomboidal, umbo
sub-apical; carinal lateral horn-shaped, with angle ex
tending beyond carina. Scalpellum stearnsii var. inerme Annandale, 1916a: 293. Scalpellum stearnsii var. inerme Annandale, 1916a: 293. Scalpellum stearnsii forma typica Broch, 1931: 16. Scalpellum stearnsii forma typica Broch, 1931: 16. Scalpellum stearnsii Pilsbry, 1890a: 96. – 1890b: 441, pl. IV, figs 1–5. – 1907a: 14, pl. 4, figs 1–5. – Nilsson-Cantell 1921: 175. – 1934:
33. – Broch 1922: 235, fig. 6. – Hiro 1933: 22, fig. 4, pl. 1, figs 5,
5a. – 1939a: 237. – Zevina 1981: 98, fig. 68. – Rosell 1991: 15. –
Jones 1992: 146, figs 1, 2. – Liu and Ren 2007: 226, fig. 95. – Chan
et al. 2009b: 89, figs 73–76. – Chan et al. 2010: 24, figs 2C, 18A–G. Scalpellum stearnsi: Gruvel, 1905: 44, fig. 46. – Gale 2016: fig. 5, table 1. Description. Capitulum subtriangular, covered by mem
brane. Tergum triangular, occludent and basal margins
slightly convex; scutum quadrangular, basal and upper
latus margins concave; upper latus pentagonal; rostrolatus
quadrangular; inframedian latus broad, rectangular, umbo
close to basal margin; carinolatus horn-shaped with slight
ly deep transverse furrows, umbo at basi-carinal angle;
carina convex, umbo apical. Cirrus I with rami unequal,
anterior ramus broader than posterior ramus; both rami
densely covered with long setae. Maxilla subtriangular,
with dense long setae; maxillule notched, with numerous
setae; mandible with three teeth, inferior angle with large
cuspidate setae. Measurements of specimen: height of ca Material examined. – Tanimbar Island: 1 specimen,
MNHN-IU-2019-4888, Stn. CP 71, 08°38'S, 131°44'E,
477–480 m depth, 2 November 1991; 1 specimen,
MNHN-IU-2019-4889, Stn. CC 58, 08°19'S, 132°02'E,
457–461 m depth, 31 October 1991; 6 specimens,
MNHN-IU-2019–4890, Stn. CC 42, 07°53'S, 132°42'E,
350–354 m depth, 28 October 1991; 3 specimens, zse.pensoft.net 662 Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) Figure 11. Scalpellum sp. (MNHN-UI-2019-4929). а. Scalpellum stearnsi Pilsbry, 1890
Figure 12 Carina distinctly bent near middle, umbo dis
tant from apex. Cirrus I with anterior ramus oval (12-seg
ments), posterior ramus slender, long (15-segments); cir
ri II–VI slender, long, rami almost equal length; cirrus
VI with short caudal appendages (six segments). Max
illule not notched, with numerous setae; mandible with
six teeth excluding inferior angle. Measurements of five
specimens: height of capitulum 31.69–52.00 mm, width
28.20–40.11 mm, thickness 11.19–18.56 mm; peduncle
length 28.29–86.59 mm, width 11.39–17.94 mm. Scalpellum stearnsi has a low period of larval develop
ment (Ozaki et al. 2008) and a slow growth rate (Yusa
et al. 2018). This can result in the broad geographical
distribution of this species. Recently, Lin et al. (2020)
examined the diversity and genetic differentiation of
populations of S. stearnsi from the East China Sea, West
Philippine Basin, Sulu Sea and Caroline Trenches, which
resulted in four distinct clades of S. stearnsi. Distribution. Indo-west Pacific: Indian Ocean, off Nico
bar Island; Indonesia: Java Sea, Bali Straits; north west & zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 663 Figure 12. Scalpellum stearnsi Pilsbry, 1890 (MNHN-IU-2019-4888). а. left lateral view, capitulum and peduncle; b. right lateral
view, capitulum and peduncle; c. rostral view, capitulum and peduncle; d. carinal view. Scale bar: 14 mm (a–d). Figure 12. Scalpellum stearnsi Pilsbry, 1890 (MNHN-IU-2019-4888). а. left lateral view, capitulum and peduncle; b. right lateral
view, capitulum and peduncle; c. rostral view, capitulum and peduncle; d. carinal view. Scale bar: 14 mm (a–d). Type genus. Meroscalpellum Zevina, 1978b: 1346. Type genus. Meroscalpellum Zevina, 1978b: 1346. Subfamily MEROSCALPELLINAE Zevina, 1978 Material examined. – Tanimbar Island: 2 specimens,
MNHN-IU-2019-4903, Stn. CP 71, 08°38'S, 131°44'E,
477–480 m depth, 2 November 1991; 5 specimens,
MNHN-IU-2019-4904, Stn. CC 21, 05°14'S, 133°00'E,
688–694 m depth, 25 October 1991 attached to glass rope
sponge; 1 specimen, MNHN-IU-2019-4905, Stn. CC 40,
07°46'S, 132°31'E, 443–468 m depth, 28 October 1991; 5
specimens, MNHN-IU-2019-4906, Stn. CC 57, 08°19'S,
131°53'E, 603–620 m depth, 31 October 1991. Meroscalpellinae Zevina, 1978b: 1343. Diagnosis. Capitulum with 14 or 13 plates, reduced in
differing stages or proportions; carina with two umbo
positions; females considered rarer than hermaphrodites;
males sac‑like, usually without plates, rarely with two or
four reduced plates. Diagnosis. Capitulum with plates buried or obscure;
plates reduced in size; tegum reduced, forming four-point
ed star; scutum small; peduncle armed with distinct cir
cles of large, calcareous scales. Type genus. Meroscalpellum Zevina, 1978b: 1346. Scalpellum persona Annandale, 1916a: 295, pl. IV, fig. 3; pl. V, figs 7,
8; pl. VI, figs 3–5. – Calman 1918b: 120. – Nilsson-Cantell 1934:
44, pl. 5, fig. 2. Alcockianum Zevina, 1978b: 1345. Description. Capitulum brownish, large, ovoid, inflated,
with 13 capitular plates, including a vestigial rostrum,
plates embedded and mostly concealed by thick, opaque
membrane. Scutum small, widely separated from all re
maining plates except tergum, margins not excavated or
deeply concave; tergum reduced in form as four-pointed
star, with two rays greatly and two rays slightly produced. Carina reduced in size, apex approaching terga, widely
separated from remaining plates. Peduncle cylindrical,
similar length to capitulum, with large calcareous scales
arranged in alternating rows. Cirrus I with anterior ramus
oval (8-segments), posterior ramus slender, long (12-seg
ments); cirri II–VI slender, long, rami almost equal length;
cirrus VI with caudal appendages; caudal appendages 1/3
length of cirrus VI, 15-segmented, tapering distally. Penis
rather short, smooth, pointed. Maxilla bilobed, dense setae
on margin. Maxillule relatively large, with broad, shallow
excavation on lower margin occupying more than half mar
gin, remainder of margin obliquely subtruncate; mandible
with three main teeth in addition to inner angle, which is
variously divided, broad as a whole. Measurements of five Diagnosis. Plates strongly reduced; partly obscured un
der heavy cuticle; tergum double branched, depressed at
base; scutum triangular; upper lateral triangular; mid‑lat
eral very small, umbo apical; caudal appendages very
long; hermaphrodites. Type species. Scalpellum alcockianum Annandale, 1905:
82; type locality: Gulf of Manaar and Andaman Sea. zse.pensoft.net – Jones 1992: 151–154, figs 4, 5. Alcockianum persona Zevina, 1978b: 1345. – 1981: 150–151, fig. 107.
– Jones 1992: 151–154 figs 4 5 Scalpellum persona Annandale, 1916a: 295, pl. IV, fig. 3; pl. V, figs 7,
8; pl. VI, figs 3–5. – Calman 1918b: 120. – Nilsson-Cantell 1934:
44, pl. 5, fig. 2.
Graviscalpellum persona Foster, 1980: 527, figs 1e, 2.
Alcockianum persona Zevina, 1978b: 1345. – 1981: 150–151, fig. 107.
– Jones 1992: 151–154, figs 4, 5. Graviscalpellum persona Foster, 1980: 527, figs 1e, 2. Genus Alcockianum Zevina, 1978 Alcockianum Zevina, 1978b: 1345. Alcockianum Zevina, 1978b: 1345. Alcockianum persona (Annandale, 1916) Figure 13 Scalpellum persona Annandale, 1916a: 295, pl. IV, fig. 3; pl. V, figs 7,
8; pl. VI, figs 3–5. – Calman 1918b: 120. – Nilsson-Cantell 1934:
44, pl. 5, fig. 2. Graviscalpellum persona Foster, 1980: 527, figs 1e, 2. – Jones 1992: 151–154, figs 4, 5. zse.pensoft.net 664 Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) Figure 13. Alcockianum persona (Annandale, 1916) (MNHN-IU-2019-4903). а. left lateral view, capitulum and peduncle; b. right
lateral view, capitulum and peduncle; c. rostral view, capitulum and peduncle; d. carinal view. Scale bar: 9 mm (a–d). Figure 13. Alcockianum persona (Annandale, 1916) (MNHN-IU-2019-4903). а. left lateral view, capitulum and peduncle; b. right
lateral view, capitulum and peduncle; c. rostral view, capitulum and peduncle; d. carinal view. Scale bar: 9 mm (a–d). 69, taf. II, fig. 4. – Hiro 1937 b: 392. – Tarasov and Zevina 1957: 144,
figs 46, 47. – Zevina 1969: 68. – 1970: 257–259, figs 5, 6. Scalpellum chitinosum Hoek, 1907: 73. Scalpellum curiosum Hoek, 1907: 49. Scalpellum japonicum biramosum Pilsbry, 1911: 68, fig. 4. – Weltner
1922: 69, taf. II, fig. 5. – Nilsson-Cantell 1938: 7. Scalpellum japonicum metapleurum Pilsbry, 1907b: 360. Annandaleum japonicum biramosum Chan et al., 2009b: 98–99, fig. 80. – Chan et al. 2010: 17–18, figs 2A, 14, 15. Annandaleum japonicum. – Newman & Ross, 1971: 122. – Zevina
1981: 166–167. – Jones et al. 2001: 251. – Young 2001: 465, fig. 8. – 2007: 23, fig. 22. specimens: height of capitulum 17.71–35.06 mm, width
12.91–28.06 mm, thickness 4.96–18.20 mm; length of pe
duncle 13.42–28.67 mm, width 6.66–10.79 mm. Scalpellum chitinosum Hoek, 1907: 73. Scalpellum curiosum Hoek, 1907: 49. Scalpellum japonicum biramosum Pilsbry, 1911: 68, fig. 4. – Weltner
1922: 69, taf. II, fig. 5. – Nilsson-Cantell 1938: 7. Distribution. Indonesian Seas, eastern Australia, New Zea
land; 109–915 m depth (Jones 1992). In this study, Alcock
ianum persona was found at Tanimbar Island, Indonesia. Scalpellum japonicum metapleurum Pilsbry, 1907b: 360. Annandaleum japonicum biramosum Chan et al., 2009b: 98 99, fig. 80. – Chan et al. 2010: 17–18, figs 2A, 14, 15. – Chan et al. 2010: 17–18, figs 2A, 14, 15. Type лocality. Timor Sea (10°22'30"S, 120°7'30"E);
109–366 m depth: bottom temp. 13.1 °C (Jones 1992). Type лocality. Timor Sea (10°22'30"S, 120°7'30"E);
109–366 m depth: bottom temp. 13.1 °C (Jones 1992). Annandaleum japonicum. – Newman & Ross, 1971: 122. – Zevina
1981: 166–167. Alcockianum persona (Annandale, 1916) – Jones et al. 2001: 251. – Young 2001: 465, fig. 8. – 2007: 23, fig. 22. Genus Annandaleum Newman & Ross, 1971 Material examined. – Tanimbar Island: 4 specimens,
MNHN-IU-2019-4932, Stn. CP 53, 08°18'S, 131°41'E,
1026–1053 m depth, 30 October 1991. Annandaleum Newman & Ross, 1971: 122. – Zevina 1978b: 1346. Diagnosis.Capitulum of female with 14 partially calcified
plates; tergum in shape of inverted V; scutum with moder
ately long apicolateral arm, 1/4 to 1/2 length of tergal mar
gin; basal margin of scutum entire; upper latus pentagonal
to triangular or subrectangular, with or without short de
pending arm; carinolatus as high as or slightly higher than
wide, umbo at basicarinal angle; inframedian latus higher
than wide, vase-shaped, umbo submedial to basal; rostro
latus wider than high, commonly less than twice height of
inframedian latus; caudal appendage relatively long. Diagnosis. Capitulum with 14 partly calcified plates; ca
rina with flat roof; apex of tergum curved towards carina;
rostral latus trapezoidal in shape; peduncle short. Description. Capitulum yellowish, with 14 plates sepa
rated by broad, chitinous spaces. Peduncle short, curved,
scales large, not numerous. Scutum long, narrow; umbo
apical, divided into two segments, occludent segment
large, increasing in width from upper to lower part, oth
er segment very narrow. Tergum divided into occludent
and carinal segments, with triangular, chitinous portion
between, umbo apical, distinctly recurved. Cirrus I un
equal, anterior ramus oval, posterior ramus slender long;
cirrus VI with caudal appendages. Maxillule slightly
notched, two long and one short setae on upper side;
mandible with three teeth excluding inferior angle; la
brum cutting edge straight, very fine teeth on cutting
edge. Measurements of the four specimens: height of ca Type species. Scalpellum subflavum Annandale, 1906. Type species. Scalpellum subflavum Annandale, 1906. Type species. Scalpellum subflavum Annandale, 1906. Type species. Scalpellum subflavum Annandale, 1906. Scalpellum japonicum Hoek, 1883: 67, pl. III, figs 9, 10. – Annandale
1906: 131, fig. 3. – Pilsbry 1911: 66, pl. II, figs 1–3. – Weltner 1922: Annandaleum japonicum (Hoek, 1883) Figure 14 zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 665 Figure 14. Annandaleum japonicum (Hoek, 1883) (MNHN-UI-2019-4932). а. left lateral view showing the capitulum and pedun
cle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 2 mm (a–d). Figure 14. Annandaleum japonicum (Hoek, 1883) (MNHN-UI-2019-4932). а. left lateral view showing the capitulum and pedun
cle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale bar: 2 mm (a–d). Material examined. – Tanimbar Island: 1 specimen,
MNHN-IU-2019-4931, Stn. CP 54, 08°21'S, 131°43'E,
836–869 m depth, 30 October 1991, attached to gas
tropod shell. pitulum 9.56–12.73 mm, width 4.67–7.13 mm, thickness
1.85–3.21 mm; length of peduncle 3.37–4.77 mm, width
1.83–3.27 mm. Distribution. Eastern Indian Ocean; Northwest and
Western Central Pacific; Malay Archipelago; Japan; Tai
wan; Indonesia; attached to shell of gastropod, gorgoni
ans, rocks; 805–6,810 m depth (Jones and Hosie 2016). In this study, Annandaleum japonicum was found at Ta
nimbar Island, Indonesia. Diagnosis. Capitulum regularly oval; carina simply
bowed; tergum almost triangular; scutum subtriangular;
upper latus large, perfectly calcified part irregularly tri
angular, constricted above; rostral latus quadrangular;
peduncle at least half as long as capitulum. Description. Capitulum compressed; 13 plates complete
ly covered by fine, hairless membrane. Peduncle half
length of capitulum, stout, cylindrical, armed with small,
transversely elongated plates. Scutum subtriangular, lat
eral margin excavated with tooth above excavation blunt,
short, simple; tergum almost triangular, scutal margin ex
cavated, but not very boldly, occludent margin slightly,
regularly convex outwards. Carina simply bowed, umbo
subterminal, in contact with terga above or just entering
between them. Cirrus I unequal, anterior ramus oval,
posterior ramus slender, long; cirri II–VI slender, long,
rami almost equal lengths; cirrus VI with long, slender
caudal appendages. Maxillule slightly notched, two ma
jor setae on upper side; mandible with four teeth. Mea
surements of specimen: height of capitulum 18.88 mm,
width 11.41 mm, thickness 6.30 mm; length of peduncle
11.45 mm, width 4.89 mm. Type locality. Pacific, Northwest (off Nagoya, south of
Japan); 1,017 m depth (Shalaeva and Boxshall 2014). Type locality. Pacific, Northwest (off Nagoya, south of
Japan); 1,017 m depth (Shalaeva and Boxshall 2014). Annandaleum laccadivicum (Annandale, 1906)
Figure 15 Scalpellum laccadivicum Annandale, 1906b: 393. – 1908: pl. I, figs 3, 4. – 1913: 235; 1916b: 129, pl. vii, fig. 6. – Calman 1918b: 124. – Hiro
1933: 31, text figs 7, 8, pl. I, figs 11–11b. – 1937b: 392. – Stubbings
1936: 26. – Nilsson-Cantell 1938: 25. Scalpellum subflavum Annandale, 1906b: 397. – Newman and Ross
1971: 122. Scalpellum polymorphum Hoek, 1907: 80, pl. 7, figs 9–11. – Weltner
1922: 72. Scalpellum molliculum Pilsbry, 1911: 68, pl. 10, figs 4, 5. Annandaleum laccadivicum Rosell, 1991: 16, fig 1a. – Zevina 1981: 170, fig. 121. – Jones et al. 2001: 251. – Chan et al. 2009b: 102–103. zse.pensoft.net 666 Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) Figure 15. Annandaleum laccadivicum (Annandale, 1906) (MNHN-IU-2019-4931). а. left lateral view showing the capitulum and
peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal
view. Scale bar: 5 mm (a–d). Figure 15. Annandaleum laccadivicum (Annandale, 1906) (MNHN-IU-2019-4931). а. left lateral view showing the capitulum and
peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal
view. Scale bar: 5 mm (a–d). Distribution. Gulf of Oman, Arabian Sea, Sri Lanka,
Japan (Chan et al. 2009b). In this study, Annandaleum
laccadivicum was found at Tanimbar Island, Indonesia. ly formed, thin, smooth, translucent plates, eight formed
like a Greek lambda. Description. Capitulum with 14 plates. Peduncle
shorter than capitulum, cylindrical, rather slender, ex
panded at base. Tergum lambda-shaped, with promi
nent tooth on carinal margin just above point where
apex of carina approaches it. Scutum shaped similarly,
but occludent branch much stouter, with vertical ridge
running nearer to lateral than occludent margin. Cirrus
I unequal, anterior ramus oval, posterior ramus slender,
long; cirri II–VI slender, long, with rami almost equal
length; cirrus VI with long, slender caudal appendag
es. Maxillule not notched, two major setae on upper
side; mandible with four teeth including inner angle. Measurements of five specimens: height of capitulum
16.61–24.27 mm, width 8.73–14.49 mm, thickness
3.17–6.31 mm; length of peduncle 5.54–15.27 mm,
width 3.73–6.26 mm. Type locality. Laccadive Sea, 2,077 m depth; on living
shells of Dentalium (Annandale 1906). Annandaleum lambda (Annandale, 1910)
Figure 16 Scalpellum lambda Annandale, 1910b: 115. – 1916a: pl. VII, figs 6, 6a,
pl. VIII, figs 12–15. Scalpellum longius Annandale, 1913: 234. Scalpellum longius Annandale, 1913: 234. Annandaleum lambda. – Newman & Ross, 1971: 122. – Jones, 1992:
154, fig. 6. Material examined. – Tanimbar Island: 10 specimens,
MNHN-IU-2019-4915, Stn. CP 52, 08°03'S, 131°48'E,
1244–1266 m depth, 30 October 1991; 1 specimen,
MNHN-IU-2019-4913, Stn. CP 53, 08°18'S, 131°41'E,
1026–1053 m depth, 30 October 1991; 1 specimen, MNHN-
IU-2019-14, Stn. CP 89, 08°39'S, 131°08'E, 1058–1084 m
depth, 5 November 1991, attached to pumice. Distribution. Indo-Pacific from off Zanzibar, Indian
Ocean, eastern Australia; Sumbawa, Indonesia; Malay
Arch.; SW of Calatagan Pt, Philippines; S Japan (S of
Honda I.), SW Pacific; attached to shells of bivalves;
234–2077 m depth (Jones and Hosie 2016). In this
study, Annandaleum lambda was found at Tanimbar Is
land, Indonesia. Diagnosis. Capitulum narrow; carinal margin strongly
curved; laterally compressed; bearing fourteen imperfect zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 667 Figure 16. Annandaleum lambda (Annandale, 1910) (MNHN-IU-2019-4915). а. left lateral view showing the capitulum and pe
duncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal
view. Scale bar: 6 mm (a–d). Figure 16. Annandaleum lambda (Annandale, 1910) (MNHN-IU-2019-4915). а. left lateral view showing the capitulum and pe
duncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal
view. Scale bar: 6 mm (a–d). 549–552 m depth, 31 October 1991; 1 specimen, MNHN-
IU-2019-4898, stn. CP 71, 08°38'S, 131°44'E, 477–
480 m depth, 2 November 1991; 6 specimens, MNHN-
IU-2019-4901, stn. CP 72, 08°36'S, 131°33'E, 676–699 m
depth, 2 November 1991. Type locality. Eastern Indian Ocean (13°54'15"N,
94°02'15"E), Investigator Stn. 372; 1,176 m depth
(Jones 1992). Genus Litoscalpellum Newman & Ross, 1971 Diagnosis. Capitulum compressed, oval, notched above;
surface covered with very short velvety pubescence, ar
eas of valves defined by grooves, lines of growth strongly
marked; carina flattened. Litoscalpellum Newman & Ross, 1971: 108. – Zevina 1978b: 1344. –
Liu and Ren 1985: 196. – 2007: 228. Diagnosis. Capitulum with 14 plates, mostly not reduced;
tergum with straight basal margin or shallow notch; scu
tum with straight, smooth basal margin; upper latus trian
gular or elongate, commonly with slightly hollowed-out
basal margin, rarely with deep notch; inframedian latus
narrow, triangular or quadrilateral, umbo apical; caudal
appendages present. Description. Capitulum with 14 plates, cartilaginous thick. Terqum irregularly triangular, apex projecting above cari
na, recurved. Scutum triangular, basal width more than half
height, apex overlapping tergum. Carina evenly curved,
apex projecting freely for short distance, touching or enter
ing between terga, its base rounded, widely separated from
carinal latera; roof strongly convex. Upper latus triangular
with base convex. Rostrum triangular; inframedian latus
small, triangular, base deeply embedded. Peduncle with
large transverse plates, not overlapping (widely spaced). Cirrus I unequal, anterior ramus oval, posterior ramus slen
der, long; cirrus VI with caudal appendages. Maxillule with
oral edge sinuous, a large seta at its proximal third, another
at its distal end; mandible with four teeth. Measurements
of five specimens: height of capitulum 16.94–39.25 mm,
width 11.39–28.28 mm, thickness 5.20–19.89 mm; length
of peduncle 15.02–52.02 mm, width 5.63–16.87 mm. Type species. Litoscalpellum fissicarinatum Newman &
Ross, 1971: 108, pl. XC, text-fig. 55. Type species. Litoscalpellum fissicarinatum Newman &
Ross, 1971: 108, pl. XC, text-fig. 55. Litoscalpellum recurvirostrum (Hoek, 1883)
Figure 18 Scalpellum recurvirostrum Hoek, 1883: 77–79, pl. III, figs 11, 12,
pl. VIII, figs 9, 10. Arcoscalpellum recurvirostrum. – Newman & Ross, 1971: 79–80, fig. 39. Litoscalpellum recurvirostrum. – Zevina, 1974: 214. – 1981: 127, 128,
fig. 88. – Shalaeva and Boxshall 2014: 13, fig. 6. Scalpellum recurvirostrum Hoek, 1883: 77–79, pl. III, figs 11, 12,
pl. VIII, figs 9, 10. Distribution. Indian Ocean, Antarctic and Southern
(South of the Kerguelen Islands); known depth range 195
to 274 m (Shalaeva and Boxshall 2014). In this study,
Litoscalpellum recurvirostrum was found at Kei Islands
and Tanimbar Island, Indonesia. Arcoscalpellum recurvirostrum. – Newman & Ross, 1971: 79–80, fig. 39. Litoscalpellum recurvirostrum. – Zevina, 1974: 214. – 1981: 127, 128,
fig. 88. – Shalaeva and Boxshall 2014: 13, fig. 6. Material examined. – Kei Islands: 1 specimen, MNHN-
IU-2019-4934, Stn. CP 05, 05°49'S, 132°18'E, 296–
299 m depth, 22 October 1991; 1 specimen, MNHN-
IU-2019-4936, Stn. CP 09, 05°23'S, 132°29'E, 368–389 m
depth, 23 October 1991. Type locality. Between Kerguelen and Heard Islands;
52°4'S, 71°22'E; depth: 270 m; bottom temperature:
1.8 °C; bottom: rocks (Hoek 1883). Type locality. Between Kerguelen and Heard Islands;
52°4'S, 71°22'E; depth: 270 m; bottom temperature:
1.8 °C; bottom: rocks (Hoek 1883). – Tanimbar Island: 4 specimens, MNHN-IU-2019-4935,
Stn. CP 83, 09°23'S, 131°00'E, 285–297 m depth, 4 Novem
ber 1991; 16 specimens, MNHN-IU-2019-4937, Stn. CP 69,
08°42'S, 131°53'E, 356–368 m depth, 2 November 1991. Litoscalpellum juddi (Calman, 1918) Figure 17 Figure 17 Scalpellum juddi Calman, 1918b: 116, text-figs 5–7. Scalpellum regulus Calman, 1918b: 113, text-fig. 4. Litoscalpellum juddi Zevina, 1981:135–136, fig. 94. Scalpellum juddi Calman, 1918b: 116, text-figs 5–7. Scalpellum regulus Calman, 1918b: 113, text-fig. 4. Litoscalpellum juddi Zevina, 1981:135–136, fig. 94. Scalpellum juddi Calman, 1918b: 116, text-figs 5–7. Scalpellum regulus Calman, 1918b: 113, text-fig. 4. Litoscalpellum juddi Zevina, 1981:135–136, fig. 94. Material examined. – Tanimbar Island: 5 specimens,
MNHN-IU-2019-4896, stn. CC 56, 08°16'S, 131°59'E, Distribution. In this study, Litoscalpellum juddi was
found at Tanimbar Island, Indonesia. zse.pensoft.net 668 Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) Figure 17. Litoscalpellum juddi (Calman, 1918) (MNHN-UI-2019-4898). а. left lateral view showing the capitulum and peduncle;
b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale
bar: 11 mm (a–d). Figure 17. Litoscalpellum juddi (Calman, 1918) (MNHN-UI-2019-4898). а. left lateral view showing the capitulum and peduncle;
b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale
bar: 11 mm (a–d). Type locality. 11°0'S, 121°30'E (Java-Australia), 720 m
depth (Calman 1918b). Cirrus I with rami unequal, anterior ramus oval, posterior
ramus slender, long, segments very hairy. Maxilla bilobed;
maxillule notched, two large setae above notch; mandible
with three teeth, inferior angle pectinated. Measurements
of five specimens: height of capitulum 16.15–26.05 mm,
width 9.24–16.59 mm, thickness 4.45–8.18 mm; length of
peduncle 7.25–20.90 mm, width 4.13–8.95 mm. zse.pensoft.net zse.pensoft.net Litoscalpellum walleni Newman & Ross, 1971: 116, pl. X E text-fig. 60. et al.: Deep-sea barnacles from the Karubar expedition (1991) 670 376 – Poltarukha 2013: 52–53, fig. 1. –Shalaeva and Boxshall 2014:
16, fig. 9. 376 – Poltarukha 2013: 52–53, fig. 1. –Shalaeva and Boxshall 2014:
16, fig. 9. angle serrate along basal margin with four spines. Mea
surements of specimen: height of capitulum 14.56 mm,
width 8.09 mm, thickness 2.50 mm; length of peduncle
7.52 mm, width 3.89 mm. angle serrate along basal margin with four spines. Mea
surements of specimen: height of capitulum 14.56 mm,
width 8.09 mm, thickness 2.50 mm; length of peduncle
7.52 mm, width 3.89 mm. Material examined. – Tanimbar Island: 2 specimens,
MNHN-IU-2019-4922, Stn. CC 21, 05°14'S, 133°00'E,
688–694 m depth, 25 October 1991, attached to glass
rope sponge. Distribution. Southeast Pacific Ocean (Newman and
Ross 1971). In this study, Litoscalpellum walleni was
found at Tanimbar Island, Indonesia. Distribution. Southeast Pacific Ocean (Newman and
Ross 1971). In this study, Litoscalpellum walleni was
found at Tanimbar Island, Indonesia. Diagnosis. Capitulum triangular; plates ornamented with
radial ridges, covered by sparsely hirsute integument; ca
rina with roof deeply grooved; scutum with apex over
lapping tergum; inframedian latus triangular, very small. Type locality. northeast of Peter I Island, southeast Pa
cific Ocean; 65°50'S, 88°56'W; depth: 4,502 m (Newman
and Ross 1971). Type locality. northeast of Peter I Island, southeast Pa
cific Ocean; 65°50'S, 88°56'W; depth: 4,502 m (Newman
and Ross 1971). Desciption. Capitulum yellowish, with 13 fully calci
fied plates. Peduncles short with scales slightly over
lapping in the middle part. Scutum with pit for comple
mental males, above shallow pit for adductor muscle. Carina wide in lower part, ribbed in upper part. Upper
latus with straight sides; rostrum appearing externally
as inverted triangle. Cirrus I unequal, anterior ramus
oval, posterior ramus slender, long; cirrus VI with very
short caudal appendages. Maxillule with notch between
two or three stout setae at upper angle, group of more
slender setae on cutting edge; mandible with three
teeth excluding inferior angle; labrum cutting edge
slightly concave, numerous pointed teeth on cutting
edge. Measurements of two specimens: height of ca
pitulum 12.44–13.88 mm, width 6.97–7.52 mm, thick
ness 2.77–3.47 mm; length of peduncle 2.99–3.15 mm,
width 2.94–3.24 mm. Subfamily ARCOSCALPELLINAE Zevina, 1978 Arcoscalpellinae Zevina, 1978b: 1346. –1981: 184. – Liu and Ren
2007: 238. Diagnosis. Capitulum with plates packed very closely; ca
rina slightly convex; umbo of scutum and tergum apical. Diagnosis. Capitulum with plates packed very closely; ca
rina slightly convex; umbo of scutum and tergum apical. Type genus. Arcoscalpellum Hoek, 1907: 57. Genus Amigdoscalpellum Zevina, 1978 Arcoscalpellum Hoek, 1907: 85 (in part). – Newman and Ross 1971:
42 (part). Arcoscalpellum Hoek, 1907: 85 (in part). – Newman and Ross 1971:
42 (part). Amigdoscalpellum Zevina, 1978b: 1349. – Foster 1980: 527. – Liu and
Ren 1985: 206. – 2007: 251. Amigdoscalpellum Zevina, 1978b: 1349. – Foster 1980: 527. – Liu and
Ren 1985: 206. – 2007: 251. Distribution. Indian Ocean, Eastern and Western; Pacif
ic, Southwest and Western Central; Atlantic, Southeast
and Eastern central; 110–2,397 m depth (Shalaeva and
Boxshall 2014). In this study, Amigdoscalpellum costel
latum was found at Tanimbar Island, Indonesia. Diagnosis. Capitulum of female or hermaphrodite with 13
or 14 completely calcified plates; scutum and tergum with
umbones apical; carina evenly bent, umbo apical or sub
apical; middle latera narrowly triangular, baton-like, small,
umbo apical, not reaching upper latera; carinolatera typi
cally with umbo orientated in middle part of carinal area,
not extending beyond rim of capitulum; caudal appendag
es usually uni-articulate, but possibly absent or extended to
seven segments; basically females with dwarf males. Type locality. 1. Station 135: Island of Tristan da Cunha;
depth: 110 m, 137 m, 183 m, 274 m, 1,006 m, 1,829 m,
2,012 m; bottom: rocky, shells. 2. Station 164a: off Syd
ney, 34°13'S, 151°38'E; depth: 750 m; bottom: grey
ooze. 3. Station 169: off East Cape, Auckland, 37°34'S,
179°22'E; depth: 1,280 m; bottom temperature: 4.2 °C;
bottom: grey ooze (Hoek 1883). Type species. Scalpellum manum Zevina, 1973: 843,
figs 1–7; type locality: Indian Ocean, 12°18'S, 112°43'E. Type species. Scalpellum manum Zevina, 1973: 843,
figs 1–7; type locality: Indian Ocean, 12°18'S, 112°43'E. Scalpellum elongatum Hoek, 1883: 93, pl. IV, figs 8, 9. – Nilsson-Can
tell 1928: 8, fig. 4. – 1931: 3, text-fig.1. – 1938: 7.
non Scalpellum (Arcoscalpellum) elongatum. – Steenstrup, 1837: 409.
Scalpellum (Scalpellum) elongatum. – Stubbings, 1936: 25, text-fig. 10.
Scalpellum (Arcoscalpellum) costellatum Withers, 1935: 279.
Arcoscalpellum buccinum Newman & Ross, 1971: 55, pl. VIII, figs 22, 23.
Arcoscalpellum costellatum. – Foster, 1978: 56, pl. 7B, C, fig. 33.
Amigdoscalpellum costellatum. – Foster, 1980: 527–529, fig. 3J. – Zevi
na 1981: 270–271, fig. 204. – Jones et al. 1990: 5 – Jones 2012: 371, Material examined. – Kei Islands: 1 specimen, MNHN-
IU-2019-4921, Stn. CP 20, 05°15'S, 132°59'E, 769–
809 m depth, 25 October 1991. Litoscalpellum walleni Newman & Ross, 1971: 116, pl. X E text-fig. 60. Diagnosis. Capitulum plates separated, covered by fine
membrane; carina slightly angularly bent; upper latus
trapeziform; inframedian latus triangular; peduncle with
scattered, calcareous scales. Material examined. – Tanimbar Island: 1 specimen,
MNHN-IU-2019-4927, Stn. CP 91, 08°44'S, 131°05'E,
884–891 m depth, 5 November 1991. Diagnosis. Capitulum with 14 plates; inframedian latus
triangular, very small, narrow; carina with flat roof; ros
trum minute; scutum lacking prominent apicolateral arm;
maxilla subtriangular, slightly trilobate; maxilulle slight
ly notched with teeth diverging laterally from cutting
edge; caudal appendage multi-articulate, slightly shorter
than first segment of cirrus VI. Description. Capitulum with 14 plates, lines of growth not
distinct, plates separated by broad, membranous interspac
es. Scutum elongate quadrangular, broader at base than
in upper half; umbo apical, slightly projecting outwards. Tergum triangular; carina bowed; upper latus trapeziform;
inframedian latus very small, triangular. Peduncle cylindri
cal with numerous calcareous scales scattered over surface. zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 669 Figure 18. Litoscalpellum recurvirostrum (Hoek, 1883) (MNHN-UI-2019-4934). а. left lateral view showing the capitulum and
peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal
view. Scale bar: 6 mm (a–d). Figure 18. Litoscalpellum recurvirostrum (Hoek, 1883) (MNHN-UI-2019-4934). а. left lateral view showing the capitulum and
peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal
view. Scale bar: 6 mm (a–d). Figure 19. Litoscalpellum walleni Newman & Ross, 1971 (MNHN-UI-2019-4927). а. left lateral view showing the capitulum and
peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal
view. Scale bar: 4 mm (a–d). Figure 19. Litoscalpellum walleni Newman & Ross, 1971 (MNHN-UI-2019-4927). а. left lateral view showing the capitulum and
peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal
view. Scale bar: 4 mm (a–d). Description. Capitulum elongate, higher than broad;
plates ornamented with growth lines. Carina bowed,
roof slightly convex, bounded by angles. Tergum trian
gular; scutum with lateral margin concave; upper latus
roughly quadrangular; carinal latus higher than wide; rostral latus quadrangular, slightly broader than high;
rostrum minute, narrow, essentially rectangular. Cirrus
I unequal, anterior ramus shorter, broader than posterior
ramus; both rami densely covered with long setae. Man
dible with four teeth including inferior angle, inferior zse.pensoft.net Pitriana, P. Scalpellum tenue Hoek, 1883: 119, pl. IV, figs 20, 21, pl. X, fig. 6. –
Gruvel 1912: 345. – Nilsson-Cantell 1938: 8.
non Scalpellum tenue Annandale, 1906a: 142.
Amigdoscalpellum tenue. – Zevina 1981: 292–293, fig. 220 (2). – Sha
laeva and Boxshall 2014: 17, fig. 10. Amigdoscalpellum tenue (Hoek, 1883)
Figure 21 Figure 21 Scalpellum elongatum Hoek, 1883: 93, pl. IV, figs 8, 9. – Nilsson-Can
tell 1928: 8, fig. 4. – 1931: 3, text-fig.1. – 1938: 7. non Scalpellum (Arcoscalpellum) elongatum. – Steenstrup, 1837: 409. Scalpellum (Scalpellum) elongatum. – Stubbings, 1936: 25, text-fig. 10. Scalpellum (Arcoscalpellum) costellatum Withers, 1935: 279. Arcoscalpellum buccinum Newman & Ross, 1971: 55, pl. VIII, figs 22, 23. Arcoscalpellum costellatum. – Foster, 1978: 56, pl. 7B, C, fig. 33. Amigdoscalpellum costellatum. – Foster, 1980: 527–529, fig. 3J. – Zevi
na 1981: 270–271, fig. 204. – Jones et al. 1990: 5 – Jones 2012: 371, Scalpellum elongatum Hoek, 1883: 93, pl. IV, figs 8, 9. – Nilsson-Can
tell 1928: 8, fig. 4. – 1931: 3, text-fig.1. – 1938: 7. Amigdoscalpellum tenue. – Zevina 1981: 292–293, fig. 220 (2). – Sha
laeva and Boxshall 2014: 17, fig. 10. Amigdoscalpellum tenue. – Zevina 1981: 292–293, fig. 220 (2). – Sha
laeva and Boxshall 2014: 17, fig. 10. Material examined. – Kei Islands: 1 specimen, MNHN-
IU-2019-4921, Stn. CP 20, 05°15'S, 132°59'E, 769–
809 m depth, 25 October 1991. zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 671 Figure 20. Amigdoscalpellum costellatum (Withers, 1935) (MNHN-IU-2019-4922). а. left lateral view showing the capitulum and
peduncle; b. right lateral view showing the capitulum and peduncle; c. carinal view; d. rostral view showing the capitulum and
peduncle. Scale bar: 3 mm (a–d). Figure 20. Amigdoscalpellum costellatum (Withers, 1935) (MNHN-IU-2019-4922). а. left lateral view showing the capitulum and
peduncle; b. right lateral view showing the capitulum and peduncle; c. carinal view; d. rostral view showing the capitulum and
peduncle. Scale bar: 3 mm (a–d). Figure 21. Amigdoscalpellum tenue (Hoek, 1883) (MNHN-IU-2019-4921). а. left lateral view showing the capitulum and pedun
cle; b. right lateral view showing the capitulum and peduncle; c. carinal view showing the capitulum and peduncle; d. rostral view
showing the capitulum and peduncle. Scale bar: 2 mm (a–d). Figure 21. Amigdoscalpellum tenue (Hoek, 1883) (MNHN-IU-2019-4921). а. left lateral view showing the capitulum and pedun
cle; b. right lateral view showing the capitulum and peduncle; c. carinal view showing the capitulum and peduncle; d. rostral view
showing the capitulum and peduncle. Scale bar: 2 mm (a–d). Diagnosis. Capitulum with 13 fully calcified plates; cari
na slightly bowed, umbo at apex; upper latus trapeziform;
inframedian latus small, narrow; carinal latus not project
ing beyond carina; peduncle short, cylindrical. Description. Catherinum rossi (Rao & Newman, 1972)
Figure 22 Figure 22 Arcoscalpellum rossi Lakshmana Rao & Newman, 1972: 82, fig. 7. Arcoscalpellum rossi Lakshmana Rao & Newman, 1972: 82, fig. 7. Catherinum rossi. – Zevina, 1978b: 1348. – Liu and Ren 1985: 2 Catherinum rossi. – Zevina, 1978b: 1348. – Liu and Ren 1985: 203,
fig. 12, pl. 2, figs 11–14. – 2007: 250, fig. 108. – Chan et al. 2009b:
113, figs 92–94. Distribution. Indian Ocean, Antarctic and Southern (North
East of Prince Edward Island); known depth 2,516 m (Sha
laeva and Boxshall 2014). In this study, Amigdoscalpellum
tenue was found at Kei Islands, Indonesia. Material examined. – Tanimbar Island: 1 specimen,
MNHN-IU-2019-4933, Stn. DW 02, 05°47'S, 132°13'E,
209–240 m depth, 22 October 1991. Type locality. 46°46'S, 45°31'E; depth: 2,475 m; bottom
temperature: 1.5 °C; bottom: globigerina ooze (Hoek 1883). Diagnosis. Capitulum smooth, elongate, 14 fully calci
fied plates; inframedia latus rectangular, not constricted;
rostrum large, ovo-triangular; caudal appendages short. Amigdoscalpellum tenue (Hoek, 1883)
Figure 21 Capitulum yellowish, elongate-oval shape;
surface with distinct lines of growth. Carina large, sim
ply bowed. Scutum with umbo at apex, slightly recurved,
projecting slightly over tergum; tergum triangular, stout, zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 672 umbo sub-basicarinal, not extending beyond carinal mar
gin; caudal appendages long. broad, apex recurved, scutal margin almost straight. Up
per latus quadrangular, apex slightly projecting over scu
tum. Rostral latus quadrangular, scutal and basal margins
parallel. Carinal latus quadrangular, carinal margin almost
straight. Cirrus I unequal, anterior ramus oval, posterior
ramus more slender. Maxillule with notch between two
or three stout setae at upper angle, a group of more slen
der setae on cutting edge; mandible with three teeth ex
cluding inferior angle; labrum with numerous blunt teeth
on straight, cutting edge. Measurements of specimen:
height of capitulum 7.26 mm, width 3.74 mm, thickness
1.40 mm; length of peduncle 3.00 mm, width 1.99 mm. Type species. Scalpellum recurvitergum Gruvel, 1902;
type locality: Atlantic Ocean, SW of Azores. Genus Catherinum Zevina, 1978 Description. Capitulum long, narrow, sparsely covered
with hairs, plates separated by narrow, chitinous inter
spaces, marked with growth lines. Occludent margin
strongly convex; carinal margin irregularly straight; apex
slightly retroverted towards carinal side. Carina long,
simply bowed; roof flat; parietes well developed towards
distal half of plate. Tergum triangular, occludent margin
short, convex, scutal and basal margins almost straight, Acroscalpellum Hoek, 1907: 85. – Newman and Ross 1971: 42 (part). –
Liu and Ren 1985: 202. Catherinum Zevina, 1978b: 1348. Acroscalpellum Hoek, 1907: 85. – Newman and Ross 1971: 42 (part). –
Liu and Ren 1985: 202.
Catherinum Zevina, 1978b: 1348. Acroscalpellum Hoek, 1907: 85. – Newman and Ross 1971: 42 (part). –
Liu and Ren 1985: 202. Catherinum Zevina, 1978b: 1348. Catherinum Zevina, 1978b: 1348. Diagnosis. Capitulum with 13 or 14 plates; inframedian
latus very narrow, bacilliform, umbo sub-medial; umbo
of upper latus apical or sub-apical; carinal latus broad, Figure 22. Catherinum rossi (Rao & Newman, 1972) (MNHN-IU-2019-4933). а. left lateral view showing the capitulum and pe
duncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal
view. Scale bar: 3 mm (a–d). Figure 22. Catherinum rossi (Rao & Newman, 1972) (MNHN-IU-2019-4933). а. left lateral view showing the capitulum and pe
duncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal
view. Scale bar: 3 mm (a–d). zse.pensoft.net 673 Zoosyst. Evol. 96 (2) 2020, 649–698 Type species. Scalpellum planum Hoek, 1883; type lo
cality: 42°42'S, 134°10'E; depth: 4,755 m; bottom tem
perature: 0.2 °C; bottom: red clay. carinal margin concave. Scutum with umbo apical, over
lapping occludent margin of tergum. Upper latus triangu
lar; carinal latus twice as long as broad; inframedian latus
rectangular; rostral latus nearly rectangular in outline;
rostrum large, elongate triangular, broad above, pointed
below. Cirrus I unequal, anterior ramus oval, posterior
ramus more slender; cirrus VI with caudal appendages. Maxillule not notched stout spine along the cutting edge;
mandible with three teeth excluding inferior angle. Mea
surements of specimen: height of capitulum 9.67 mm,
width 5.52 mm, thickness 2.58 mm; length of peduncle
2.79 mm, width 2.29 mm. Type species. Scalpellum planum Hoek, 1883; type lo
cality: 42°42'S, 134°10'E; depth: 4,755 m; bottom tem
perature: 0.2 °C; bottom: red clay. carinal margin concave. Scutum with umbo apical, over
lapping occludent margin of tergum. Upper latus triangu
lar; carinal latus twice as long as broad; inframedian latus
rectangular; rostral latus nearly rectangular in outline;
rostrum large, elongate triangular, broad above, pointed
below. Cirrus I unequal, anterior ramus oval, posterior
ramus more slender; cirrus VI with caudal appendages. Maxillule not notched stout spine along the cutting edge;
mandible with three teeth excluding inferior angle. Mea
surements of specimen: height of capitulum 9.67 mm,
width 5.52 mm, thickness 2.58 mm; length of peduncle
2.79 mm, width 2.29 mm. Planoscalpellum distinctum (Hoek, 1883)
Figure 23 Scalpellum distinctum Hoek, 1883: 111–112, pl. VI, figs 10, 11. – 1907: ii Scalpellum distinctum Hoek, 1883: 111–112, pl. VI, figs 10, 11. – 1907:
83, pl. VII, fig. 12 – Nilsson-Cantell 1927: 750, text-fig. 4. 83, pl. VII, fig. 12 – Nilsson-Cantell 1927: 750, text-fig. 4. Planoscalpellum distinctum. – Zevina, 1981: 186–187. – Foster and
Buckeridge 1995 a: 360–361, fig. 7(A–C) – Shalaeva and Boxshall
2014: 36, fig. 22. Distribution. East China Sea, Pacific Ocean, Taiwan
(Chan et al. 2009b). In this study, Catherinum rossi was
found at Tanimbar Island, Indonesia. Material examined. – Tanimbar Island: 2 specimens,
MNHN-IU-2019-4928, Stn. DW 49, 08°00'S, 132°59'E,
206–210 m depth, 29 October 1991. Type locality. Stn. 3, Hess Guyot (17°53.2'N,
174°24.8'W), 1,692–1,735 m depth (Sigsbee beam trawl);
Stn. 1, Allison Guyot (18°31.0'N, 179°36.0'W), 1,413–
1,645 m depth (otter trawl) (Rao and Newman 1972). Diagnosis. Capitulum with 13 plates separated from each
other by distinct chitinous interspaces; scutum almost tri
angular; upper latus hexagonal, large. Description. Capitulum flattened, elongate-oval shape;
plates with distinct growth lines, separated from each
other by rather broad chitinous interspaces. Scutum tri
angular, occludent margin arched, umbo of scutum api
cal, apex slightly turned upwards. Tergum triangular,
occludent margin not very long. Carina simply bowed. Upper latus irregularly hexagonal, rostral latus irreg
ularly quadrangular, infra-median latus in the form of
wine-glass, carinal latus irregularly quadrangular. Pe Teloscalpellum ecaudatum (Calman, 1918)
Figure 24 Scalpellum ecaudatum Calman, 1918b: 106, text-fig. 2. Teloscalpellum ecaudatum. – Zevina, 1981: 365, fig. 282. – Chan 2009:
55, figs 1E, 7. Material examined. – Kei Islands: 1 specimen, MNHN-
IU-2019-4917, Stn. CP 35, 06°08'S, 132°45'E, 390–
502 m depth, 27 October 1991. – Tanimbar Island: 1 specimen, MNHN-IU-2019-4918,
Stn. CP 59, 08°20'S, 132°11'E, 399–405 m depth, 31 Oc
tober 1991. Material examined. – Kei Islands: 1 specimen, MNHN-
IU-2019-4917, Stn. CP 35, 06°08'S, 132°45'E, 390–
502 m depth, 27 October 1991. Distribution. Pacific, Western Central; known depth
range 1,302 to 2,745 m (Shalaeva and Boxshall 2014). In this study, Planoscalpellum distinctum was found at
Tanimbar Island, Indonesia. – Tanimbar Island: 1 specimen, MNHN-IU-2019-4918,
Stn. CP 59, 08°20'S, 132°11'E, 399–405 m depth, 31 Oc
tober 1991. Type locality. 2°33'S, 144°4'E; depth: 1,926 m; bot
tom temperature: 2.1 °C; bottom: Globigerina ooze
(Hoek 1883). Type locality. 2°33'S, 144°4'E; depth: 1,926 m; bot
tom temperature: 2.1 °C; bottom: Globigerina ooze
(Hoek 1883). Diagnosis. Capitulum compressed, elongated; 14 plates,
sculptured with radiating ribs; inframedian latus triangu
lar; rostro-lateral plates very low; penis and caudal ap
pendages absent. Type species. Scalpellum spicatum Zevina, 1975. Type species. Scalpellum spicatum Zevina, 1975. duncle short, with very small, numerous, calcareous
scales. Cirrus I unequal, anterior ramus oval, posterior
ramus more slender; cirrus VI with short caudal append
ages. Maxillule notched, three stout spines on the up
per side; mandible with three teeth, excluding inferior
angle. Measurements of two specimens: height of ca
pitulum 6.80–7.90 mm, width 3.68–4.36 mm, thickness
1.24–1.26 mm; length of peduncle 1.39–2.49 mm, width
1.80–2.04 mm. Genus Planoscalpellum Zevina, 1978 Arcoscalpellum Hoek, 1907: 85 (part). Planoscalpellum Zevina, 1978b: 1347. Diagnosis. Upper latus with umbo on scutal margin; in
framedian latus with umbo apical or sub-basal; carinal
latus with umbo on carinal part, may be protruding. Figure 23. Planoscalpellum distinctum (Hoek, 1883) (MNHN-IU-2019-4928). а. left lateral view showing the capitulum and pe
duncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal
view. Scale bar: 2 mm (a–d). Figure 23. Planoscalpellum distinctum (Hoek, 1883) (MNHN-IU-2019-4928). а. left lateral view showing the capitulum and pe
duncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal
view. Scale bar: 2 mm (a–d). zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 674 Genus Teloscalpellum Zevina, 1978 Arcoscalpellum Hoek, 1907: 85 (sectio, part.). – Newman and Ross
1971: 42 (part.); Section V. – Pilsbry 1907: 47 (part.). Description. Capitulum with all plates strongly ribbed,
lines of growth marked by fine, inconspicuous striation. Tergum with occludent margin straight, carinal margin
convex, but straight concave near apex. Scutum with
occludent margin convex, other margins straight, apex
slightly overlapping tergum. Carina evenly curved with
flat roof bordered by strong ridges; upper latus quadran
gular; rostrum small, triangular, overlapped at sides by
rostral latera; inframedian latus very narrow. Peduncle
shorter than capitulum, covered with strong scales. Cir Teloscalpellum Zevina, 1978b: 1350. – Liu and Ren 1985: 212. –
2007: 257. Diagnosis. Capitulum with 13 or 14 plates; carinal lateral
umbo at basi-carinal angle, angle not extending beyond
carinal margin; inframedian latus triangular or rod-like-
shaped, umbo apical or sub-apical; caudal appendage
multi-segmented. Figure 24. Teloscalpellum ecaudatum (Calman, 1918) (MNHN-IU-2019-4917). а. left lateral view showing the capitulum and
peduncle; b. right lateral view showing the capitulum and peduncle; c. carinal view; d. rostral view showing the capitulum and
peduncle. Scale bar: 5 mm (a–d). Figure 24. Teloscalpellum ecaudatum (Calman, 1918) (MNHN-IU-2019-4917). а. left lateral view showing the capitulum and
peduncle; b. right lateral view showing the capitulum and peduncle; c. carinal view; d. rostral view showing the capitulum and
peduncle. Scale bar: 5 mm (a–d). zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 675 ty: 5°42'S, 132°25'E; depth: 236 m; bottom: mud; it was
found attached to an arm of a Comatula or Pentacrinus. ty: 5°42'S, 132°25'E; depth: 236 m; bottom: mud; it was
found attached to an arm of a Comatula or Pentacrinus. rus I with unequal rami (anterior ramus: 11 segmented,
posterior ramus: 13 segmented). Maxilla globular with
serrulate setae; maxillule not notched; mandible with
three major teeth, lower margin with three to four den
ticles. Measurements of two specimens: height of capit
ulum 12.07–18.59 mm, width 6.17–11.47 mm, thickness
2.68–5.41 mm; length of peduncle 6.30–8.33 mm, width
3.21–5.41 mm. Trianguloscalpellum balanoides (Hoek, 1883)
Figure 25 Scalpellum balanoides Hoek, 1883: 129, pl. V, fig. 15, pl. X, fig. 11, pl. XI,
figs 1–3. – Broch 1922: 242, fig. 10 – Weltner 1922: 63, taf. II, fig. 7,
taf. III, fig. 8. – Nilsson-Cantell 1931: 2. – Hiro 1937b: 42, fig. 33. Scalpellum gonionotum Pilsbry, 1907b: 360. – Pilsbry 1911: 65, pl. IX,
figs 2–4. – Hiro 1937a: 43. Trianguloscalpellum balanoides. – Zevina, 1978b: 1349. – 1981: 294,
fig. 221. – Liu and Ren 1985: 210, fig. 16, pl. XI, figs 15–17. – 2007:
256–257, fig. 111. – Rosell 1991: 20–22, fig. 2c, d. – Huang 1994:
517. – 2001: 318. – Jones et al. 2001: 253. –Young 2001: 464, fig. 7. – Jones 2007: 292. – Chan et al. 2009b: 116, figs 95, 96. – Shalaeva
and Boxshall 2014: 40, fig. 26. Scalpellum balanoides Hoek, 1883: 129, pl. V, fig. 15, pl. X, fig. 11, pl. XI,
figs 1–3. – Broch 1922: 242, fig. 10 – Weltner 1922: 63, taf. II, fig. 7,
taf. III, fig. 8. – Nilsson-Cantell 1931: 2. – Hiro 1937b: 42, fig. 33. Scalpellum gonionotum Pilsbry, 1907b: 360. – Pilsbry 1911: 65, pl. IX,
figs 2–4. – Hiro 1937a: 43. Trianguloscalpellum balanoides. – Zevina, 1978b: 1349. – 1981: 294,
fig. 221. – Liu and Ren 1985: 210, fig. 16, pl. XI, figs 15–17. – 2007:
256–257, fig. 111. – Rosell 1991: 20–22, fig. 2c, d. – Huang 1994:
517. – 2001: 318. – Jones et al. 2001: 253. –Young 2001: 464, fig. 7. – Jones 2007: 292. – Chan et al. 2009b: 116, figs 95, 96. – Shalaeva
and Boxshall 2014: 40, fig. 26. Distribution. Java Sea, Indonesia; Philippines (Chan
2009). In this study, Teloscalpellum ecaudatum was
found at Kei Islands and Tanimbar Island, Indonesia. Type locality. Java Sea; 7°35'S, 114°30'30"E; 132–315 m
depth (Calman 1918b). Type locality. Java Sea; 7°35'S, 114°30'30"E; 132–315 m
depth (Calman 1918b). Genus Trianguloscalpellum Zevina, 1978b Carina very short, simply,
not very strongly, bowed, umbo at apex. Upper latus tri
angular, carinal margin absent. Rostral latus quadrangu
lar, umbo at apex of angle formed by rostral and scutal
margins. Infra-median latus elongate, triangular, umbo
apical. Carinal latus very large, convex, umbo at top of
carinal margin. Cirrus I unequal, posterior ramus more
slender than anterior ramus; cirrus VI with caudal ap
pendages. Maxillule distinctly notched, two stout spines
on upper side; mandible with three teeth excluding inferi
or angle. Measurements of specimen: height of capitulum
8.20 mm, width 4.40 mm, thickness 1.75 mm; length of
peduncle 5.06 mm, width 2.10 mm. Material examined. – Tanimbar Island: 1 specimen,
MNHN-IU-2019-4908, Stn. CP 91, 08°44'S, 131°05'E,
884–891 m depth, 5 November 1991. Diagnosis. Capitulum with 14 plates covered by mem
brane, covered by very long hairs; carina simply bowed,
umbo at apex, roof flat; upper latus triangular; rostrum
very narrow stripe distinctly visible at surface. Description. Capitulum small, long hairs covering
plates. Scutum elongate, convex, more than twice as
long as broad. Tergum large, elongate rhomboid, umbo
at apex. Carina simply bowed, roof flat. Upper latus al
most triangular, carinal-basal margin arched. Rostrum
small, linear-shaped. Rostral latus quadrangular, basal
and scutal margins parallel. Infra-median latus triangu
lar, umbo apical. Carinal latus with irregular shape, umbo
near apex. Peduncle short. Cirrus I with rami unequal, an
terior ramus oval, posterior ramus more slender; cirrus VI
with caudal appendages. Maxillule not notched, two stout
spines on upper side; mandible with three teeth excluding
inferior angle. Measurements of the specimen: height of
capitulum 12.18 mm, width 6.50 mm, thickness 3.74 mm;
length of peduncle 4.10 mm, width 3.43 mm. Description. Capitulum small, long hairs covering
plates. Scutum elongate, convex, more than twice as
long as broad. Tergum large, elongate rhomboid, umbo
at apex. Carina simply bowed, roof flat. Upper latus al
most triangular, carinal-basal margin arched. Rostrum
small, linear-shaped. Rostral latus quadrangular, basal
and scutal margins parallel. Infra-median latus triangu
lar, umbo apical. Carinal latus with irregular shape, umbo
near apex. Peduncle short. Cirrus I with rami unequal, an
terior ramus oval, posterior ramus more slender; cirrus VI
with caudal appendages. Maxillule not notched, two stout
spines on upper side; mandible with three teeth excluding
inferior angle. Measurements of the specimen: height of
capitulum 12.18 mm, width 6.50 mm, thickness 3.74 mm;
length of peduncle 4.10 mm, width 3.43 mm. Distribution. Genus Trianguloscalpellum Zevina, 1978b Arcoscalpellum Hoek, 1907: 85 (part). – Pilsbry 1907a: 47. – Newman
and Ross 1971: 42 (part). Trianguloscalpellum Zevina, 1978b: 1349. – Liu and Ren 1985: 205. Arcoscalpellum Hoek, 1907: 85 (part). – Pilsbry 1907a: 47. – Newman
and Ross 1971: 42 (part). Material examined. – Kei Islands: 1 specimen, MNHN-
IU-2019-4923, Stn. CP 16, 05°17'S, 132°50'E, 330–
350 m depth, 24 October 1991, attached to crinoid. –
Tanimbar
Island:
2
specimens,
MNHN-
IU-2019-4924, Stn. CP 86, 09°26'S, 131°13'E, 223–225
m depth, 4 November 1991. Material examined. – Kei Islands: 1 specimen, MNHN-
IU-2019-4923, Stn. CP 16, 05°17'S, 132°50'E, 330–
350 m depth, 24 October 1991, attached to crinoid. Trianguloscalpellum Zevina, 1978b: 1349. – Liu and Ren 1985: 205. –
Tanimbar
Island:
2
specimens,
MNHN-
IU-2019-4924, Stn. CP 86, 09°26'S, 131°13'E, 223–225
m depth, 4 November 1991. Diagnosis. Capitulum of female or hermaphrodite with
13 or 14 completely calcified plates; tergum and scutum
with umbones apical; middle latera triangular, sometimes
quadrangular, with apical umbo reaching upper latera; ca
rinolatera triangular or subtriangular with apical umbo;
caudal appendages distinctly long. Diagnosis. Capitulum with 13 fully calcified plates, cov
ered by thin membrane; carina and extremely short, sim
ply bowed without distinct roof; umbo of carina at apex;
upper latus triangular; carinal latus almost as high as ca
rina, umbo apical, carinal latus very large, almost similar
size to tergum. Type species. Scalpellum balanoides Hoek, 1883: 129,
pl. V, fig. 15, pl. X, fig. 11, pl. XI, figs 1–3; type locali Figure 25. Trianguloscalpellum balanoides (Hoek, 1883) (MNHN-IU-2019-4923). а. left lateral view showing the capitulum and
peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal
view. Scale bar: 2 mm (a–d). Figure 25. Trianguloscalpellum balanoides (Hoek, 1883) (MNHN-IU-2019-4923). а. left lateral view showing the capitulum and
peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal
view. Scale bar: 2 mm (a–d). zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 676 figs 1F, 9A–H. – Jones 2012: 371, 376. – Shalaeva and Boxshall
2014: 44, fig. 29. Description. Capitulum flat, narrow at base, same breadth
as peduncle. Scutum about twice as long as broad; occlu
dent margin almost straight, umbo at apex. Tergum trian
gular, the same size as scutum. Genus Trianguloscalpellum Zevina, 1978b Pacific, Western Central; South and East
China Sea; South of Sumatra, Banda Sea, Indonesia;
Vietnam; Philippines; Taiwan; South of Japan; attached
to crinoids, hydroids; 220–1,097 m depth (Jones et al. 2001; Chan et al. 2009b; Shalaeva and Boxshall 2014). In
this study, Trianguloscalpellum balanoides was found at
Kei Islands and Tanimbar Island, Indonesia. Type locality. 5°42'S, 132°25'E; depth: 232 m; bottom:
mud; attached to arm of a Comatula or Pentacrinus
(Hoek 1883). Distribution. Borneo, Indonesia; Pacific, Western Cen
tral and Southeast; Australia; Taiwan; Antarctica; at
tached to gastropod shells, rocks; 1,502–1,965 m depth
(Chan et al. 2010; Jones 2012; Shalaeva and Boxshall
2014). In this study, Trianguloscalpellum hirsutum was
found at Tanimbar Island, Indonesia. Trianguloscalpellum hirsutum (Hoek, 1883)
Figure 26 Scalpellum hirsutum Hoek, 1883: 88, pl. IV, fig. 19. – Gruvel 1905: 66,
fig. 74. – Pilsbry 1907a: 25. Scalpellum hirsutum Hoek, 1883: 88, pl. IV, fig. 19. – Gruvel 1905: 66,
fig. 74. – Pilsbry 1907a: 25. Arcoscalpellum hirsutum. – Newman & Ross, 1971: 62–64, fig. 28. Trianguloscalpellum hirsutum. – Zevina, 1981: 309, fig. 233. – Chan
et al. 2009b: 119–121, figs 100–102. – Chan et al. 2010: 13, Arcoscalpellum hirsutum. – Newman & Ross, 1971: 62–64, fig. 28. Type locality. Pacific, Western Central (Moluccas Sea);
0°48'S, 120°58'E; depth: 252 m; bottom temperature:
2.4 °C; bottom: rock (Hoek 1883). Trianguloscalpellum hirsutum. – Zevina, 1981: 309, fig. 233. – Chan
et al. 2009b: 119–121, figs 100–102. – Chan et al. 2010: 13, Figure 26. Trianguloscalpellum hirsutum (Hoek, 1883) (MNHN-IU-2019-4908). а. left lateral view showing the capitulum and
peduncle; b. right lateral view showing the capitulum and peduncle; c. carinal view; d. rostral view showing the capitulum and
peduncle. Scale bar: 5 mm (a–d). Figure 26. Trianguloscalpellum hirsutum (Hoek, 1883) (MNHN-IU-2019-4908). а. left lateral view showing the capitulum and
peduncle; b. right lateral view showing the capitulum and peduncle; c. carinal view; d. rostral view showing the capitulum and
peduncle. Scale bar: 5 mm (a–d). zse.pensoft.net 677 Zoosyst. Evol. 96 (2) 2020, 649–698 Genus Verum Zevina, 1978 Material examined. – Tanimbar Island: 1 specimen,
MNHN-IU-2019-4930, Stn. CP 86, 09°26'S, 131°13'E,
223–225 m depth, 4 November 1991. Arcoscalpellum Pilsbry, 1907: 47 (part). – Newman and Ross 1971:
42 (part). Verum Zevina, 1978b: 1348. Arcoscalpellum Pilsbry, 1907: 47 (part). – Newman and Ross 1971:
42 (part). Verum Zevina, 1978b: 1348. Arcoscalpellum Pilsbry, 1907: 47 (part). – Newman and Ross 1971:
42 (part). Verum Zevina, 1978b: 1348. Diagnosis. Capitulum with 13 plates closely locked to
gether, surface smooth with slightly prominent ridges;
carina simply bowed with flat roof; upper latus trapezi
form; inframedian latus elongate, narrow; carinal latus
with umbo projecting beyond carina. Verum Zevina, 1978b: 1348. Diagnosis. Capitulum of female or hermaphrodite, with
13–14 completely calcified plates; tergum with carinal
margin straight or slightly to moderately recurving; scutum
with or without short apico-lateral arm, umbo apical; carina
with apical or subapical umbo, evenly curved or angular
ly flexed; upper latera sub-triangular or pentagonal, umbo
apical or subapical; middle latera narrow, triangular or
quadrangular, umbo basal or sub-basal; carinolatera short,
umbo basal to sub-basocarinal, usually not or slightly pro
jecting beyond rim of capitulum; caudal appendages short. Description. Capitulum elongate-oval, flat with distinct
ridges and furrow. Scutum slightly convex, umbo apical, oc
cludent margin arched. Tergum triangular, umbo apical, dis
tinctly recurved. Rostral latus irregularly quadrangular; infra
median latus narrow, umbo near base; carinal latus quadran
gular. Peduncle slightly conical, short, scales near capitulum
larger than those near base. Cirrus I unequal, anterior ramus
oval, posterior ramus more slender; cirri II to VI very long,
rami subequal, segments elongate; cirrus VI with caudal ap
pendages. Maxillule not notched, large spine on upper side,
cutting edge almost straight; mandible with three large teeth
excluding inferior angle. Measurements of specimen: height
of capitulum 10.33 mm, width 6.09 mm, thickness 2.08 mm;
length of peduncle 4.62 mm, width 2.24 mm. Type species. Scalpellum zenkevichi Zevina, 1972: 44,
fig. 3; type locality: Pacific Ocean, 24°27'S, 70°42'E. Scalpellum australicum Hoek, 1883: 118, pl. V, fig. 11. – Weltner 1922:
63. – Nilsson-Cantell 1938: 7.
Verum australicum. – Zevina, 1981: 223–224, fig. 160. – Jones et al.
1990: 5. – Jones 1992: 156–160, figs 7–8. – Jones 2012: 371, 376. –
Shalaeva and Boxshall 2014: 52, fig. 36. Scalpellum novae-zelandiae Hoek, 1883: 124, pl. V, figs 7, 8. – 1907:
100, pl. VIII, figs 4, 4a; 1913: 14. – Weltner 1897: 249. – 1922: 71.
– Annandale 1905: 83. – 1906b: 389. – 1908: pl. 5, fig. 7. – 1913:
231. – Broch 1927a: 540, fig. 512.
Scalpellum Novae-Zealandiae. – Gruvel, 1905: 88, fig. 99.
Scalpellum (Scalpellum) novae-zelandiae. – Calman, 1918b: 123.
Arcoscalpellum novaezelandiae. – Foster, 1978: 65, pl. 8D, fig. 39. Verum carinatum (Hoek, 1883)
Figure 28 Scalpellum carinatum Hoek, 1883: 76, pl. III, figs 7, 8. – Pilsbry 1907a:
53, fig. 18. – Gruvel 1920: 20. – Barnard 1925: 3. – Broch 1953: 7. – Weisbord 1977: 243–244, pl. 27, figs 3, 4. Scalpellum imperfectum Pilsbry, 1907a: 75, fig. 30, pl. IV, figs 15–18. –
Annandale 1913: 233. – Barnard 1924: 47. – MacDonald 1929: 537. – Broch 1953: 9. – Stubbings 1961: 11, text-fig. 2. – 1967: 234. Meroscalpellum imperfectum. – Newman & Ross, 1971: 119, text-fig. 62. Verum carinatum. – Zevina, 1981: 225–226, fig. 163. – Shalaeva and
Boxshall 2014: 53, fig. 37. Scalpellum carinatum Hoek, 1883: 76, pl. III, figs 7, 8. – Pilsbry 1907a:
53, fig. 18. – Gruvel 1920: 20. – Barnard 1925: 3. – Broch 1953: 7. – Weisbord 1977: 243–244, pl. 27, figs 3, 4. Scalpellum imperfectum Pilsbry, 1907a: 75, fig. 30, pl. IV, figs 15–18. –
Annandale 1913: 233. – Barnard 1924: 47. – MacDonald 1929: 537. – Broch 1953: 9. – Stubbings 1961: 11, text-fig. 2. – 1967: 234. Meroscalpellum imperfectum. – Newman & Ross, 1971: 119, text-fig. 62. Verum carinatum. – Zevina, 1981: 225–226, fig. 163. – Shalaeva and
Boxshall 2014: 53, fig. 37. Distribution. Atlantic, excluding polar areas; Pacific,
Southeast. Known depth range 600 to 2,400 m (Shalaeva
and Boxshall 2014). In this study, Verum carinatum was
found at Kei Islands, Indonesia. Boxshall 2014: 53, fig. 37. Material examined. – Kei Islands: 1 specimen, MNHN-
IU-2019-4920, Stn. DW 18, 05°18'S, 133°01'E, 205–
212 m depth, 24 October 1991. Type locality. Station 135, near the Island of Tristan da
Cunha; depth: 1,800 m; bottom: rock, shells (Hoek 1883). Verum australicum (Hoek, 1883)
Figure 27 Figure 27 Scalpellum australicum Hoek, 1883: 118, pl. V, fig. 11. – Weltner 1922:
63. – Nilsson-Cantell 1938: 7. Verum australicum. – Zevina, 1981: 223–224, fig. 160. – Jones et al. 1990: 5. – Jones 1992: 156–160, figs 7–8. – Jones 2012: 371, 376. –
Shalaeva and Boxshall 2014: 52, fig. 36. Scalpellum australicum Hoek, 1883: 118, pl. V, fig. 11. – Weltner 1922:
63. – Nilsson-Cantell 1938: 7. Distribution. Indian Ocean, Western; Pacific Western
Central and Southeast; Zanzibar; known depth range 463–
2,561 m (Shalaeva and Boxshall 2014). In this study, Ver
um australicum was found at Tanimbar Island, Indonesia. Verum australicum. – Zevina, 1981: 223–224, fig. 160. – Jones et al. 1990: 5. – Jones 1992: 156–160, figs 7–8. – Jones 2012: 371, 376. –
Shalaeva and Boxshall 2014: 52, fig. 36. Figure 27. Verum australicum (Hoek, 1883) (MNHN-IU-2019-4930). а. left lateral view showing the capitulum and peduncle;
b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale
bar: 3 mm (a–d). Figure 27. Verum australicum (Hoek, 1883) (MNHN-IU-2019-4930). а. left lateral view showing the capitulum and peduncle;
b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale
bar: 3 mm (a–d). zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 678 Type locality. between New Guinea and Australia (12°08'S,
145°10'E): Challenger station 184: depth: 2,561 m: bottom
temperature: 1.8 °C; substrate grey ooze (Hoek 1883). latus flat, irregular pentagonal; rostrum narrow, elon
gated; rostral lateral convex with rostral margin short;
inframedian latus wine-glass-shaped; carinal latus flat,
large. Peduncle short, calcareous scales distinct. Cirrus
I unequal, anterior ramus broader than posterior ramus;
cirri II to VI long, rami equal; cirrus VI with caudal
appendages. Maxillule not notched, two large spines
on upper side, cutting edge almost straight; mandible
with three large teeth excluding inferior angle. Mea
surements of specimen: height of capitulum 9.14 mm,
width 4.53 mm, thickness 1.74 mm; length of peduncle
2.54 mm and width 2.20 mm. Verum novaezelandiae (Hoek, 1883) Diagnosis. Capitulum with 14 plates separated by chitin
ous interspaces, surface smooth; carina bowed, angularly
bent, with flat roof; upper latus irregular pentagonal; in
framedian latus large, wine-glass-shaped. Figure 29 Description. Capitulum elongate, plates covered by
thin, chitinous membrane. Scutum elongated, apex
pointed, occludent margin very convex. Tergum flat,
triangular, apex very recurved, occludent margin very
arched. Carina with umbo at top of flat roof. Upper rcoscalpellum novaezelandiae. – Foster, 1978: 65, pl. 8D, fig. 39. Figure 28. Verum carinatum (Hoek, 1883) (MNHN-IU-2019-4920). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale
bar: 3 mm (a–d). Figure 28. Verum carinatum (Hoek, 1883) (MNHN-IU-2019-4920). а. left lateral view showing the capitulum and peduncle; b. right lateral view showing the capitulum and peduncle; c. rostral view showing the capitulum and peduncle; d. carinal view. Scale
bar: 3 mm (a–d). zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 679 Figure 29. Verum novaezelandiae (Hoek, 1883) (MNHN-IU-2019-4919). а. left lateral view showing the capitulum and peduncle;
b. right lateral view showing the capitulum and peduncle; c. carinal view; d. rostral view showing the capitulum and peduncle. Scale
bar: 3 mm (a–d). Figure 29. Verum novaezelandiae (Hoek, 1883) (MNHN-IU-2019-4919). а. left lateral view showing the capitulum and peduncle;
b. right lateral view showing the capitulum and peduncle; c. carinal view; d. rostral view showing the capitulum and peduncle. Scale
bar: 3 mm (a–d). non Scalpellum Novae-Zelandiae. – Gruvel, 1902: 54, pl. 2, figs 12, 13,
15. – Gruvel 1912: 346 (= V. parazelandiae Young, 1998b). Verum novaezelandiae. – Zevina, 1978b: 1348. – Zevina 1981: 228,
fig. 165. – Chan et al. 2009b: 17, figs 1I, 12A–H, 13A–D. – Chan et
al. 2010: 17, figs 11–13. – Shalaeva and Boxshall 2014: 53, fig. 39. 13.46 mm, width 6.98 mm, thickness 2.99 mm; length of
peduncle 6.02 mm, width 3.23 mm. non Scalpellum Novae-Zelandiae. – Gruvel, 1902: 54, pl. 2, figs 12, 13,
15. – Gruvel 1912: 346 (= V. parazelandiae Young, 1998b). Verum novaezelandiae. – Zevina, 1978b: 1348. – Zevina 1981: 228,
fig. 165. – Chan et al. 2009b: 17, figs 1I, 12A–H, 13A–D. – Chan et
al. 2010: 17, figs 11–13. – Shalaeva and Boxshall 2014: 53, fig. 39. Distribution. Suborder VERRUCOMORPHA, Pilsbry 1916 Description. Capitulum flatted, elongate, plates separat
ed by narrow, chitinous interspaces. Scutum quadrangu
lar, umbo apical, occludent margin arched. Tergum tri
angular with umbo slightly produced apex. Carina with
bowed, flat roof, increasing little in width from upper to
lower. Upper latus irregularly trapeziform; rostral latus
with umbo at top of rostral margin; infra-median latus
irregular, quadrangular, elongate, narrow; carina latus
quadrangular. Cirrus I unequal, anterior ramus broad
er than posterior ramus; cirrus VI with short caudal ap
pendages. Maxillule slightly notched, two large spines
on upper side; mandible with three large teeth excluding
inferior angle; labrum slightly convex, with numerous
teeth. Measurements of specimen: height of capitulum Verrucomorpha Pilsbry, 1916: 14. – Newman et al. 1969: 281. – New
man and Ross 1971: 135. – Newman 1987: 8. – 1996: 501. – Bucker
idge 1994: 89. – Young 1998: 74. Verrucomorpha Pilsbry, 1916: 14. – Newman et al. 1969: 281. – New
man and Ross 1971: 135. – Newman 1987: 8. – 1996: 501. – Bucker
idge 1994: 89. – Young 1998: 74. Diagnosis. Shell asymmetrical with four plates (carina, ros
trum, tergum and scutum), base membranous or calcareous. Verum novaezelandiae (Hoek, 1883) Indo-west Pacific: Eastern Africa, Gulf of
Aden, eastwards to New Zealand; Philippines; Taiwan; at
tached to sunken wood, gorgonians, glassy spicule of hex
actinellid sponges; 822–4850 m depth (Shalaeva and Box
shall 2014; Jones and Hosie 2016). In this study, Verum
novaezelandiae was found at Tanimbar Island, Indonesia. Material examined. – Tanimbar Island: 1 specimen,
MNHN-IU-2019-4919, Stn. CP 87, 08°47'S, 130°49'E,
1017–1024 m depth, 5 November 1991. Diagnosis. Capitulum with 13 plates covered by very
thin, chitinous membrane; carina simply bowed, flat roof
with umbo apical; upper latus quadrangular or rather
pentagonal, lower edge being truncated; inframedian la
tus elongate, quadrangular; carinal latus large, elongate,
umbo near base; peduncle short. Type locality. 37°34'S, 179°22'E; depth: 1,260 m; bottom
temperature: 4.2 °C; bottom: grey ooze (Hoek 1883). Family VERRUCIDAE Darwin, 1854 Verrucidae Darwin, 1854: 495. – Gruvel 1905: 169. – Withers 1935:
323. – Newman et al. 1969: 281. – Newman and Ross 1971: 135. –
Buckeridge 1994: 89. – Young 1998: 74. Verrucidae Darwin, 1854: 495. – Gruvel 1905: 169. – Withers 1935:
323. – Newman et al. 1969: 281. – Newman and Ross 1971: 135. –
Buckeridge 1994: 89. – Young 1998: 74. zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 680 Diagnosis. Scutum and tergum without depressor mus
cles; movable only on one side; other side immovable,
united with rostrum and carina. face with numerous articular ridges. Movable tergum large,
quadrangular; surface with strongly developed, curved ax
ial articular ridge. Carina and rostrum irregular quadrangu
lar, with carina higher, rostrum broader. Fixed tergum with
two parts: (1) triangular portion very narrow at apex, slight
ly broader in its inferior (2) flat and broad part at a rear por
tion of shell. Fixed scutum pointed with distinctly beaked
apex; composed of broader, nearly flat, triangular portion
and narrower inflected portion, only widening towards
its inferior. Base of shell elongatedly oval-shaped. Cirrus
I with rami very unequal (anterior ramus: 12-segmented,
posterior ramus: 28-segmented); cirrus VI with caudal ap
pendages. Maxilla bilobed, fringed with setae, except on
the notch; maxillule widely notched, horizontally elongat
ed, two large spines above notch, numerous dense setae at
notch; mandible with three teeth excluding inferior angle;
labrum slightly concave, conical teeth on cutting margin. Genus Alteriverruca Pilsbry, 1916 Verruca Section D Altiverruca Pilsbry, 1916: 40. Altiverruca Pilsbry, 1916: 40 – Newman et al. 1969: 282. – Newman
and Ross 1971: 135. – Ren 1984b: 172. – Buckeridge 1994: 92. –
Young 1998: 77. Altiverruca navicula (Hoek, 1913)
Figure 30 Distribution. Pacific Ocean (Chan et al. 2010). In this
study, Altiverruca navicula was found at Tanimbar Is
land, Indonesia. Verruca navicula Hoek, 1913: 134, figs 4–6. – Nilsson-Cantell 1927:
778, figs a–f. Altiverruca navicula. – Buckeridge 1994: 100, fig. 5. – Chan et al. 2009b: 280, figs 245–247. – Chan et al. 2010: 29, figs 2E, 21, 22. Verruca navicula Hoek, 1913: 134, figs 4–6. – Nilsson-Cantell 1927:
778, figs a–f. Type locality. (1) 9°3.4'S, 119°56.7'E; depth 959 m; bot
tom: globigerina ooze. (2) 3°37.7'S, 131°26.4'E; depth
924 m; bottom: fine grey mud (Hoek 1913). Altiverruca navicula. – Buckeridge 1994: 100, fig. 5. – Chan et al. 2009b: 280, figs 245–247. – Chan et al. 2010: 29, figs 2E, 21, 22. Material examined. – Tanimbar Island: 3 specimens,
MNHN-IU-2019-4941, Stn. CP 91, 08°44'S, 131°05'E,
884–891 m depth, 5 November 1991, attached to pedun
cle of Alcockianum persona (Annandale, 1916). Diagnosis. Verrucids with erect form; bases of plates not
inflected. Operculum close to vertical, myophore absent. Diagnosis. Verrucids with erect form; bases of plates not
inflected. Operculum close to vertical, myophore absent. Type species. Verruca hoeki Pilsbry, 1907. Newmaniverruca albatrossiana (Pilsbry, 1912)
Figure 31 Operculata tribe Symetrica Gruvel, 1905: 189. Balanomorpha Pilsbry, 1916: 47 (Balanidae sensu Darwin, 1854). Operculata tribe Symetrica Gruvel, 1905: 189. Balanomorpha Pilsbry, 1916: 47 (Balanidae sensu Darwin, 1854). Verruca albatrossiana Pilsbry, 1912: 292. – 1916: 47. – Buckeridge
1994: 91, fig. 1a–f. – Buckeridge 1997: 129. Verruca grex Hoek, 1913: 142, pl. XI, figs 7–13, pl. XIII, figs 11–13. Verruca (Euverruca) albatrossiana. – Broch, 1931: 45. Verruca (Verruca) albatrossiana. – Ren, 1984: 168, fig. 2, pl. 1 (7–11). Newmaniverruca albatrossiana. – Young, 1998: 77. – Chan 2009: 74,
figs 2G, 22A–I. Verruca albatrossiana Pilsbry, 1912: 292. – 1916: 47. – Buckeridge
1994: 91, fig. 1a–f. – Buckeridge 1997: 129. Verruca grex Hoek, 1913: 142, pl. XI, figs 7–13, pl. XIII, figs 11–13. Verruca (Euverruca) albatrossiana. – Broch, 1931: 45. Verruca (Verruca) albatrossiana. – Ren, 1984: 168, fig. 2, pl. 1 (7–11). N
i
lb t
i
Y
1998 77
Ch
2009 74 Verruca albatrossiana Pilsbry, 1912: 292. – 1916: 47. – Buckeridge
1994: 91, fig. 1a–f. – Buckeridge 1997: 129. Diagnosis. Peduncle absent; shells bilaterally symmetri
cal on either side of rostrocarinal axis; shell with parietal
plates of carina, rostrum and one to three pairs of lateral
plates, all separate from one another, variously fused or
totally concrescent; base membranous or calcareous; her
maphroditic or with dwarf males. erruca grex Hoek, 1913: 142, pl. XI, figs 7–13, pl. XIII, figs 11–13 Verruca (Euverruca) albatrossiana. – Broch, 1931: 45. Verruca (Verruca) albatrossiana. – Ren, 1984: 168, fig. 2, pl. 1 (7–11). Newmaniverruca albatrossiana. – Young, 1998: 77. – Chan 2009: 74, Newmaniverruca albatrossiana. – Young, 1998: 77. – Chan 2009: 74,
figs 2G, 22A–I. Material examined. – Kei Islands: 1 specimen, MNHN-
IU-2019-4942, Stn. CC 21, 05°14'S, 133°00'E, 688–694 m
depth, 25 October 1991, attached to glass rope sponge. Family BATHYLASMATIDAE Newman & Ross, 1971 Bathylasmatidae Newman & Ross, 1971: 138 (part.). – Newman and
Ross 1976: 37, 45 (part.). Bathylasmatidae Newman & Ross, 1971: 138 (part.). – Newman and
Ross 1976: 37, 45 (part.). Bathylasmatidae Newman & Ross, 1971: 138 (part.). – Newman and
Ross 1976: 37, 45 (part.). Bathylasmatinae Newman & Ross, 1976: 37, 45. – Buckeridge 1983:
68. – Newman 1996: 502. – Buckeridge 1999: 522. – Jones 2000:
231, tables 1, 2. Diagnosis. Rostrum and fixed scutum with unusual length;
rostrum low, fixed scutum larger than fixed tergum. Bathylasmatinae Newman & Ross, 1976: 37, 45. – Buckeridge 1983:
68. – Newman 1996: 502. – Buckeridge 1999: 522. – Jones 2000:
231, tables 1, 2. Description. Movable plates parallel to base, wall of
parietal vertically ribbed; fixed scutum without internal
pit. Movable scutum with crescentic ridge and longi
tudinal striations; movable tergum with articular ribs
and diagonal rib. Apices of fixed scutum and tergum
contiguous. Carina occupying carino-rostral wall, api
ces marginal. Cirrus I with rami unequal and serrulate
setae; cirrus VI with caudal appendages. Maxilla glob
ular, with fringing setae; maxillule notched, two large
setae on upper side; mandible with three teeth exclud
ing inferior angle. Diagnosis. Shell with four or six plates; wall solid or per
meated by single row of chitin-filled longitudinal canals;
radii absent; one or both rami of cirri I and cirri II some
times antenniform; labrum without notch in crest. Genus Newmaniverruca Young, 1998 Verruca Section B: Verruca, Group of Verruca alba Pilsbry, 1916: 25 (part.). Verruca Section B: Verruca, Group of Verruca calotheca Pilsbry, 1916:
30 (part.). Verruca (Verruca): Foster, 1979: 68 (part.). – Zevina 1987: 1812 (part. – Buckeridge 1994: 90 (part.). Newmaniverruca Young, 1998: 77. Verruca Section B: Verruca, Group of Verruca alba Pilsbry, 1916: 25 (part.). Verruca Section B: Verruca, Group of Verruca calotheca Pilsbry, 1916:
30 (part.). Diagnosis. Shell not depressed; carina and rostrum inter
locking with single rib from each plate; movable plates
large, scutum with four articular ribs, tergum with six articu
lar ribs, growth lines very distinct; caudal appendages long. Newmaniverruca Young, 1998: 77. Description. Shell yellowish. Movable scutum elongately
triangular, apex distinctly beaked, projecting freely; sur Diagnosis. Shell with four plates (carina, rostrum, scu
tum and tergum); operculum parallel to base. Description. Shell yellowish. Movable scutum elongately
triangular, apex distinctly beaked, projecting freely; sur Figure 30. Altiverruca navicula (Hoek, 1913) (MNHN-IU-2019-4941). а. left lateral view; b. right lateral view; c. upper lateral
view; d. lower lateral view. Scale bar: 7 mm (a–d). Figure 30. Altiverruca navicula (Hoek, 1913) (MNHN-IU-2019-4941). а. left lateral view; b. right lateral view; c. upper lateral
view; d. lower lateral view. Scale bar: 7 mm (a–d). zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 681 Type locality. East of Luzon, Philippines (Pilsbry 1912). Type species. Verruca albatrossiana Pilsbry, 1912: 292. – 1916: 47; type locality: East of Luzon, Philippines. Hexelasminae Newman & Ross, 1976: 37, 46. Hexelasminae Newman & Ross, 1976: 37, 46. Diagnosis. Shell with six plates, infiltrated by chitin-filled
tubes; basis calcareous; scuta parallel to basis; tergum
with distinct spur; cirrus II resembling cirrus I more than
cirrus III. Distribution. Indo-west Pacific: NW Australia; South
China Sea; China; Philippines; attached to cidaroid spines
(e.g. Stereocidaris indica philippinensis Mortensen,
1928), 345–620 m depth (Jones and Hosie 2016). In this
study, Newmaniverruca albatrossiana was found at Kei
Islands, Indonesia. Type genus. Hexalasma Hoek, 1913. Figure 32 Figure 32 Hexelasma arafurae Hoek, 1913: 251, pl. 25, figs 12–16. – Utinomi
1965: 11. – Newman and Ross 1971: 155. – 1976: 46. – Foster 1981:
fig. 6F–H. – Jones 2000: 246, fig. 51, tables 28–31. Aaptolasma arafura. – Foster, 1978: 79. Type genus. Hexalasma Hoek, 1913. Figure 31. Newmaniverruca albatrossiana (Pilsbry, 1912) (MNHN-IU-2019-4942). а. left lateral view; b. right lateral view; c. up
per lateral view; d. lower lateral view. Scale bar: 3 mm (a–d). Figure 31. Newmaniverruca albatrossiana (Pilsbry, 1912) (MNHN-IU-2019-4942). а. left lateral view; b. right lateral view; c. up
per lateral view; d. lower lateral view. Scale bar: 3 mm (a–d). zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 682 Genus Hexelasma Hoek, 1913 apex beaked, carinal margin rounded, growth-ridges less
distinct than on scutum. Cirrus I with unequal rami (an
terior ramus: 8-segmented; posterior ramus: 12-segment
ed), dense long setae on surface areas. Cirrus II with equal
rami, dense long setae. Cirri IV–VI with equal rami with
numerous segments; segments almost without exception
furnished with two pairs very long, stiff, needle-like spines
along inner faces. Measurements of specimen: basal length
of shell 14.32 mm, orifice length 8.00 mm, carinal height
12.39 mm, orifice width 6.49 mm, basal width 13.76 mm. Hexelasma Hoek, 1913: 244 (part.). – Utinomi 1965: 13. – Jones 2000:
240, figs 51, 64, tables 28–30. Aaptolasma Newman & Ross, 1971: 158. Diagnosis. Parietes solid; radii absent; plates with a ca
rina, paired carinolatera and latera and a rostrum without
any sign of fusion; caudal appendages absent. Type species. Hexelasma velutinum Hoek, 1913: 246
(part.); type locality: Indonesia, 6°08'S, 121°19'E (Sibo
ga Station 105). Distribution. Arafura Sea, Indonesia; 205–560 m depth
(Jones and Hosie 2016). In this study, Hexelasma arafu
rae was found at Kei Islands, Indonesia. Hexelasma arafurae Hoek, 1913
Figure 32 Type locality. Indonesia; Siboga stat. 262; 5°54'S, 132°49'E;
560 m depth; bottom: solid bluish-grey mud (Hoek 1913). Superfamily BALANOIDEA Leach, 1817 Balanidae Leach, 1817: 68 – Pilsbry 1916: 48 – Newman et al. 1969:
284 – Newman and Ross 1976: 38, 49 – Newman 1993: 408. Balaninae – Darwin, 1854: 175. Balanoidea – Newman, 1996: 502. Material examined. – Kei Islands: 2 specimens, MNHN-
IU-2019-4851, Stn. DW 18, 05°18'S, 133°01'E, 205–
212 m depth, 24 October 1991. Diagnosis. Shell wall composed of four or six plates
(rostrum, carina and one to three pairs of laterals); pa
rietes solid or tubiferous, when tubiferous rarely sec
ondarily filled; radii solid or tubiferous. Basis com
monly calcareous, solid or permeated by tubes, rarely
membranous; when basis calcareous, internal surfaces
of compartments commonly with uniform ribs; when
calcareous commonly forming complex interdigitations
with wall. Opercular plates occlude aperture; articu
lations between the pairs generally shallow or fused. Cirrus I with rami subequal or grossly unequal; cirri II Diagnosis. Shell with very wide orifice; alae broadly-tri
angular with summits oblique; scutum folded longitudi
nally, articular ridge slightly prominent, deep pit for ad
ductor muscle; tergum beaked, with prominent articular
ridge, spur very broadly rounded; inner side of scutum
and tergum orange-coloured. Description. Shell yellowish, conical, with six plates. Or
ifice diamond-shaped; scutum triangular elongated with
protruding growth-ridges; tergum smaller than scutum, Figure 32. Hexelasma arafurae Hoek, 1913 (MNHN-IU-2019-4851). а. view from above; b. right lateral view. Scale bar: 4 mm (a, b). exelasma arafurae Hoek, 1913 (MNHN-IU-2019-4851). а. view from above; b. right lateral view. Scale bar: 4 mm (a, b). Figure 32. Hexelasma arafurae Hoek, 1913 (MNHN-IU-2019-4851). а. view from above; b. right lateral view. zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 683 and III with rami never antenniform; cirrus III resem
bling II more than IV; caudal appendages absent; penis
with basidorsal point. Labrum thin, never bullate; crest
with pronounced medial incision; mandible quadri- or
quinquedentoid; second and following teeth with one or
more subsidiary cusps; fifth tooth often vestigial; inferi
or angle commonly molariform. Diagnosis. Shell firm, strong, with six thick compart
ments; parietes with or without pores; radii solid with
summits parallel to basal margin of parietes and dentic
ulated sutural margins; basis calcareous, elongated along
carino-rostral axis, boat-shaped; orifice smooth, not
dentated; scutum with simple growth ridges; penis with
basidorsal point; attached to gorgonians or antipatharians. Type species. Conopea elongata Say, 1822; type locality:
eastern Florida, USA. Type species. Conopea elongata Say, 1822; type locality:
eastern Florida, USA. Conopea cymbiformis (Darwin, 1854)
Figure 33 Diagnosis. Shell with four or sixplates; parietes solid,
rarely tubiferous; tubes uniformly or irregularly arranged;
radii solid; basis commonly calcareous, rarely tubiferous. Balanus cymbiformis Darwin, 1854: 221, pl. 3, fig. 5a, b. – Broch 1931:
85, fig. 29a, b. – Nilsson-Cantell 1938: 55, pl. 2, fig. 3. – Utinomi
1962: 219, fig. 2. – Dong et al. 1982: 103. Balanus proripiens Hoek, 1913: 228, pl. 24, figs 1–3, pl. 24, figs 1–3. –
Nilsson-Cantell 1921: 331, fig. 70c, d. Pyrgoma jedani Hoek, 1913: 262, pl. 27, figs 3–8. Conopea cymbiformis. – Newman & Ross, 1976: 55. Subfamily ARCHAEOBALANINAE Newman &
Ross, 1976 Pyrgoma jedani Hoek, 1913: 262, pl. 27, figs 3–8. Conopea cymbiformis. – Newman & Ross, 1976: 55. Archaeobalaninae Newman & Ross, 1976: 38, 49. Material examined. – Kei Islands: 9 specimens,
MNHN-IU-2019-4823, Stn. DW 30, 05°39'S, 132°56'E,
111–118 m depth, 26 October 1991; 1 specimen, MNHN-
IU-2019-4824, Stn. DW 22, 05°22'S, 133°01'E, 82 m
depth, 25 October 1991. Diagnosis. Shell with four or six plates; parietes solid or
tubiferous; when tubiferous, tubes uniformly arranged in
single row; interlaminate figures simple; basis calcareous
or membranous, when membranous wall solid. Type genus. Archaeobalanus Menesini, 1971: 19. Type genus. Archaeobalanus Menesini, 1971: 19. Diagnosis. Shell irregularly conical; orifice oval with
swollen border; basis irregularly cup-formed; scutum and
tergum triangular, not calcified together; spur of tergum
feebly developed. Conopea cymbiformis (Darwin, 1854) Archaeobalanidae Newman & Ross, 1976: 38, 49. Genus Conopea Say, 1822 Conopea Say, 1822: 323. Description. Shell yellowish with orange rust-brown in
proximal areas. Carina, carinolatera and latera with pale
orange-brown and rust red-brown longitudinal stripes,
latter may have oblique white spots. Radii with pale or
ange-brown and rust red-brown horizontal striation. Oper Conoplea Gray, 1825: 98, 103. Balaninus Costa, 1839: 181. Balanus Section B Darwin, 1854: 216. Patella-Balanus Hoek, 1913: 160, 162, 221. Figure 33. Conopea cymbiformis (Darwin, 1854) (MNHN-IU-2019-4823). а. left lateral view; b. right lateral view; c. view from
above; d. basal view. Scale bar: 3 mm (a–d). Figure 33. Conopea cymbiformis (Darwin, 1854) (MNHN-IU-2019-4823). а. left lateral view; b. right lateral view; c. view from
above; d. basal view. Scale bar: 3 mm (a–d). zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 684 Conopea navicula. – Newman & Ross, 1976: 55. cular plates with scutum pink-brown, transparent; tergum
transparent white. Shell may appear longer and lower, due
to elongation of carina and rostrum or low and comparative
ly shorter, due to development of rostrum alone or more up
right and comparatively higher, with neither carina nor ros
trum elongated. Cirrus I with unequal rami (anterior ramus:
7-segmented; posterior ramus: 12-segmented). Cirri II–VI
with equal rami, numerous segments. Penis very long, del
icate hairs scattered over surface, a few more disposed near
tip. Labrum deeply notched, two small teeth on each side of
notch. Mandibles with five teeth, inferior angle not distinct
ly separated from fifth; distance between tips of first and
second teeth slightly more than that between those of sec
ond and third teeth; third tooth larger; fourth and fifth small
er than others. Maxillule with straight edge and numerous
large setae. Measurements of specimen: basal length of shell
7.73 mm, orifice length 4.60 mm, carinal height 8.70 mm,
orifice width 3.88 mm, basal width 4.94 mm. Material examined. – Tanimbar Island: 12 specimens,
MNHN-IU-2019-4821, Stn. CP 82, 09°32'S, 131°02'E,
215–219 m depth, 4 November 1991 attached to gorgo
nian; 4 specimens, MNHN-IU-2019-4822, Stn. DW 49,
08°00'S, 132°59'E, 206–210 m depth, 29 October 1991,
attached to gorgonian. Diagnosis. Shell with parietes and basis not porose; cari
no-lateral compartments very narrow, almost same width
from top to bottom; radii with smooth sutural edges; scu
tum externally striated longitudinally. Description. Specimens covered with coenosarc of coral,
except orifice. Balanus navicula Darwin, 1854: 221, pl. 3, fig. 6a–d. – Hoek 1913:
223, pl. 22, fig. 26, pl. 23, figs 1–3. – Stubbings 1936: 48. – Utinomi
1962: 74, fig 1. – Dong et al. 1982: 103.
Acasta spinitergum Foster, 1982: 209, fig. 4d. Genus Conopea Say, 1822 Easily recognisable species due to narrow
carino-lateral plate, which is nearly same width at top as
bottom; scutum externally longitudinally striated; pari
etal plates studded with calcareous points. Parietal plates
pearly white, solid, superficially appearing to possess
longitudinal tubes, growth lines horizontal. Alae moder
ately developed. Basis calcareous. Size small. Rostrum
well developed, concave, lying at angle of ~ 45°. Later
als very well developed. Carino lateral parietes thin, ra
dii and alae well developed. Carina tall, about half width
of rostrum. External surfaces of all parietes with very
small, calcareous studs, regularly spaced, arranged along
horizontal growth lines. Opercular plates sunk down
into orifice. Cirrus I with unequal rami (anterior ramus:
5-segmented; posterior ramus: 7-segmented). Cirrus II
with unequal rami (anterior ramus: 6-segmented; pos
terior ramus: 9-segmented). Cirri III–VI with subequal
rami more slender, longer, with segments more elongate. Penis very long, tapering towards tip, bearing few, very
minute hairs. Maxilulle with straight edge with numer
ous large setae. Mandibles with five teeth and inferior
angle. Measurements of four specimens: basal length of Distribution. Indo-west Pacific: Indian Ocean; Gulf of
Aden, India, east to Fiji and NW to Indonesia, N Aus
tralia, Malay Arch.; China; Philippines; S Japan; Fiji Is;
attached to coenosarc of gorgonians or antipatharians; lit
toral–453 m depth (Jones and Hosie 2016). In this study,
Conopea cymbiformis was found at Kei Islands, Indonesia. Type locality. Near Madras, India; attached to a gorgo
nian (Darwin 1854). Solidobalanus auricoma (Hoek, 1913)
Figure 35 Figure 35 Type locality. Madras, India; attached to gorgonian (Dar
win 1854). Balanus (Solidobalanus) auricoma Hoek, 1913: 198, pl. XVIII, figs 20–
22, pl. XIX, figs 1–7. – Broch 1922: 323, fig. 62. – 1931: 71. – Foster
1978: 100, fig. 60. – Rosell 1981: 303. Solidobalanus (Solidobalanus) auricoma. – Newman & Ross, 1976: 50. Solidobalanus auricoma. – Foster ,1981: 364, fig. 2G. – Rossel 1991: 38. Genus Solidobalanus Hoek, 1913 Solido-Balanus Hoek, 1913: 159, 192. Balanus (Solidobalanus). – Pilsbry, 1916: 220. Solidobalanus. – Newman & Ross, 1976: 23, 50. Solido-Balanus Hoek, 1913: 159, 192. Material examined. – Tanimbar Island: 2 specimens,
MNHN-IU-2019-4842, Stn. CP 45, 07°54'S, 132°47'E,
302–305 m depth, 29 October 1991, shell only; 10 spec
imens, MNHN-IU-2019-4841, Stn. DW 49, 08°00'S,
132°59'E, 206–210 m depth, 29 October 1991, attached
to sea urchin spines; 1 specimen, MNHN-IU-2019-4838,
Stn. CP 82, 09°32'S, 131°02'E, 215–219 m depth, 4 No
vember 1991, attached to antipatharian; 7 specimens,
MNHN-IU-2019-4847, Stn. CP 85, 09°22'S, 131°14'E,
240–245 m depth, 4 November 1991, attached to spines
of sea urchin; 63 specimens, MNHN-IU-2019-4850, Stn. CP 86, 09°26'S, 131°13'E, 223–225 m depth, 4 Novem
ber 1991, attached to spines of sea-urchins. Diagnosis. Shell parietes solid, six plates; radii solid,
well developed, with denticulate sutural edges; basis cal
careous, solid; complemental male, when present, in pit
of rostral plate of hermaphrodite. Type species. Balanus auricoma Hoek, 1913: 198, pl. XVIII, figs 20–22, pl. XIX, figs 1–7; type locality: Ter
nate, Indonesia (Siboga station 136). Conopea navicula (Darwin, 1854)
Figure 34 Balanus navicula Darwin, 1854: 221, pl. 3, fig. 6a–d. – Hoek 1913:
223, pl. 22, fig. 26, pl. 23, figs 1–3. – Stubbings 1936: 48. – Utinomi
1962: 74, fig 1. – Dong et al. 1982: 103. Acasta spinitergum Foster, 1982: 209, fig. 4d. Balanus navicula Darwin, 1854: 221, pl. 3, fig. 6a–d. – Hoek 1913:
223, pl. 22, fig. 26, pl. 23, figs 1–3. – Stubbings 1936: 48. – Utinomi
1962: 74, fig 1. – Dong et al. 1982: 103. Acasta spinitergum Foster, 1982: 209, fig. 4d. Acasta spinitergum Foster, 1982: 209, fig. 4d. Figure 34. Conopea navicula (Darwin, 1854) (MNHN-IU-2019-4822). a. left lateral view; b. orifice view. Scale bar: 1 mm (a, b). onopea navicula (Darwin, 1854) (MNHN-IU-2019-4822). a. left lateral view; b. orifice view. Scale bar: 1 mm (a, b). zse.pensoft.net 685 Zoosyst. Evol. 96 (2) 2020, 649–698 shallow furrow without infolded sides; conspicuous teeth
on anterior margins of fourth cirri absent. shallow furrow without infolded sides; conspicuous teeth
on anterior margins of fourth cirri absent. shell 2.23–4.22 mm, orifice length 1.09–2.02 mm, carinal
height 2.04–3.09 mm, orifice width 0.94–1.59 mm, basal
width 1.79–2.80 mm. shell 2.23–4.22 mm, orifice length 1.09–2.02 mm, carinal
height 2.04–3.09 mm, orifice width 0.94–1.59 mm, basal
width 1.79–2.80 mm. Type species. Balanus auricoma Hoek, 1913: 198, pl. XVIII, figs 20–22, pl. XIX, figs 1–7; type locality: Ter
nate, Indonesia (Siboga station 136). Type species. Balanus auricoma Hoek, 1913: 198, pl. XVIII, figs 20–22, pl. XIX, figs 1–7; type locality: Ter
nate, Indonesia (Siboga station 136). Distribution. Indo-west Pacific, from Gulfs of Aden
and Persia, India, Malaysia, Indonesia, Gulf of Siam, to
southern Japan; 45–220 m depth (Jones and Hosie 2016). In this study, Conopea navicula was found at Tanimbar
Island, Indonesia. Solidobalanus pseudauricoma (Broch, 1931)
Figure 36 Balanus (Solidobalanus) pseudauricoma Broch, 1931. – Utinomi 1949:
97, fig. 4. Diagnosis. Shell flatly-conical, colour reddish basally,
whitish apically; radii narrow, summits slightly oblique;
opercular plates with golden hairs along occludent mar
gins; scutum with articular ridge slightly prominent, ad
ductor ridge absent; tergum narrow, scutal margin dis
tinctly dentated. Solidobalanus (Solidobalanus) pseudauricoma. – Newman & Ross,
1976: 51. – Jones 2007: 294. Material examined. – Kei Islands: 46 specimens,
MNHN-IU-2019-4818, Stn. CP 25, 05°30'S, 132°52'E,
336–346 m depth, 26 October 1991, 30 specimens at
tached to gorgonian, 16 specimens attached to cor
al; 6 specimens, MNHN-IU-2019-4825, Stn. CP 27,
05°33'S, 132°51'E, 304–314 m depth, 27 October 1991;
1 specimen, MNHN-IU-2019-4826, Stn. CP 05, 05°49'S,
132°18'E, 296–299 m depth, 22 October 1991, attached
to crinoid; 13 specimens, MNHN-IU-2019-4827, Stn. CP 16, 05°17'S, 132°50'E, 330–350 m depth, 24 October
1991; 2 specimens, MNHN-IU-2019-4828, Stn. DW 22,
05°22'S, 133°01'E, 82 m depth, 25 October 1991, 1 spec
imen attached to gorgonian stem; 8 specimens, MNHN-
IU-2019-4830, Stn. CP 05, 05°49'S, 132°18'E, 296–299
m depth, 22 October 1991, 5 specimens attached to gor
gonian, 1 specimen attached to crinoid. Material examined. – Kei Islands: 46 specimens,
MNHN-IU-2019-4818, Stn. CP 25, 05°30'S, 132°52'E,
336–346 m depth, 26 October 1991, 30 specimens at
tached to gorgonian, 16 specimens attached to cor
al; 6 specimens, MNHN-IU-2019-4825, Stn. CP 27,
05°33'S, 132°51'E, 304–314 m depth, 27 October 1991;
1 specimen, MNHN-IU-2019-4826, Stn. CP 05, 05°49'S,
132°18'E, 296–299 m depth, 22 October 1991, attached
to crinoid; 13 specimens, MNHN-IU-2019-4827, Stn. CP 16, 05°17'S, 132°50'E, 330–350 m depth, 24 October
1991; 2 specimens, MNHN-IU-2019-4828, Stn. DW 22,
05°22'S, 133°01'E, 82 m depth, 25 October 1991, 1 spec
imen attached to gorgonian stem; 8 specimens, MNHN-
IU-2019-4830, Stn. CP 05, 05°49'S, 132°18'E, 296–299
m depth, 22 October 1991, 5 specimens attached to gor
gonian, 1 specimen attached to crinoid. Description. Shell with plates ribbed longitudinally. Shell colour brownish-pink to dull rose-pink, ribs tend
ing to white, colour often faded with specimens appear
ing uniform white. Parietes of carinolatera very narrow,
with single, conspicuous, longitudinal ridge. Scutum
with occludent margin straight, surface indistinctly
ridged, pit for adductor muscle scarcely visible. Tergum
short, narrow, scutal margin straight, unusually distinctly
dentated, carinal margin short, convex, depressor muscle
crests moderately well developed. Opercular plates with
long, golden setae fringing occludent margins, especial
ly distally. Subgenus Solidobalanus Hoek, 1913 p
– Kei Islands: 1 specimen, MNHN-IU-2019-4849,
Stn. CP 05, 05°49'S, 132°18'E, 296–299 m depth, 22
October 1991, attached to lateral plate of Chirona te
nuis; 1 specimen, MNHN-IU-2019-4844, Stn. CP
12, 05°23'S, 132°37'E, 413–436 m depth, 23 October
1991, attached to antipatharian; 6 specimens, MNHN-
IU-2019-4848, Stn. CP 16, 05°17'S, 132°50'E, 330–
350 m depth, 24 October 1991, attached to dead gorgo
nian, spines of sea-urchins, arm of crinoid; 3 specimens,
MNHN-IU-2019-4840, Stn. CP 35, 06°08'S, 132°45'E,
390–502 m depth, 27 October 1991; 1 specimen, p
– Kei Islands: 1 specimen, MNHN-IU-2019-4849,
Stn. CP 05, 05°49'S, 132°18'E, 296–299 m depth, 22
October 1991, attached to lateral plate of Chirona te
nuis; 1 specimen, MNHN-IU-2019-4844, Stn. CP
12, 05°23'S, 132°37'E, 413–436 m depth, 23 October
1991, attached to antipatharian; 6 specimens, MNHN-
IU-2019-4848, Stn. CP 16, 05°17'S, 132°50'E, 330–
350 m depth, 24 October 1991, attached to dead gorgo
nian, spines of sea-urchins, arm of crinoid; 3 specimens,
MNHN-IU-2019-4840, Stn. CP 35, 06°08'S, 132°45'E,
390–502 m depth, 27 October 1991; 1 specimen, Solido-Balanus Hoek, 1913: 159, 192. Balanus (Solidobalanus). – Pilsbry, 1916: 220. Solidobalanus. – Newman & Ross, 1976: 23, 50. Solido-Balanus Hoek, 1913: 159, 192. Balanus (Solidobalanus). – Pilsbry, 1916: 220. Solidobalanus. – Newman & Ross, 1976: 23, 50. Solidobalanus. – Newman & Ross, 1976: 23, 50. Diagnosis. Parietes and radii rather thick, smooth, with
out pores; basis flat, calcareous; rostrum not very elongat
ed; radii with strongly septate sutural edges; tergal spur
narrow; crests for lateral depressor muscles of scutum
absent; adductor ridge weak or absent; tergum flat or with Figure 35. Solidobalanus auricoma (Hoek, 1913) (MNHN-IU-2019-4850). a. upper lateral view from above; b. left lateral view; c. right lateral view; d. basal view. Scale bar: 2 mm (a–d). Figure 35. Solidobalanus auricoma (Hoek, 1913) (MNHN-IU-2019-4850). a. upper lateral view from above; b. left lateral view; c. right lateral view; d. basal view. Scale bar: 2 mm (a–d). zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 686 MNHN-IU-2019-4843, Stn. CP 36, 06°05'S, 132°44'E,
210–268 m depth, 27 October 1991. Striatobalanus amaryllis (Darwin, 1854)
Figure 37 Figure 37 Figure 37 Distribution. Manado Bay (Indonesia); Japan (Broch
1931–1932). In this study, Solidobalanus pseudauricoma
was found at Kei Islands and Tanimbar Island, Indonesia. Distribution. Manado Bay (Indonesia); Japan (Broch
1931–1932). In this study, Solidobalanus pseudauricoma
was found at Kei Islands and Tanimbar Island, Indonesia. alanus amaryllis var. (a), var. (b) Darwin, 1854: 279, pl. 7, fig. 6a–c , p
,i
g
, p
,i
g
y
217. – Nilsson-Cantell 1925: 329. – Hiro 1936: 624. – 1939c: 243. Type locality. Manado Bay, Indonesia (1°31'N,
124°47'E), 500 m depth; Japan (32°25'N, 128°33'E),
400 m depth (Broch 1931–1932). – Stubbings 1936: 174. – Utinomi 1962: 216. – 1969: 88. Balanus amaryllis dissimilis Lanchester, 1902: 369, pl. XXXIV, figs 3–3C, with var. clarovittata Lanchester, 1902: 370. Balanus amaryllis var. a (= Balanus roseus Lamarck, 1818). – Gruvel
1905: 250, with var. b (niveus). – Gruvel, 1905: 250 (= Balanus
amaryllis var. b Darwin, 1854: 279)). Balanus amaryllis var. a (= Balanus roseus Lamarck, 1818). – Gruvel
1905: 250, with var. b (niveus). – Gruvel, 1905: 250 (= Balanus
amaryllis var. b Darwin, 1854: 279)). Remarks. In the type description, Broch (1931–1932)
commented that the specimens were white, without
stripes. However, several of the specimens collected by
KARUBAR had a pale pink tinge and one specimen
(from station DW22) had pale brownish-pink parietes
with small, narrow ellipsoidal whitish spots, the latter ori
entated longitudinally, thus producing a reticulated effect. Radii whitish with pink tinge along distal borders. Balanus (Chirona) amaryllis. – Nilsson-Cantell 1921: 329, pl. 3, fig. 9. – Rosell 1981: 302. Balanus (Chirona) amaryllis. – Nilsson-Cantell 1921: 329, pl. 3, fig. 9. – Rosell 1981: 302. Balanus amaryllis forma euamaryllis. – Broch, 1922: 321. Balanus amaryllis forma euamaryllis. – Broch, 1922: 321. Balanus amaryllis forma laevis Broch, 1931: 67, fig. 24. Balanus amaryllis forma laevis Broch, 1931: 67, fig. 24. Balanus sp. Dong & Mao, 1956: 290, fig. 8. Chirona (Striatobalanus) amaryllis. – Newman & Ross, 1976: 50 Chirona (Striatobalanus) amaryllis. – Newman & Ross, 1976: 50. Chirona amaryllis. – Foster, 1981: 350. Solidobalanus pseudauricoma (Broch, 1931)
Figure 36 Cirri I–II with rami slightly unequal, covered
with setae; cirri III–VI longer, more slender, dense setae
on inner face. Mandible with four teeth, second to fourth
with accessory cusps, lower angle molariform with three
blunt cusps in series, lower edge with row of stiff setae. Measurements of five specimens: basal length of shell
6.11–8.18 mm, orifice length 4.18–5.83 mm, carinal
height 4.11–6.30 mm, orifice width 2.58–3.19 mm, basal
width 4.51–5.96 mm. –
Tanimbar
Island:
11
specimens,
MNHN-
IU-2019-4839, Stn. CP 86, 09°26'S, 131°13'E, 223–225 m
depth, 4 November 1991, several specimens attached to
spines of sea urchins (associated with Solidobalanus au
ricoma (Hoek, 1913)). –
Tanimbar
Island:
11
specimens,
MNHN-
IU-2019-4839, Stn. CP 86, 09°26'S, 131°13'E, 223–225 m
depth, 4 November 1991, several specimens attached to
spines of sea urchins (associated with Solidobalanus au
ricoma (Hoek, 1913)). Diagnosis. Shell with smooth, glossy white plates, co
loured stripes absent; internal plates with thick, solid, fine
ly ribbed longitudinally; base non-porous, radially ribbed. Distribution. Banda Sea (Moluccas, Indonesia); SW
Australia; New Zealand; New Caledonia; Philippines to
southern Japan; Malaysian water; Gulf of Oman, Persia. 27–502 m depth (Jones and Hosie 2016). In this study,
Solidobalanus auricoma was found at Kei Islands and
Tanimbar Island, Indonesia. Description. Shell plates white, stripes absent. Sever
al specimens with pale pink tinge, one with pale brown
ish-pink parietes with small, narrow ellipsoidal whitish
spots, latter orientated longitudinally producing reticulated
effect. Radii whitish, pink tinge along distal borders. Scutal
growth lines without longitudinal striations; articular ridge
absent; pit for adductor muscle small, round. Tergum with Type locality. Ternate anchorage; 27 m depth; bottom:
mud and stone; numerous specimens on the surface of
pieces of rock (Hoek 1913). Figure 36. Solidobalanus pseudauricoma (Broch, 1931) (MNHN-IU-2019-4818). a. left lateral view; b. orifice view; c. basal view;
d. right lateral view. Scale bar: 4 mm (a–d). Figure 36. Solidobalanus pseudauricoma (Broch, 1931) (MNHN-IU-2019-4818). a. left lateral view; b. orifice view; c. basal view;
d. right lateral view. Scale bar: 4 mm (a–d). zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 687 Chirona (Striatobalanus). – Newman & Ross, 1976: 23, 50 (as subgenus). Striatobalanus. – Jones, 2004: 150. Chirona (Striatobalanus). – Newman & Ross, 1976: 23, 50 (as subgenus). Striatobalanus. – Jones, 2004: 150. shallow, wide furrow running from apex to base. Cirrus
I with unequal (anterior ramus: 7-segmented; posterior
ramus: 15-segmented). Solidobalanus pseudauricoma (Broch, 1931)
Figure 36 Cirrus II with rami subequal (an
terior ramus: 11-segmented; posterior ramus: 12-segment
ed). Cirri I and II with very dense, long setae on surface
areas. Cirri III–VI with rami slightly subequal, rounded. Penis sturdy, not long. Labrum with very shallow notch,
three or four irregularly arranged, blunt teeth on each side. Maxillule with distinct, narrow notch with two large se
tae on upper side. Mandibles with five teeth, second and
third bifid and fifth is rudimentary. Measurements of five
specimens: basal length of shell 6.23–12.08 mm, orifice
length 4.63–9.62 mm, carinal height 3.48–13.22 mm, ori
fice width 3.24–6.33 mm, basal width 5.66–9.56 mm. shallow, wide furrow running from apex to base. Cirrus
I with unequal (anterior ramus: 7-segmented; posterior
ramus: 15-segmented). Cirrus II with rami subequal (an
terior ramus: 11-segmented; posterior ramus: 12-segment
ed). Cirri I and II with very dense, long setae on surface
areas. Cirri III–VI with rami slightly subequal, rounded. Penis sturdy, not long. Labrum with very shallow notch,
three or four irregularly arranged, blunt teeth on each side. Maxillule with distinct, narrow notch with two large se
tae on upper side. Mandibles with five teeth, second and
third bifid and fifth is rudimentary. Measurements of five
specimens: basal length of shell 6.23–12.08 mm, orifice
length 4.63–9.62 mm, carinal height 3.48–13.22 mm, ori
fice width 3.24–6.33 mm, basal width 5.66–9.56 mm. Diagnosis. Parietes and radii without pores; radii usual
ly narrow; scutum striated longitudinally with adductor
ridge; tergum furrow deep; cirrus I with rami very un
equal in length. Type species. Balanus amaryllis var. (a), var. (b) Darwin,
1854: 279, pl. 7, fig. 6a–c. Type species. Balanus amaryllis var. (a), var. (b) Darwin,
1854: 279, pl. 7, fig. 6a–c. Type locality. Unknown. Type locality. Unknown. Genus Striatobalanus Hoek, 1913 – Nilsson-Cantell 1921: 302. Balanus tenuis. – Dong et al.. 1982: 97, fig. A–C. Description. Shell conical; tips of rostrum and cari
na slightly curved inwards. Orifice large, pentagonal,
toothed. Colour yellowish-white, with slightly darker
longitudinal lines on main parts of plates. Radii with
very oblique summits, broadest a little distance from
the orifice, narrower towards basis. Alae broader than
radii, summits rounded. Specimen without scutum, ter
gum and soft parts. Measurements of specimen: basal
length of shell 16.76 mm, orifice length 9.13 mm, cari
nal height 9.89 mm, orifice width 7.24 mm, basal width
15.14 mm. Chirona tenuis. – Zevina et al.. 1992: 74, fig. 50. Striatobalanus tenuis. – Jones. 2004: 152. – Liu and Ren 2007: 363,
fig. 161. – Chan 2009: 74, fig. 2H and fig. 23A–G. Material examined. – Kei Islands: 5 specimens, MNHN-
IU-2019-4820, Stn. CC 10, 05°21'S, 132°30'E, 329–
389 m depth, 23 October 1991; 1 specimen (shell only),
MNHN-IU-2019-4835, Stn. CP 16, 05°17'S, 132°50'E,
330–350 m depth, 24 October 1991. – Tanimbar Island: 1 specimen, MNHN-IU-2019-4832,
Stn. DW 49, 08°00'S, 132°59'E, 206–2010 m depth, 29 Oc
tober 1991; 1 specimen (shell only), MNHN-IU-2019-4833,
Stn. CP 65, 09°14'S, 132°27'E, 174–176 m depth, 1 Novem
ber 1991; 4 specimens (shell only), MNHN-IU-2019-4834,
Stn. CP 79, 09°16'S, 131°22'E, 239–250 m, 3 November
1991; 1 specimen (only shell), MNHN-IU-2019-4813,
Stn. CP 85, 09°22'S, 131°14'E, 240–245 m, 4 November
1991, attached to shell of gastropod; 2 specimens, MNHN-
IU-2019-4819, Stn. CP 86, 09°26'S, 131°13'E, 223–225 m
depth, 4 November 1991, attached to gastropod shells. Distribution. Arafura Sea, Indonesia. Indo-west Pa
cific: South and East Africa, Indian Ocean; Australia
(N); Singapore; Malay Arch.; Cambodia; Gulf of Thai
land; Mouth of Bassac; Vietnam (Bay of Nhatrang;
Lien Chien, Tourane; Codor Is); Hong Kong; S China
Sea; China; Philippines; Taiwan; Japan (S); attached to
rocks, gastropod shells, rusting iron, fouling species;
sublittoral, 5–500 m depth (Jones and Hosie 2016). In
this study, Striatobalanus amaryllis was found at Ta
nimbar Island, Indonesia. Diagnosis. Shell colour whitish to yellowish; sur
face smooth, glossy; orifice pentagonal shaped, deep
ly toothed; radii narrow, summits very oblique, slightly
concave; basis solid; scutum with longitudinal striations;
tergum with short, rather broad spur. Type locality. Unknown. Genus Striatobalanus Hoek, 1913 Balanus amaryllis. – Dong et al., 1982: 95. Balanus amaryllis euamaryllis Dong et al., 1982: 96, fig. A–C. Balanus Section F (part.) Darwin, 1854: 277. Chirona Gray, 1825: 37. – Pilsbry, 1916: 204. Balanus (Striato-Balanus) Hoek, 1913: 159, 179. Balanus Section F (part.) Darwin, 1854: 277. Chirona Gray, 1825: 37. – Pilsbry, 1916: 204. Balanus (Striato-Balanus) Hoek, 1913: 159, 179. Chirona amaryllis: Zevina et al., 1992: 72, fig. 49. Striatobalanus amaryllis. – Jones, 2004: 150. – Chan et al. 2009b: 222,
190–192. Figure 37. Striatobalanus amaryllis (Darwin, 1854) (MNHN-IU-2019-4814). a. orifice view; b. right lateral view. Scale bar: 3 mm (a, b). triatobalanus amaryllis (Darwin, 1854) (MNHN-IU-2019-4814). a. orifice view; b. right lateral view. Scale bar: 3 mm (a, b zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 688 1916: 216. – Barnard 1924: 74. – Nilsson-Cantell 1925: 34, fig. 13,
pl. 1, figs 5, 6. – 1927: 785. – 1938: 46. – Broch 1931: 70. – Hiro
1937b: 439, fig. 24. – Utinomi 1962: 216. – 1968: 174. – 1969: 88,
fig. 6. – Utinomi and Kikuchi 1966: 6. 1916: 216. – Barnard 1924: 74. – Nilsson-Cantell 1925: 34, fig. 13,
pl. 1, figs 5, 6. – 1927: 785. – 1938: 46. – Broch 1931: 70. – Hiro
1937b: 439, fig. 24. – Utinomi 1962: 216. – 1968: 174. – 1969: 88,
fig. 6. – Utinomi and Kikuchi 1966: 6. 1916: 216. – Barnard 1924: 74. – Nilsson-Cantell 1925: 34, fig. 13,
pl. 1, figs 5, 6. – 1927: 785. – 1938: 46. – Broch 1931: 70. – Hiro
1937b: 439, fig. 24. – Utinomi 1962: 216. – 1968: 174. – 1969: 88,
fig. 6. – Utinomi and Kikuchi 1966: 6. Material examined. – Tanimbar Island: 1 specimen
(only shell), MNHN-IU-2019-4814, Stn. CP 65, 09°14'S,
132°27'E, 174–176 m depth, 1 November 1991, attached
to broken sea urchin shell. Balanus albus Hoek, 1913: 185, pl. 16, figs 12, 13, pl. 17, figs 1–6. –
Stubbings 1936: 41, fig. 18. Diagnosis. Shell conical; orifice large, pentagonal,
toothed; shell colour pinkish, lighter transparent stripes
run longitudinally over surface; radii very narrow, reddish,
upper margins very oblique; alae broader, lighter colour. Chirona (Striatobalanus) tenuis. – Newman & Ross, 1976: 50. – Rosell
1989: 33, pl. 10g. – 1991: 38. Balanus (Chirona) tenuis. – Ren & Liu, 1978: 161, fig. 22, pl. 7,
fi
il
ll figs 6–10. Subfamily MEGATREMAOTINAE Holthuis, 1982 striations; tergum slightly beaked, exhibiting traces of lon
gitudinal striations, spur short, slightly narrow, depressor
crests distinctly developed. Cirri I to VI with rami slight
ly equal in length, transparent and orange-coloured. Penis
rather long, sparse, minute hairs on the surface, tapering to
wards tip, distinctly hairy at tip. Maxillule with edge slight
ly straight, all the setae similar sized. Mandibles with five
teeth, fourth and fifth small. Measurements of five speci
mens basal length of shell 18.92–28.30 mm, orifice length
13.78–16.29 mm, carinal height 18.02–20.24 mm, orifice
width 10.15–13.70 mm, basal width 20.51–28.03 mm. striations; tergum slightly beaked, exhibiting traces of lon
gitudinal striations, spur short, slightly narrow, depressor
crests distinctly developed. Cirri I to VI with rami slight
ly equal in length, transparent and orange-coloured. Penis
rather long, sparse, minute hairs on the surface, tapering to
wards tip, distinctly hairy at tip. Maxillule with edge slight
ly straight, all the setae similar sized. Mandibles with five
teeth, fourth and fifth small. Measurements of five speci
mens basal length of shell 18.92–28.30 mm, orifice length
13.78–16.29 mm, carinal height 18.02–20.24 mm, orifice
width 10.15–13.70 mm, basal width 20.51–28.03 mm. Bosciinae Newman & Ross, 1976: 59. Megatrematinae Holthuis, 1982: 319. Bosciinae Newman & Ross, 1976: 59. Bosciinae Newman & Ross, 1976: 59. Megatrematinae Holthuis, 1982: 319. Megatrematinae Holthuis, 1982: 319. Megatrematinae Holthuis, 1982: 319. Diagnosis. Wall of four plates wholly concrescent; oper
cular valves normal; tergum with weakly developed lat
eral depressor muscle crests or crests lacking; when shell
concrescent, sheath with paired sulci. Type genus. Megatrema Sowerby, 1823, by original des
ignation (Holthuis 1982). Type genus. Megatrema Sowerby, 1823, by original des
ignation (Holthuis 1982). Distribution. Arafura Sea, Indonesia; Indo-west Pacif
ic: S Africa; Persian Gulf; Indian Ocean; Australia (N);
Singapore; Malay Arch.; Vietnam; Hong Kong; S Chi
na Sea; E China Sea; Philippines; Taiwan; Japan (S); W
Pacific; attached to crabs, gastropod, bivalve, shells sol
itary coral, bark of coconut, gorgonians, antipatharians,
stones; 7–551 m depth (Jones and Hosie 2016). In this
study, Striatobalanus tenuis was found at Kei Islands and
Tanimbar Island, Indonesia. Family PYRGOMATIDAE Gray, 1825 Balanidae Leach, 1817: 68 (part.). – Darwin 1854: 33 (part.). Balaninae. – Nilsson-Cantell, 1921: 306 (part.). Pyrgomatidae Gray, 1825: 102. – Newman 1996: 503. Pyrgomatinae. – Ross & Newman, 1973: 149. Type locality. Pliocene (Piacenzian Stage); Gournes,
Iraklion nomarchia, Island of Crete, Greece, 35°06'N,
25°47'E; host coral unknown (Ross and Pitombo 2002). Pyrgomatinae. – Ross & Newman, 1973: 149. Tribe PYRGOMINI Ross & Pitombo, 2002 Pyrgominini Ross & Pitombo, 2002: 58. Pyrgominini Ross & Pitombo, 2002: 58. Type genus. Pyrgomina Baluk & Radwanski, 1967. Type genus. Pyrgomina Baluk & Radwanski, 1967. Striatobalanus tenuis (Hoek, 1883)
Figure 38 Figure 38 Description. Shell with six plates, conical; orifice large,
distinctly toothed; radii well-developed; basis thin, ribbed,
solid. Scutum triangular, not elongated, with longitudinal Balanus tenuis Hoek, 1883: 154, pl. 13, figs 29–33. – 1913: 190, pl. 17, figs 14–19, pl. 18, fig. 1. – Gruvel 1905: 247, fig. 275. – Pilsbry Figure 38. Striatobalanus tenuis (Hoek, 1883) (MNHN-IU-2019-4820). a. orifice view; b. left lateral view; c. right lateral view;
d. basal view. Scale bar: 10 mm (a–d). Figure 38. Striatobalanus tenuis (Hoek, 1883) (MNHN-IU-2019-4820). a. orifice view; b. left lateral view; c. right lateral view;
d. basal view. Scale bar: 10 mm (a–d). zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 689 Subfamily MEGATREMAOTINAE Holthuis, 1982 Genus Pyrgomina Baluk & Radwanski, 1967 Type locality. West of Mindoro, Philippines; 6°8'N,
121°19'E; depth: 275 m, at coral-bottom (Hoek 1913). Pyrgomina Baluk & Radwanski, 1967b: 691, pl. 1–2. Type species. Pyrgomina seguenzai Baluk & Radwanski,
1967a: 485. Pyrgomina sp. Diagnosis. Wall of four plates wholly concrescent; pa
rietes solid or tubiferous; when tubiferous, tubes occur
between outer lamina and sheath or between external ribs
of wall; interlaminate figures complex, essentially arbo
rescent; radii solid; basis calcareous, rarely tubiferous,
membranous in Pyrgopsella. Material examined. – Tanimbar Island: 1 specimen,
MNHN-IU-2019-4817, Stn. CP 86, 09°26'S, 131°13'E,
223–225 m depth, 4 November 1991, attached to coral. Figure 39. Pyrgomina sp. (MNHN-UI-2019-4817). a. orifice view; b. left lateral view; c. right lateral view. Scale bar: 2 mm (a–c). Figure 39. Pyrgomina sp. (MNHN-UI-2019-4817). a. orifice view; b. left lateral view; c. right lateral view Pyrgomina sp. (MNHN-UI-2019-4817). a. orifice view; b. left lateral view; c. right lateral view. Scale bar: 2 mm (a–c zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 690 Diagnosis. Shell purple-pinkish, tall, conical; oriface
oval; base permeated by pores, expanded out of the coral;
tergum and scutum subtriangular. Diagnosis. Shell purple-pinkish, tall, conical; oriface
oval; base permeated by pores, expanded out of the coral;
tergum and scutum subtriangular. lower side only; alae not cleft; basis with single tubif
erous; scutum with conspicuous adductor ridge; tergum
with well-developed depressor muscle crests, growth
lines in tergum spur display an obvious change in di
rection; second maxilla with smooth anterior margin of
distal lobe, acuminate setae with enlarged, modified tips. Cirrus III with inner face of endopod with pinnate setae,
rarely bifurcate (complex) setae. Description. Shell stands exserted on the coral, externally
furnished with ribs, lower part rounded with radiating ribs;
oriface oval, small and narrow. Scutum triangular, basal
margin curved; tergum triangular with narrow spur. Cirrus
I with rami unequal (anterior ramus: 10-segmented; pos
terior ramus: 15-segmented); posterior ramus of cirrus IV
without spines. Maxilla globular; maxillule not notched,
eight large setae on straight cutting edge; mandible with
five major teeth. Measurements of specimen: basal length
of shell 7.46 mm, orifice length 2.42 mm, carinal height
10.45 mm, orifice width 1.41 mm, basal width 6.15 mm. Type species. Balanus amphitrite Darwin, 1854: 240
(part.), pl. 5, fig. 2a–d, i–k, m–o; type locality: Natal,
South Africa. Type species. Balanus amphitrite Darwin, 1854: 240
(part.), pl. 5, fig. 2a–d, i–k, m–o; type locality: Natal,
South Africa. Amphibalanus amphitrite (Darwin, 1854)
Figure 40 Distribution. Pyrgomina sp. was found at Tanimbar Is
land, Indonesia. Balanus amphitrite Darwin, 1854: 240 (part.), pl. 5, fig. 2a-d, i-k, m-o. –
Weltner 1897: 264. – Hoek 1913: 167. – Pilsbry 1916: 89. – Zevina
et al. 1992: 89, fig. 61. – Puspasari et al. 2001: 7. Balanus amphitrite Darwin, 1854: 240 (part.), pl. 5, fig. 2a-d, i-k, m-o. –
Weltner 1897: 264. – Hoek 1913: 167. – Pilsbry 1916: 89. – Zevina
et al. 1992: 89, fig. 61. – Puspasari et al. 2001: 7. Balanus amphitrite var. (1) communis Darwin, 1854: 240, pl. 5, fig. 2e, h, l. Balanus amphitrite communis. – Nilsson-Cantell, 1921: 311, fig. 64. Subfamily AMPHIBALANINAE Pitombo, 2004 Balanus amphitrite amphitrite. – Harding, 1962: 274, pl. 1a–g, pl. 2a–k. – Dong et al. 1982: 90, fig. A-E. – Rosell 1981: 302. – Dong et al. 1982: 90, fig. A-E. – Rosell 1981: 302. Balanus amphitrite var. Hawaiiensis. – Stubbings, 1963b: 15. – Dong et al. 1982: 90, fig. A-E. – Rosell 1981: 302. Amphibalaninae Pitombo, 2004: 263. Balanus amphitrite var. Hawaiiensis. – Stubbings, 1963b: 15. Amphibalanus amphitrite. – Pitombo, 2004: 263, 274, figs 2A, B, 7A, Amphibalanus amphitrite. – Pitombo, 2004: 263, 274, figs 2A, B, 7A, Amphibalanus amphitrite. – Pitombo, 2004: 263, 274, figs 2A, B, 7A,
B, 8C. – Chan et al. 2009b: 241. – Chen et al. 2014: 1071. – Pochai
et al. 2017: 27, fig. 9. – Pitriana et al. 2020: 42, fig. 21. Amphibalanus amphitrite. Pitombo, 2004: 263, 274, figs 2A, B, 7A,
B, 8C. – Chan et al. 2009b: 241. – Chen et al. 2014: 1071. – Pochai
et al. 2017: 27, fig. 9. – Pitriana et al. 2020: 42, fig. 21. Diagnosis. Shell with four or six plates; parietal tubes
with one or more rows, commonly transverse septa; radii
with transverse teeth on sutural edge with denticles on
lower side only; alae not cleft; basis with single tubif
erous; scutum with conspicuous adductor ridge; tergum
with well-developed depressor muscle crests, growth
lines in tergum spur display an obvious change in direc
tion; second maxilla with smooth anterior margin of dis
tal lobe, acuminate setae with enlarged, modified tips. Material examined. – Tanimbar Island: 4 specimens,
MNHN-IU-2019-4815, Stn. CP 52, 08°03'S, 131°48'E,
1244–1266 m depth, 30 October 1991. Diagnosis. Primary parietal tubes with transverse septa;
exterior of shell with longitudinal purple striations, hori
zontal striations absent; tergum short with wide spur; cirri
III–VI with erect teeth below posterior angles of distal;
cirrus III without complex setae. Family BALANIDAE Leach, 1817 Balanus amphitrite var. (1) communis Darwin, 1854: 240, pl. 5, fig. 2e, h, l. Balanus amphitrite communis. – Nilsson-Cantell, 1921: 311, fig. 64. Balanidae Leach, 1817: 68. – Gray 1825: 104. – Darwin 1854: 33
(part.). – Pilsbry 1916: 48. – Nilsson-Cantel 1921: 306. – Newman
and Ross 1976: 59. – Foster 1978: 95. – Buckeridge 1983: 103. –
Newman 1996: 503. – Pitombo 2004: 262. – Chan et al. 2009b: 229. Balanus amphitrite forma hawaiiensis Broch, 1922: 314, fig. 56 (part.). Balanus amphitrite hawaiiensis. – Hiro, 1937c: 432, figs 20, 21. Balanus amphitrite cochinensis Nilsson-Cantell, 1938b: 43, fig. 11a–e. Balanus amphitrite var. fluminensis Oliveira, 1941: 21, pl. 4, fig. 4, pl. 5, figs 1, 2, pl. 8, figs 1–5. Diagnosis. Shell with four or six plates; parietes tubif
erous; tubes arranged in single uniform row formed be
tween inner and outer lamina, supplementary tubes may
form basally; interlaminate figures complex, arborescent;
radii solid or tubiferous, more-or-less developed; basis
calcareous, commonly tubiferous; caudal appendages ab
sent; penis commonly with basidorsal point. Balanus amphitrite var. aeratus Oliveira, 1941: 22, pl. 4, fig. 5, pl. 9, i Balanus amphitrite var. aeratus Oliveira, 1941: 22, pl. 4, fig. 5, pl. 9,
figs 1–4. figs 1–4. Balanus amphitrite herzi Rogers, 1949: 28, pl. 1, figs 6, 12–15. Balanus amphitrite franciscanus Rogers, 1949: 29, pl. 1, figs 5, 7, 16–19. Balanus amphitrite var. columnarius Tarasov & Zevina, 1957: 179, 184,
fig. 68 a–e. Balanus amphitrite denticulata Henry, 1959: 192, pl. 1, fig. 5, pl. 3, i Balanus amphitrite denticulata Henry, 1959: 192, pl. 1, fig. 5, pl. 3,
fig. 7, upper row right. Balanus amphitrite amphitrite. – Harding, 1962: 274, pl. 1a–g, pl. 2a–k. zse.pensoft.net Genus Amphibalanus Pitombo, 2004 Amphibalanus Pitombo, 2004: 263. Description. Shell six-plated, conical, round; external
ly smooth, white with groups of well-spaced, dark pur
ple vertical stripes; horizontal striations on shell surface
absent. Interior of paries with single row of tubes; radii
solid, wide; alae with summits moderately oblique; basis Diagnosis. Shell with four or six plates; parietal tubes
with one or more rows, commonly transverse septa; radii
with transverse teeth on sutural edge, with denticles on zse.pensoft.net zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 649–698 691 Figure 40. Amphibalanus amphitrite (Darwin, 1854) (MNHN-IU-2019-4815). a. orifice view; b. right lateral view; c. basal view. Scale bar: 4 mm (a–c). Figure 40. Amphibalanus amphitrite (Darwin, 1854) (MNHN-IU-2019-4815). a. orifice view; b. right lateral view; c. basal view. Scale bar: 4 mm (a–c). Remarks. Known as an important fouling species of
ships and marine installations. The suggestion of an
ti-fouling paint on the bases of the specimens examined
suggests that these specimens were probably knocked off
the ship during trawling operations, explaining the great
depth at which these specimens were collected, as the
normal depth range is 0–9 m. porose, calcareous. Scutum externally striped, occludent
margin toothed, lateral depressor muscle pit small. Ter
gum with spur wider than long, less than its own width
from basi-scutal angle. Cirrus I with unequal rami (ante
rior ramus: 10-segmented; posterior ramus: 16-segment
ed). Cirrus II with slightly equal rami (anterior ramus:
10-segemented; posterior ramus: 12-segmented). Cirrus
III with rami subequal (anterior ramus: 12-segmented;
posterior ramus: 13-segmented. Cirri IV–VI with rami
subequal, longer, more slender. Penis moderately long,
tapering distally, sparse, minute hairs on surface, distinct
ly hairy distally. Labrum deeply notched, numerous teeth
on each side. Maxilla without notch below upper pair of
spines, nine spines between upper and lower pairs, few
short spines on inferior angle; lower pair of spines usually
on slight prominence, which may be sometimes moder
ately strong or strong. Mandible with five teeth, inferi
or angle with second tooth bifid. Measurements of four
specimens: basal length of shell 5.27–12.10 mm, orifice
length 3.81–7.89 mm, carinal height 3.64–5.65 mm, or
ifice width 2.72–5.22 mm, basal width 4.62–12.03 mm. porose, calcareous. Scutum externally striped, occludent
margin toothed, lateral depressor muscle pit small. Ter
gum with spur wider than long, less than its own width
from basi-scutal angle. Cirrus I with unequal rami (ante
rior ramus: 10-segmented; posterior ramus: 16-segment
ed). Genus Amphibalanus Pitombo, 2004 Cirrus II with slightly equal rami (anterior ramus:
10-segemented; posterior ramus: 12-segmented). Cirrus
III with rami subequal (anterior ramus: 12-segmented;
posterior ramus: 13-segmented. Cirri IV–VI with rami
subequal, longer, more slender. Penis moderately long,
tapering distally, sparse, minute hairs on surface, distinct
ly hairy distally. Labrum deeply notched, numerous teeth
on each side. Maxilla without notch below upper pair of
spines, nine spines between upper and lower pairs, few
short spines on inferior angle; lower pair of spines usually
on slight prominence, which may be sometimes moder
ately strong or strong. Mandible with five teeth, inferi
or angle with second tooth bifid. Measurements of four
specimens: basal length of shell 5.27–12.10 mm, orifice
length 3.81–7.89 mm, carinal height 3.64–5.65 mm, or
ifice width 2.72–5.22 mm, basal width 4.62–12.03 mm. Type locality. Natal, on a piece of bamboo (Darwin 1854). Discussion Prior to the Karubar expedition, 24 species of barnacles
had been collected from the Kei Islands and Aru Island by
the Siboga expedition (Hoek 1913). Other pertinent refer
ence works to the barnacles from these islands are Jones
et al. (2001) and Jones and Hosie (2016), who recorded
15 species from the Kei Islands and Aru Island. In addition to the works of Hoek (1913), Jones et al. (2001) and Jones and Hosie (2016), Broch (1931–1932)
reported on 67 species of barnacles collected by the Dan
ish expedition to the Kei Islands (1922) and deposited in
the Zoological Museum of Copenhagen University. In his
report, only four species, Euscalpellum rostratum (Dar
win, 1851), Lepas (Anatifa) anatifera Linnaeus, 1758,
Conchoderma virgatum Spengler, 1789 and Acasta den
tifer (Broch, 1922), were explicitly collected in the Kei Is
lands. The other barnacle species recorded were collected
at other places along the route of this expedition, such as
Lampung Bay, Krakatau, Java Sea, Sunda Strait, Makas
sar Strait, Tual, Banda Neira, Ambon and Saparua Bay. Distribution. Cosmopolitan in tropical and subtropical
waters. Bermuda & SE USA to Brazil; England and West
Europe to South coast of Africa Red, Black and Medi
terranean Seas; Suez Canal; SE Africa; Indian Ocean;
Australia; Singapore; Malaysia; Réam (Cambodia); Gulf
of Siam; Vietnam; Condor Island; Tang Trien (South An
nam); Cauda Nhatrang; Hongay, Tonkin; S China Sea;
Hong Kong; China; Bohai Sea; Taiwan; Philippines;
Japan; South Honsyu, Kyusyu & Ryukyu Island; Vlad
ivostok; Hawaii; central California to SW Mexico; foul
ing species; lower littoral to sublittoral (Jones and Hosie
2016). Recently, also recorded from Ambon Island and
Saparua Island, Indonesia (Pitriana et al. 2020). In this
study, Amphibalanus amphitrite was found at Tanimbar
Island, Indonesia. The lists of Hoek (1913), Broch (1931–1932), Jones
et al. (2001) and Jones and Hosie (2016) record a total of
25 species from the Kei Islands, Aru Island and Tanimbar
Island. The results currently recorded herein reveal that
40 species are now recorded from these Islands. Of the 40 species herein, ten are recorded in previ
ous studies: Megalasma striatum Hoek, 1883 (listed in
Hoek (1913)); Scalpellum stearnsi Pilsbry, 1890 (listed
in Hoek (1913)); Annandaleum japonicum (Hoek, 1883) Type locality. Natal, on a piece of bamboo (Darwin 1854). Type locality. Natal, on a piece of bamboo (Darwin 1854). zse.pensoft.net zse.pensoft.net 692 Pitriana, P. Discussion et al.: Deep-sea barnacles from the Karubar expedition (1991) The results of the studies of barnacles from the Phil
ippines and eastern Indonesian waters reconfirm the In
do-Malayan region as the epicentre of marine biodiversity. (listed in Hoek (1913)); Annandaleum laccadivicum (An
nandale, 1906) (listed in Hoek (1913)); Regioscalpellum
moluccanum (Hoek, 1883) (listed in Hoek (1913)); Al
tiverruca navicula (Hoek, 1913) (listed in Hoek (1913)
and Buckeridge (1994)); Newmaniverruca albatrossiana
(Pilsbry, 1912) (listed in Hoek (1913) and Buckeridge
(1994)); Hexelasma arafurae Hoek, 1913 (listed in Hoek
(1913) and Jones and Hosie (2016)); Solidobalanus au
ricoma (Hoek, 1913) (listed in Hoek (1913) and Jones et
al. (2001)); Striatobalanus tenuis (Hoek, 1883) (listed in
Hoek (1913) and Jones and Hosie (2016)).The remaining
30 species can, therefore, be considered as new records
for the Kei Islands and Tanimbar Island. Acknowledgements The first author would like to thank Prof. Frank Riedel
from the Freie Universität Berlin for his general support
and Bani Wahyudi and Abdul Basit for their support and
comments on picture editing. Thanks to Bernhard Schuri
an for supporting the morphological work at the Museum
für Naturkunde Berlin. We would like to thank Dr. Martin
Husemann, Prof. Benny Kwok Kan Chan and anonymous
reviewer for valuable comments on the manuscript.i The present study and previous works on the barna
cles of the Kei Islands, Aru Island and Tanimbar Island,
especially the works of Hoek (1883, 1907, 1913), Broch
(1922, 1931–1932), Buckeridge (1994, 1997), Jones et al. (2001) and Jones and Hosie (2016), enrich our knowl
edge of the barnacle fauna of these islands. This study
demonstrates once more the value of museum collections
as a resource in biodiversity science. The first author would also like to thank Prof. Keiji
Baba, Dr. Enrique Macpherson, Ferran Palero, PhD, Pau
la Martin-Lefèvre and Sébastien Soubzmaigne for the
great time working in the Crustacean Laboratory of the
Muséum National d’Histoire Naturelle (MNHN), Paris
and to Thomas von Rintelen (MfN Berlin) for providing
funds for the research visit to Paris. The result of this study also strengthens the state
ment of Hoeksema (2007) that the Indo-Malayan re
gion (which extends from East Indonesia to the Philip
pines and the Solomon Islands) is a centre of maximum
marine biodiversity. Darwin (1854) demonstrated that
this area had greater species richness than elsewhere
in the world at the time. He named it the East Indian
Archipelago (including the Philippines, Borneo, New
Guinea, Sumatra, Java, Malacca and the eastern coast
of India) and categorised it as his third province of bar
nacles. In this province, he found 37 barnacle species,
the largest number known at that time, compared with
the other provinces. The second author would like to acknowledge Dr
Alain Crosnier (retired, MNHN) for his generous support
to enable her study of the KARUBAR cirripede collec
tion in Paris. This study was funded by the Ministry of Research,
Technology and Higher Education, the Republic of Indo
nesia within the Program for Research and Innovation in
Science and Technology (RISET-Pro), World Bank Loan
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lands. Pacific Science 3(1): 93–99. https://doi.org/10.5134/174435
Utinomi H (1962) Studies on the cirripedian fauna of Japan. VIII
Thoracic cirripeds from Western Kyusu. Publications of the Seto
Marine Laboratory 10(2): 211–239. [figs. 1–12.] https://doi. org/10.5134/175313 Utinomi H (1949) Further notes on cirripeds from the Ogasawara Is
lands. Pacific Science 3(1): 93–99. https://doi.org/10.5134/174435 Utinomi H (1962) Studies on the cirripedian fauna of Japan. VIII
Thoracic cirripeds from Western Kyusu. Publications of the Seto
Marine Laboratory 10(2): 211–239. [figs. 1–12.] https://doi. org/10.5134/175313 Zevina GB (1972) Benthic Lepadomorpha (Cirripedia) from
the southeast Pacific. Crustaceana 22: 39–63. https://doi. org/10.1163/156854072X00660 Supplementary material 1 Zevina GB (1978a) A new classification of the family Scalpellidae Pils
bry (Cirripedia, Thoracica). Part 1. Subfamilies Lithotryinae, Calan
ticinae, Pollicipinae, Scalpellinae, Brochiinae and Scalpellopsinae. Zoologicheskii Zhurnal 57 (7): 998–1007. [in Russian] org/10.5134/175313 Zevina GB (1974) The Cirripedia Thoracica of the Kerguelen Islands. Crus
taceana 27: 209–215. https://doi.org/10.1163/156854074X00433 Utinomi H (1965) A giant Antarctic barnacle Hexelasma antarcticum
Borradaile (Cirripedia Thoracica). Scientific Reports of the Japanese
Antarctic Research Expedition 1956–1962, Series E, 25: 1–15. [figs. 1–4. [pls. 1–2]] Zevina GB (1975) Cirripedia Thoracica of the American Caribbean. Zevina GB (1975) Cirripedia Thoracica of the American Caribbean. Trudy Instituta Oceanologii Akademii, Nauk USSR 100: 233–258. [in Russian] zse.pensoft.net Pitriana, P. et al.: Deep-sea barnacles from the Karubar expedition (1991) 698 Tables S1–S39 Authors: Pipit Pitriana, Diana S. Jones, Laure Corbari,
Kristina von Rintelen Authors: Pipit Pitriana, Diana S. Jones, Laure Corbari,
Kristina von Rintelen Zevina GB (1978b) A new classification of the Scalpellidiae (Cir
ripedia, Thoracica). Part 2. Subfamilies Arcoscalpellinae and
Meroscalpellinae. Zoologicheskii Zhurnal 57(9): 1343–1352. [in Russian] Data type: Checklist table of species and morphometric
for each species Data type: Checklist table of species and morphometric
for each species Explanation note: Table S1. Checklist of barnacle spe
cies collected during the Karubar expedition (1991)
and deposited in MNHN, Paris. Tables S2–39. Mor
phometric/measurements for each species. Zevina GB (1981) Barnacles of the Suborder Lepadomorpha (Cirripe
dia, Thoracica) of the world oceans. I: Family Scalpellidae. Fauna
SSSR 127: 1–398. [in Russian] Explanation note: Table S1. Checklist of barnacle spe
cies collected during the Karubar expedition (1991)
and deposited in MNHN, Paris. Tables S2–39. Mor
phometric/measurements for each species. Zevina GB (1982) Barnacles of the suborder Lepadomorpha (Cirripe
dia, Thoracica) of the world ocean. Part 2. Guides to the fauna of
the USSR-Zoologicheskii Institut Akademy Nauk SSSR, Leningrad,
223 pp. [162 figs. [in Russian]] Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited. Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited. Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited. Zevina GB (1987) Deep-sea Verrucomorpha (Cirripedia, Thoracica) of
the Pacific. 1. The North Pacific. Zoologicheskii Zhurnal 66(12):
1812–1821. [in Russian] Zevina GB, Zvyagintsev AY, Negashev SE (1992) Usonogie raki po
berezh’ya V’etnama i ikh rol’v obrastanii [Barnacles of the Vietnam
Coast and their role in encrustation]. Vladivostok, Dal’nauka, 142
pp. [figs. 69, tables 8. zse.pensoft.net Tables S1–S39 [in Russian]] g
( )
Link: https://doi.org/10.3897/zse.96.55733.suppl1 Zullo VA, Newman WA (1964) Thoracic Cirripedia from a South East
Pacific Guyot. Pacific Science 18: 355–372. zse.pensoft.net
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https://openalex.org/W2526236365
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https://link.springer.com/content/pdf/10.1007%2Fs12560-016-9262-4.pdf
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English
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Contamination by Norovirus and Adenovirus on Environmental Surfaces and in Hands of Conscripts in Two Finnish Garrisons
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Food and environmental virology
| 2,016
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cc-by
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Satu Oristo1
• Maria Ro¨nnqvist1,2 • Mika Aho3 • Ava Sovija¨rvi3 •
Tuula Hannila-Handelberg3 • Ari Ho¨rman4 • Simo Nikkari3 • Paula M. Kinnunen2,3 •
Leena Maunula1 This study
increased our understanding of the possible transmission of
viruses via contaminated environment and hands. p
g
y p
In addition to the swab samples, 14 fecal samples were
obtained for viral analysis. In total, norovirus was present
in 9.0 % of the surface swabs in 2013, whereas enteric
adenovirus was present in 0.0 % and non-enteric aden-
ovirus in 9.4 %. In the same year, 2.6 % of the hand swabs
contained norovirus, 2.6 % enteric adenovirus, and 40.3 %
non-enteric adenovirus. Norovirus GI.6 was continually
detected on the surfaces of garrison A, and identical virus
was detected in some of the fecal samples. In garrison B,
two slightly different norovirus GII.4 strains were present
on the surfaces. The questionnaires revealed no recent Keywords Adenovirus Environmental contamination
Gastroenteritis Norovirus Questionnaire Surface swab Satu Oristo1
• Maria Ro¨nnqvist1,2 • Mika Aho3 • Ava Sovija¨rvi3 •
Tuula Hannila-Handelberg3 • Ari Ho¨rman4 • Simo Nikkari3 • Paula M. Kinnunen2,3 •
Leena Maunula1 Satu Oristo1
• Maria Ro¨nnqvist1,2 • Mika Aho3 • Ava Sovija¨rvi3 •
Tuula Hannila-Handelberg3 • Ari Ho¨rman4 • Simo Nikkari3 • Paula M. Kinnunen2,3 •
Leena Maunula1 Satu Oristo1
• Maria Ro¨nnqvist1,2 • Mika Aho3 • Ava Sovija¨rvi3 •
Tuula Hannila-Handelberg3 • Ari Ho¨rman4 • Simo Nikkari3 • Paula M. Kinnunen2,3 •
Leena Maunula1 Received: 13 July 2016 / Accepted: 21 September 2016 / Published online: 30 September 2016
The Author(s) 2016. This article is published with open access at Springerlink.com Abstract This study investigated the presence of norovirus
and adenovirus, especially enteric adenovirus, on the
environmental surfaces (n = 481) and military conscripts’
hands (n = 109) in two Finnish garrisons (A and B) in
2013 and 2014. A questionnaire study was conducted to
reveal possible correlations between viral findings on the
conscripts’ hands and their acute gastroenteritis symptoms. In addition to the swab samples, 14 fecal samples were
obtained for viral analysis. In total, norovirus was present
in 9.0 % of the surface swabs in 2013, whereas enteric
adenovirus was present in 0.0 % and non-enteric aden-
ovirus in 9.4 %. In the same year, 2.6 % of the hand swabs
contained norovirus, 2.6 % enteric adenovirus, and 40.3 %
non-enteric adenovirus. Norovirus GI.6 was continually
detected on the surfaces of garrison A, and identical virus
was detected in some of the fecal samples. In garrison B,
two slightly different norovirus GII.4 strains were present
on the surfaces. The questionnaires revealed no recent Abstract This study investigated the presence of norovirus
and adenovirus, especially enteric adenovirus, on the
environmental surfaces (n = 481) and military conscripts’
hands (n = 109) in two Finnish garrisons (A and B) in
2013 and 2014. A questionnaire study was conducted to
reveal possible correlations between viral findings on the
conscripts’ hands and their acute gastroenteritis symptoms. acute gastroenteritis cases in garrison A, but in garrison B,
where the norovirus-positive hand swabs were collected,
30.6 % of the conscripts reported of recent symptoms. In
2014, norovirus was rarely detected, but adenovirus was
again frequently present, both on the surfaces and hands. Taken together, our results suggest that gastroenteritis
outbreaks occurred in 2013, but not in 2014. Due to the low
number of hand swabs positive for enteric viruses, no
conclusions about associations between viral findings and
gastroenteritis symptoms could be drawn. Food Environ Virol (2017) 9:62–71
DOI 10.1007/s12560-016-9262-4 Food Environ Virol (2017) 9:62–71
DOI 10.1007/s12560-016-9262-4 ORIGINAL PAPER & Satu Oristo
satu.oristo@helsinki.fi Introduction Norovirus (NoV) is the leading cause of acute viral gas-
troenteritis in all age groups, as it has been reported to be
responsible for almost 20 % of all acute gastroenteritis
(AGE) cases worldwide (Ahmed et al. 2014). Several NoV
genotypes are recognized among the three genogroups (GI,
GII, GIV) that infect humans. Each genotype possesses a
characteristic set of epidemiological and clinical features
(Matthews et al. 2012; Kirby et al. 2014). Clinical mani-
festations
of
NoV
infection
are
typically
vomiting,
abdominal cramps, and diarrhea, but viral shedding can
also be asymptomatic (Teunis et al. 2015). The infectious
dose of NoV is low (Atmar et al. 2008; Teunis et al. 2008)
and the virus exploits several transmission routes. It
spreads efficiently, especially in semi-closed settings;
during a NoV outbreak in a scout camp setting, it was
estimated that 14 secondary cases occurred per every Electronic supplementary material The online version of this
article (doi:10.1007/s12560-016-9262-4) contains supplementary
material, which is available to authorized users. & Satu Oristo
satu.oristo@helsinki.fi 1
Department of Food Hygiene and Environmental Health,
Faculty of Veterinary Medicine, University of Helsinki,
Agnes Sjo¨bergin katu 2, 00790 Helsinki, Finland 2
Finnish Food Safety Authority Evira, Mustialankatu 3,
00790 Helsinki, Finland 3
Centre for Military Medicine, Tukholmankatu 8 A,
00290 Helsinki, Finland 4
Finnish Defence Command Logistics Division, Fabianinkatu
2, 00130 Helsinki, Finland 123 Food Environ Virol (2017) 9:62–71 63 primary case, when enhanced hygienic measures were not
practiced (Heijne et al. 2009). 2014), but swab samples from frequently touched objects
in the conscripts’ living quarters were also included
(Table 1; Figs. 1, 2). primary case, when enhanced hygienic measures were not
practiced (Heijne et al. 2009). Over 60 adenovirus (AdV) types are recognized to date
(Robinson et al. 2013), and in addition to respiratory dis-
ease, different AdV types are capable of causing menin-
gitis, eye infections, and gastroenteritis (Lynch et al. 2011). Respiratory AdV infections have affected the armed
forces so severely in the past that an efficient vaccine
against the most common types of AdV responsible for
respiratory disease (types 4 and 7) is routinely used in the
US Armed Forces (Radin et al. 2014). AdV types 40 and
41 are known as enteric AdVs (eAdVs), as they are the
most common types associated with gastroenteritis (Lynch
et al. 2011). Although clinical gastroenteritis due to eAdV
usually only occurs in children and immunocompromised
people (Lynch et al. Introduction 2011), they are so common in the
general population that they have been proposed as viral
markers of fecal contamination of water (Rusin˜ol et al. 2014). Surface Swab Sampling Our sampling scheme included the collection and analysis
of only swab samples, but after the surface and hand swab
sampling period was finished in 2013, we obtained 11
anonymous fecal samples from conscripts who had suf-
fered from gastroenteritis in garrison A between March 5,
and May 8, 2013 (Table 1). These samples were collected
by the health care personnel of garrison A. In garrison B,
no fecal samples were collected in 2013 but three were
obtained in 2014. In March–May 2013, we collected 132 surface swabs in
garrison A, and 135 surface swabs in garrison B, during six
visits to each garrison (Table 1). In addition, 214 surface
swabs were collected during 11 visits to garrison B in
January–February 2014. The swabbing was performed as
previously described by Ro¨nnqvist et al. (2013). Briefly, a
25 cm2 surface area (or the whole object in case it was
smaller) was swabbed with a polyester or microfiber swab
moistened in phosphate-buffered saline (PBS). The swabs
were taken from surfaces that are often touched, e.g., door
handles, flushing buttons, vending machines, and electronic
devices. Most of the sampling sites within both garrisons
were in the lavatories (76.4 % in 2013 and 74.3 % in Questionnaires All the conscripts (n = 109) who participated in the hand
swab study filled in a questionnaire, in which they reported
when they had last experienced AGE symptoms (diarrhea
and either abdominal pain, vomiting, or both) and whether
they had been in contact with other conscripts or non-
military persons who had AGE symptoms within the pre-
vious 6 days. Although the participants for the hand swab
study were selected in the sick bay waiting area in 2013,
their reason for visiting there on the sampling date was not
enquired. The hand swabs and questionnaires were col-
lected anonymously. Hand Swab Sampling We collected 28 hand swabs during two of the visits to
garrison A in April 2013, and 49 hand swabs during three
of the visits to garrison B in April–May 2013 (Table 1). The conscripts who participated in the hand swab study
were randomly selected in the sick bay waiting area. Of the
garrison A and B conscripts, 8/28 (28.6 %) and 25/49
(51.0 %), respectively, resided in the barracks from where
the surface swabs were taken. In January–February 2014,
32 hand swabs were collected during two of the visits to
garrison B. In contrast to the hand swab study performed in
2013, the garrison B conscripts who participated in 2014
were all residing in the sampled barracks. Hand swabbing
was performed similarly to the surface swabbing but using
only the microfiber swab. Both palms were swabbed for at
least 1 min. Both NoV and AdV infections are problematic for the
armed forces because these are capable of causing a
remarkable reduction in the operational efficiency of the
affected units. The aim of this study therefore was to
characterize the contamination by NoV and AdV, espe-
cially eAdV, on environmental surfaces and army con-
scripts’ hands in military garrison settings. Hand swabbing
was coupled with a questionnaire to reveal any correlation
between viral findings on conscripts’ hands and their AGE
symptoms, or other signs of a possible AGE outbreak. In
2013, the sampling was performed in March–May, when
NoV outbreaks typically occur (Kroneman et al. 2008). In
2014, the sampling was done earlier, in January–February,
in order to follow the possible transmission of NoV among
the new conscripts during their first training period. Table 1 A summary of the
different samples collected in
garrisons A and B during the
study period Swab and Fecal Sample Preparation A known amount of either murine norovirus (MuNoV)
strain MNV-1 (kindly gifted by Professor Herbert W. Virgin, Washington University, St. Louis, MO, USA) or 3 3 12 64 Food Environ Virol (2017) 9:62–71 Sample category
Year
2013
2014
Garrison A
Garrison B
Garrison B
Surface swabs
No. of swabs per sampled building
Health center
22
22
6
Barracks
110
107
208
Cafe
0
6
0
Total
132
135
214
Sampling period (no. of visits)
Mar.12–May 14 (6)
Apr.10–May 22 (6)
Jan.3–Feb.2 (11)
Hand swabs
No. of hand swabs
28
49
32
Sampling period (no. of visits)
Apr.16–Apr.23 (2)
Apr.17–May 8 (3)
Jan.29–Feb.2 (2)
Fecal samples
No. of fecal samples
11
0
3
Sampling perioda
Mar.5–May 14
–
Jan.30–Feb.4
a The 11 fecal samples collected in 2013 were available for viral analysis only after the surface and hand
swabbing period was finished in May 2013 a The 11 fecal samples collected in 2013 were available for viral analysis only after the surface and hand
swabbing period was finished in May 2013 a The 11 fecal samples collected in 2013 were available for viral analysis only after the surface and hand
swabbing period was finished in May 2013 70
50
30
10
10
30
50
70
Lavatory, door handle (n=44)
Lavatory, flushing button (n=32)
Door handle or light switch (n=17)
Game controller (n=12)
Soft drinks vending machine (n=11)
Lavatory, water tap (n=5)
Lavatory, bowl (n=3)
Remote controller (n=3)
Snacks vending machine (n=2)
Telephone (n=2)
Basement water tap (n=1)
NoV GI %
AdV %
Fig. 1 Distribution of norovirus (NoV) and adenovirus (AdV) findings over different surface swabbing sites in garrison A in 2013. All NoV
findings represented genogroup I (NoV GI) Fig. 1 Distribution of norovirus (NoV) and adenovirus (AdV) findings over different surface swabbing sites in garrison A in 2013. All NoV
findings represented genogroup I (NoV GI) 60
40
20
0
20
40
60
Lavatory, door handle (n=62)
Lavatory, flushing button (n=49)
Lavatory, bowl (n=7)
Soft drinks vending machine (n=6)
Computer keyboard and mouse (n=6)
Door handle or light switch (n=2)
Lavatory, water tap (n=2)
Wardrobe (n=1)
NoV GII %
AdV %
Fig. 2 Distribution of norovirus (NoV) and adenovirus (AdV) findings over different surface swabbing sites in garrison B in 2013. Real-Time Reverse Transcription PCR and PCR
Protocols The swab and fecal samples were screened for NoV GI and
GII by real-time reverse transcription PCR (rRT-PCR),
whereas real-time PCR (rPCR) was used for screening
AdV. All primers and probes used in this study are pre-
sented in Online Resource 1. NoV GII detection was per-
formed as previously described (Ro¨nnqvist et al. 2013), and
the same protocol was used for NoV GI, except 0.9 lM of
each GI-specific primer and 0.3 lM of GI-specific probe
were used. MuNoV and MeV were analyzed by a similar
method to NoV GII but with virus-specific primers and
probes. The QuantiTect Probe PCR kit (QIAGEN) was
used both for the detection of all AdVs and then for the
detection of eAdV in the AdV-positive samples. The 20 ll
AdV (or eAdV) reaction mix consisted of 10 ll of 2 x
QuantiTect Probe PCR Master Mix, 1.0 lM of reverse and
forward primers, 0.2 lM of probe, 0.6 ll of PCR-grade
H2O, and 5 ll of template. Initial activation was performed
at 95 C for 15 min, followed by 45 cycles of 94 C for
15 s, 55 C for 45 s, and 72 C for 45 s. Both rRT-PCR
and rPCR reactions were performed using the Rotor-Gene
3000 thermal cycler (QIAGEN). All viral findings were
immediately
reported
to
the
respective
garrisons’
personnel. Swab and Fecal Sample Preparation All NoV
findings represented genogroup II (NoV GII) 60
40
20
0
20
40
60
Lavatory, door handle (n=62)
Lavatory, flushing button (n=49)
Lavatory, bowl (n=7)
Soft drinks vending machine (n=6)
Computer keyboard and mouse (n=6)
Door handle or light switch (n=2)
Lavatory, water tap (n=2)
Wardrobe (n=1)
NoV GII %
AdV % Fig. 2 Distribution of norovirus (NoV) and adenovirus (AdV) findings over different surface swabbing sites in garrison B in 2013. All NoV
findings represented genogroup II (NoV GII) 12 Food Environ Virol (2017) 9:62–71 65 mengovirus (MeV) strain MC0 (kindly gifted by Professor
Albert Bosch, University of Barcelona, Spain) was added
directly on the surface and hand swabs to act as a process
control. Approximately every 12th swab sample was
spiked with 1.0 9 105 PCR units (PCR-u) of MuNoV in
2013, so that at least one spiked sample was included in
each nucleic acid extraction batch. In 2014, every 6th swab
sample was spiked either with 2.0 9 104 or 2.0 9 105
PCR-u of MeV. The viral particles were eluted from the
swabs by a semi-direct lysis method, and the nucleic acids
were extracted as previously described (Ro¨nnqvist et al. 2013). 10 % fecal suspensions were prepared in sterile 1 x
PBS, and nucleic acids were extracted with the QiaAmp
Mini
Viral
RNA
kit
(QIAGEN,
Hilden,
Germany)
according to the manufacturer’s instructions. and sequenced according to the Sanger sequencing method
in the Institute of Biotechnology, University of Helsinki,
Finland. NoV and AdV Detection on the Environmental
Surfaces of Garrisons A and B in 2013 In total, NoV was present in 9.0 % of the surface swabs
collected in garrisons A and B in 2013, whereas eAdV was
present in 0.0 % and non-eAdV in 9.4 %. p
NoV GI was detected in garrison A in 9.1 % (12/132) of
the surface swabs (Table 2). Most of the NoV-positive
samples were collected from the lavatories (Fig. 1), but the
difference between NoV findings for every garrison A
lavatory (10.7 %; 9/84) and other surface (6.3 %; 3/48)
was not significant. One of the sampled lavatory surfaces
tested positive for NoV GI in two consecutive visits
4 weeks apart. None of the AdV findings on the surfaces of
garrison A were confirmed as eAdV. Non-eAdV findings
on the garrison A surfaces (6.1 %; 8/132) were similarly
distributed between the lavatories and the other environ-
mental surfaces (7.1 %; 6/84 vs. 4.2 %; 2/48) as for NoV,
but none of the swabs were positive for both viruses. Non-
eAdV was once detected twice on the same lavatory sur-
face in two consecutive visits 1 week apart. Data Analyses Raw sequence data were analyzed using BioEdit software
version
7.0.5.3
(http://www.mbio.ncsu.edu/BioEdit/bioe
dit.html) and sequence identities calculated using the
Clustal Omega software version 1.2.1 (http://www.ebi.ac. uk/Tools/msa/clustalo/). The sequences were genotyped
using the RIVM norovirus genotyping tool (Kroneman
et al. 2011) (http://www.rivm.nl/mpf/norovirus/typingtool)
and NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). IBM SPSS Statistics software version 22 (IBM Corp.,
Armonk, NY, USA) and OpenEpi version 3.03a (http://
openepi.com/Menu/OE_Menu.htm) were used for statisti-
cal analyses of the results. P values \ 0.05 were consid-
ered to be statistically significant. Reverse Transcription PCR Protocols NoVs were detected in garrison B on three sampling
visits, but in contrast to garrison A, all strains belonged to
the GII genogroup (8.9 %; 12/135) (Table 3), and all the
NoV-positive swabs were collected from the lavatories
(Fig. 2). One of the sampled lavatory surfaces tested pos-
itive for NoV GII in two consecutive visits 1 week apart. As in garrison A, none of the swabs were positive for eAdV
or both NoV and AdV. Non-eAdV was again a frequent
finding (12.6 %; 17/135), both on the lavatory (10.8 %; The samples that were positive for NoVs by rRT-PCR were
subjected to reverse transcription PCR (RT-PCR) reac-
tions, performed with the QIAGEN One-Step RT-PCR kit
reagents (QIAGEN). Four different primer pairs that tar-
geted the polymerase (ORF1) and/or the capsid (ORF2)
region were used (Online Resource 1). Amplified products
were visualized on 1.5 % SeaKem LE (Lonza, Basel,
Switzerland) agarose gel with ethidium bromide staining, 12 3 3 Food Environ Virol (2017) 9:62–71 66 Table 2 Viral findings on the
surface and hand swabs
collected in garrison A during
the study period
Sampling date
Garrison A
Surface swabs (%)
Hand swabs (%)
Norovirusa
Adenovirusb
Norovirus
Adenovirus
Year 2013
Mar.12
8/30 (26.7)
3/30 (10.0)
–
–
Apr.9
2/20 (10.0)
0/20 (0.0)
–
–
Apr.16
1/21 (4.8)
4/21 (19.0)
0/16 (0.0)
7/16 (43.8)
Apr.23
0/18 (0.0)
1/18 (5.6)
0/12 (0.0)
7/12 (58.3)c
May 7
1/21 (4.8)
0/21 (0.0)
–
–
May 14
0/22 (0.0)
0/22 (0.0)
–
–
Total
12/132 (9.1)
8/132 (6.1)
0/28 (0.0)
14/28 (50.0)
a All detected noroviruses on the surfaces of this garrison belonged to genogroup I; of these norovirus-
positive samples, three were confirmed as genotype GI.6 by sequencing
b None of the adenoviruses detected on the surfaces were confirmed as adenovirus type 40/41
c Two adenovirus strains detected on the hand swabs represented adenovirus type 40/41
Table 3 Viral findings on the
surface and hand swabs in
garrison B during the study
period
Sampling date
Garrison B
Surface swabs (%)
Hand swabs (%)
Norovirusa
Adenovirusb
Norovirusa
Adenovirusb
Year 2013
Apr. these, 2.6 % (2/77) contained NoV, 2.6 % (2/77) eAdV and
40.3 % (31/77) non-eAdV. The hand swabs of garrison A were all negative for NoV
(Table 2). eAdV was, however, detected in 7.1 % (2/28)
and non-eAdV in 42.9 % (12/28) of the hand swab sam-
ples. Two of the hand swabs collected in garrison B
(4.1 %; 2/49) were positive for NoV GII (Table 3). Non-
eAdVs were present in 38.8 % (19/49) of the hand swabs. NoV GII and non-eAdV were detected in the same hand
swab sample on one occasion. Table 3 Viral findings on the
surface and hand swabs in
garrison B during the study
period Table 2 Viral findings on the
surface and hand swabs
collected in garrison A during
the study period Reverse Transcription PCR Protocols 10
6/24 (25.0)
3/24 (12.5)
–
–
Apr.17
5/25 (20.0)
2/25 (8.0)
1/27 (3.7)
8/27 (29.6)
Apr.24
0/21 (0.0)
7/21 (33.3)
0/13 (0.0)
7/13 (53.8)
May 8
0/21 (0.0)
1/21 (4.8)
1/9 (11.1)
4/9 (44.4)
May 15
1/22 (4.5)
0/22 (0.0)
–
–
May 22
0/22 (0.0)
4/22 (18.2)
–
–
Total
12/135 (8.9)
17/135 (12.6)
2/49 (4.1)
19/49 (38.8)
Year 2014
Jan.3
0/21 (0.0)
2/21 (9.5)
–
–
Jan.9
1/21 (4.8)
2/21 (9.5)
–
–
Jan.13 and Jan.16
0/44 (0.0)
1/44 (2.3)
–
–
Jan.21 and Jan.23
0/44 (0.0)
2/44 (4.5)
–
–
Jan.27, Jan.29, and Feb.2
0/64 (0.0)
2/64 (3.1)
0/32 (0.0)
6/32 (18.8)
Feb.5 and Feb.7
0/20 (0.0)
2/20 (10.0)
–
–
Total
1/214 (0.5)
11/214 (5.1)
0/32 (0.0)
6/32 (18.8)
a All detected noroviruses on the surfaces and hands in this garrison belonged to genogroup II; of these
norovirus-positive samples, five were confirmed as genotype GII.4 by sequencing
b None of the detected adenoviruses were confirmed as adenovirus type 40/41 a All detected noroviruses on the surfaces and hands in this garrison belonged to genogroup II; of these
norovirus-positive samples, five were confirmed as genotype GII.4 by sequencing b None of the detected adenoviruses were confirmed as adenovirus type 40/41 13/120) and the other surfaces (26.7 %; 4/15). Three of the
surfaces were non-eAdV-positive in two consecutive visits
(twice the computer keyboard and once a door knob in the
sick bay). these, 2.6 % (2/77) contained NoV, 2.6 % (2/77) eAdV and
40.3 % (31/77) non-eAdV. NoV and AdV Findings in the Hand Swab Samples
in Garrisons A and B in 2013 We collected a total of 77 hand swabs during two of the
visits to garrison A and three of the visits to garrison B. Of 123 Food Environ Virol (2017) 9:62–71 67 NoV and AdV Detection in the Fecal Samples
Collected in Garrison A in 2013 obtained
from
eight
garrison
A
samples
(Table 4). Regardless of the sample type (fecal or surface), all the
GI.Pb-GI.6 sequences from six samples were 100 %
identical. The two NoV GII-positive fecal samples repre-
sented different genotypes: sample F1 was a recombinant
between the pandemic variants GII.P4-New Orleans-2009
and GII.4-Sydney-2012, while sample F4 represented
genotype GII.7. After we had finished analyzing the swab samples in 2013,
we obtained 11 fecal samples for NoV and AdV analysis
(Table 1). Three (33.3 %) of the nine fecal samples col-
lected in the sick bay in the beginning of March 2013 were
found to be positive for NoV GI, and two (22.2 %) were
positive for NoV GII. All these NoV-positive fecal samples
were collected less than a week before the first surface
swabbing visit in March 12, 2013, when the number of
NoV-positive surface samples (26.7 %; 8/30) was highest. The two fecal samples that were collected later, on March
13 and May 14, were NoV negative. All 11 samples were
AdV-negative. Partial NoV capsid sequences (ORF1/2 junction) were
obtained from five garrison B samples (Table 4). The
capsid sequences of the surface samples S4, S6, and S7
were 100.0 % identical with each other, and also with the
short sequence obtained from the hand swab sample H1. This variant was identified as the NoV GII.4-Sydney-2012
by the RIVM norovirus genotyping tool (Kroneman et al. 2011). The capsid region of the other detected NoV GII.4-
Sydney-2012 variant (S5) was 97.2 % identical with the
surface samples S4, S6, and S7 but 100 % identical with
the capsid region of the garrison A fecal sample F1. The
exact variants of the samples F1 and S5 were not identified
by the RIVM norovirus genotyping tool (Kroneman et al. 2011), but according to the NCBI BLAST, they were
100 % identical with the capsid region of a recombinant
strain New Orleans 2009/Sydney 2012 (GenBank acces-
sion no. KF378731) that was detected in Italy in 2013
(Martella et al. 2013). NoV and AdV Findings in January–February 2014
(Garrison B Only) One lavatory surface tested positive for NoV GII (0.5 %;
1/214) in 2014 (Table 3). AdVs, all non-eAdVs, were
detected in 4.4 % (7/159) of the lavatory surfaces and
7.2 % (4/55) of the other surfaces (in total 5.1 %; 11/214). None of the hand swabs were positive for NoVs or eAdVs,
but non-eAdV was detected in 18.8 % (6/32). The three
fecal samples collected in 2014 were negative for NoVs
and AdVs. Detection of the Process Control Viruses In 2013, all 28 conscripts in garrison A reported themselves
as healthy (i.e., no AGE symptoms within 6 days at the
time of the hand swabbing), but 28.6 % (8/28) of them had
been in contact with another conscript who had AGE
symptoms within the previous 6 days (Table 5). In con-
trast, 30.6 % (15/49) of the conscripts in garrison B in 2013
had suffered from AGE symptoms within 6 days before
hand swabbing, and 63.3 % (31/49) of them had been in
contact with another conscript who had suffered from AGE
symptoms recently. Also, the conscripts in garrison B in
2013 had more contacts with non-military persons suffer-
ing from AGE symptoms than the conscripts in garrison A
(10.7 vs. 40.8 %; P = 0.005, Mid-P exact test) in the same
year. Recent AGE symptoms were rarer among the gar-
rison B conscripts in 2014 when compared to their coun-
terparts in 2013 (30.6 vs. 9.4 %; P = 0.025, Mid-P exact
test). The conscripts’ contacts with other people (military
or non-military) suffering from recent AGE symptoms did
not differ significantly between years 2013 and 2014 in
garrison B. The lower limit of an acceptable result for the process
control virus detection by rRT-PCR was decided to be a Ct
value \ 40. In all expect four occasions, the positive
control virus gave a positive result. We were not able to
reanalyze the samples that remained negative for the pro-
cess control viruses because no sample material remained
after the initial nucleic acid extraction. The majority of the
samples (94.1 %; data not shown) that were positive for
NoVs and/or AdVs were, however, not the ones that were
spiked with the process control viruses. Sequence Analysis These sequences were not submitted to the GenBank database
d Th ORF1/2 j
ti
f th
l
S3
d H1
198 b
d 127 b
ti
l Table 4 Genotypes of the sequenced samples a Genotype and variant, if available, according to the ORF1 sequence obtained with primers RegA and MJV12 c Genotype according to the ORF2 sequence obtained with primers GI.6RR/FF. These sequences were not submitted to the GenBank database
d The ORF1/2 junction sequences of the samples S3 and H1 were 198 bp and 127 bp, respectively Wadl et al. 2010; Fankem et al. 2014). In contrast, NoV
contamination on the surfaces was rare in January–Febru-
ary 2014. These results seem to reflect the overall NoV
situation in Finland during our study periods, because in
March–May 2013, the health authorities of Finland repor-
ted twice as many laboratory-confirmed NoV cases as they
did in January–February 2014 (THL 2015). Although the
detection of viral genome does not necessarily indicate the
presence of infectious virus, NoV is known to be relatively
stable on environmental surfaces (D´ Souza et al. 2006), so
transmission of viruses via fomites may have occurred. Also, Boxman et al. (2011) showed that even when there
was no evidence of an ongoing NoV outbreak, surface
contamination by NoV correlated with the food producing
facility’s NoV outbreak history. on his hands also had AGE symptoms within 6 days before
the hand swabbing, whereas the other conscript who gave a
NoV-positive hand swab, although not having AGE
symptoms, had to be given intravenous fluids at the time of
swabbing to treat dehydration. The conscripts that had
eAdV on their hands did not report of recent AGE symp-
toms. Non-eAdV findings were not correlated with AGE
symptoms, as expected. ype and variant, if available, according to the ORF1 sequence obtained with primers RegA and MJV12 Sequence Analysis Of the total number of samples that were NoV-positive by
rRT-PCR in 2013 and 2014 (n = 32; 25 surface swabs, two
hand swabs, and five fecal samples), 17 surface and one
hand swab sample collected in 2013, and one surface swab
sample collected in 2014 did not show a right-sized product
in any of the conventional RT-PCR-tests that targeted
different regions of the genome, so these samples were not
subjected to sequencing. Due to the low number of NoV- or eAdV-positive hand
swabs, reliable statistical analysis between these findings,
and the occurrence of AGE symptoms could not be per-
formed. However, one of the conscripts that had NoV GII Partial NoV sequences from either the polymerase
(ORF1) and/or capsid regions (ORF1/2 junction) were 12 123 3 Food Environ Virol (2017) 9:62–71 68 Table 4 Genotypes of the sequenced samples
Garrison
Sampling date (Year
2013)
Sample
code
Sample
type
Genotype
Genbank accession no. ORF1a
ORF1/2b
ORF2c
ORF1
ORF1/2
A
Mar.5
F1
Fecal
GII.P4-New
Orleans-2009
GII.4-Sydney-
2012
KT943510
Identical to
KT943512
Mar.6
F2
Fecal
GI.Pb
GI.6
GI.6
Identical to
KT943508
KT943509
Mar.6
F3
Fecal
GI.Pb
GI.6
NA
Identical to
KT943508
Identical to
KT943509
Mar.7
F4
Fecal
NA
GII.7
NA
NA
KT943513
Mar.8
F5
Fecal
GI.Pb
GI.6
NA
Identical to
KT943508
Identical to
KT943509
Mar.12
S1
Surface
swab
GI.Pb
GI.6
GI.6
KT943508
Identical to
KT943509
Mar.12
S2
Surface
swab
NA
NA
GI.6
NA
NA
Apr.9
S3d
Surface
swab
NA
GI.6
GI.6
NA
Identical to
KT943509
B
Apr.10
S4
Surface
swab
NA
GII.4-Sydney-
2012
NA
NA
KT943511
Apr.10
S5
Surface
swab
NA
GII.4-Sydney-
2012
NA
NA
KT943512
Apr.17
S6
Surface
swab
NA
GII.4-Sydney-
2012
NA
NA
Identical to
KT943511
Apr.17
S7
Surface
swab
NA
GII.4-Sydney-
2012
NA
NA
identical to
KT943511
Apr.17
H1d
Hand
swab
NA
GII.4-Sydney-
2012
NA
NA
Identical to
KT943512
a Genotype and variant, if available, according to the ORF1 sequence obtained with primers RegA and MJV12
b Genotype and variant, if available, according to the ORF1/2 junction sequence obtained either with primers JJVMF/G1SKR (samples F2, F3,
F5, S1, S3) or QNIF2D/G2SKR (samples F1, F4, S4 – S7, H1)
c Genotype according to the ORF2 sequence obtained with primers GI.6RR/FF. c Genotype according to the ORF2 sequence obtained with primers GI.6RR/FF. These sequences were not submitted to the GenBank database
d The ORF1/2 junction sequences of the samples S3 and H1 were 198 bp and 127 bp, respectively b Genotype and variant, if available, according to the ORF1/2 junction sequence obtained either with primers JJ
F5, S1, S3) or QNIF2D/G2SKR (samples F1, F4, S4 – S7, H1) Discussion P value 2 is calculated between the questionnaire results collected in garrison B in 2013 and 2014
* P values \ 0 05 were considered statistically significant a P values were calculated with mid-P exact test. P value 1 is calculated between the questionnaire results collected in garrisons A and B in 2013. P value 2 is calculated between the questionnaire results collected in garrison B in 2013 and 2014
* P values \ 0.05 were considered statistically significant Although NoV was detected on the surfaces of both
garrisons for several weeks in 2013, NoV contamination on
the same surface during two consecutive visits happened
only twice. This implies that the surfaces had been cleaned
and these surfaces were then recontaminated, either by new
cases or by prolonged shedding of NoV by the recovered or
asymptomatic cases. The spread of NoV via lavatory sur-
faces is a known risk. However, the frequent presence of
NoV and AdV on several other environmental surfaces on
the same premises suggest inadequate hygiene practices. Virus transmission via hands or fomites was therefore also
possible in other facilities, recreational or otherwise. It has
been reported that viral contamination can spread via
contaminated cleaning equipment (Fankem et al. 2014), but
it seems that the cleaning procedures in the two garrisons
we studied were adequate for inactivating and removing
NoVs from surfaces. in early March. Their suspicions were later supported by
the detection of NoV in five of the fecal samples. Also, it
was the same genotype (GI.6-GI.Pb) that was detected on
the surfaces—including a surface in the sick bay—and in
three of the fecal samples. Somewhat surprisingly, the
questionnaires collected in this garrison did not indicate
that gastroenteritis cases had occurred recently. However,
these questionnaires and the hand swabs were collected
several weeks later than most of the positive surface
samples. Moreover, most of the conscripts who participated
in the hand swab and questionnaire study were residing in
living quarters which were not swab sampled. In contrast, the questionnaires collected in garrison B
clearly indicated that gastroenteritis cases had occurred
during the study period: almost one-third (30.6 %) of the
conscripts had AGE symptoms within 6 days before the
hand swabbing, and the majority (63.3 %) of them had
been in contact with other conscripts who were suffering
from AGE. Discussion This study revealed that NoV was present on the envi-
ronmental surfaces of two Finnish garrisons for several
weeks in spring 2013. During the first visit to each gar-
rison, one quarter of the surface swabs were NoV-positive,
which is in line with other studies that have been conducted
during, or shortly after, an identified NoV outbreak
(Cheesbrough et al. 2000; Wu et al. 2005; Jones et al. 2007; After our swab sampling period was finished in May
2013, we were informed that the personnel of garrison A
had suspected a gastroenteritis outbreak, and collected 11
fecal samples from conscripts suffering from gastroenteritis 123 Food Environ Virol (2017) 9:62–71 69 Table 5 Conscripts’ reports of their recent acute gastroenteritis (AGE) symptoms (diarrhea and either abdominal pain, vomiting, or both) and
contacts with other conscripts or non-military persons suffering from AGE. Gar = garrison
Category
Year
P valuesa
2013
2014
Gar A
95 % CI
Gar B
95 % CI
Gar B
95 % CI
1
2
Conscripts who had AGE symptoms within
6 days before hand swabbing
(no./total; %)
0/28 (0.0)
0.0–14.3
15/49 (30.6)
19.4–44.6
3/32 (9.4)
2.5–25.0
\0.001*
0.025*
Conscripts who had been in contact with
another conscript who had AGE
symptoms within 6 days before hand
swabbing (no./total; %)
8/28 (28.6)
15.1–47.2
31/49 (63.3)
49.2–75.4
15/32 (46.9)
30.9–63.6
0.004*
0.156
Conscripts who had been in contact with
non-military persons who had AGE
symptoms within 6 days before hand
swabbing (no./total; %)
3/28 (10.7)
2.9–28.0
20/49 (40.8)
28.2–54.8
9/32 (28.1)
15.4–45.5
0.005*
0.257
a P values were calculated with mid-P exact test. P value 1 is calculated between the questionnaire results collected in garrisons A and B in 2013. P value 2 is calculated between the questionnaire results collected in garrison B in 2013 and 2014
* P values \ 0.05 were considered statistically significant Table 5 Conscripts’ reports of their recent acute gastroenteritis (AGE) symptoms (diarrhea and either abdominal pain, vomiting, or both) and
contacts with other conscripts or non-military persons suffering from AGE. Gar = garrison Table 5 Conscripts’ reports of their recent acute gastroenteritis (AGE) symptoms (diarrhea and either abdominal pain, vomiting, or both) and
contacts with other conscripts or non-military persons suffering from AGE. Gar = garrison a P values were calculated with mid-P exact test. P value 1 is calculated between the questionnaire results collected in garrisons A and B in 2013. Discussion Unfortunately, we were not informed if the
health care personnel of garrison B had suspected a gas-
troenteritis outbreak in spring 2013, and no fecal samples
were obtained. NoV was, however, detected in two of the
hand swabs collected in this garrison. According to Box-
man et al. (2009) and Liu et al. (2013), infected persons
often have detectable NoV on their hands, both in labora-
tory and outbreak settings. It has also been demonstrated
that NoV remains detectable on finger pads only for a
couple of hours (Liu et al. 2009), which implies that NoV
contamination on the hands of these conscripts must have
happened soon before the hand swabbing. The presence of
the same NoV GII.4 variant both on the surfaces and in a
hand swab further supported that NoV was at least one of
the causative agents of these gastroenteritis cases. In our study, the presence of eAdV did not coincide with
that of NoV. Non-eAdVs were, however, frequently pre-
sent both on the surfaces and hands. The non-eAdVs on the
surfaces were distributed between the lavatories and the
other places similar to that found for NoVs; however,
because some non-eAdVs are also excreted in feces
(Russell et al. 2006; Lynch et al. 2011; Rusin˜ol et al. 2014;
Verani et al. 2014), it is not possible to tell whether the
source of non-eAdV was contamination from feces or from
other bodily excretions. Other studies have also reported of
detecting non-eAdVs frequently on lavatory surfaces and
air (Russell et al. 2006; Verani et al. 2014). The prevalence
of non-eAdV on the hands of conscripts was somewhat
lower than that reported by Russell et al. (2006); in their
study, 69 % of conscripts with febrile respiratory AdV
illness had AdV 4 DNA on their hands. In our study, the 12 Food Environ Virol (2017) 9:62–71 70 hands illustrate route of transmission: A case study. Journal of
Food Protection, 72(8), 1753–1755. conscripts were not questioned about recent symptoms of
respiratory-or other illnesses, so based on our results we
cannot exclude the possibility that an outbreak of non-
eAdV was ongoing. Boxman, I. L., Verhoef, L., Dijkman, R., Ha¨gele, G., Te Loeke, N. A.,
& Koopmans, M. (2011). Year-round prevalence of norovirus in
the environment of catering companies without a recently
reported outbreak of gastroenteritis. Applied and Environmental
Microbiology, 77(9), 2968–2974. Discussion We conclude by stating that NoV cases occurred in
both garrisons during the study period in 2013, and the
detection of NoV on the surfaces during the same period
was frequent. This was in contrast to the 2014 results,
when both AGE cases and NoV findings on the surfaces
were rare. We were not able to draw any conclusions on
whether there was a correlation between the viral findings
on hands and AGE symptoms because of the low number
of NoV- or eAdV-positive hand swabs. Some swab
samples remained negative for the process control viruses,
which indicates that viruses are lost during sample pro-
cessing. Therefore, it is possible that some of our swabs
were false-negative for NoV and AdV. We find that
routine surface swabbing, however, provides valuable
information on the presence of both of these viruses, and
we believe that in our study, the rapidly disseminated
information of the virus-positive surfaces to the garrisons’
personnel had a role in preventing larger scale outbreaks
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Brown, D. W. (2000). Widespread environmental contamination
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Creative Commons Attribution 4.0 International License (http://crea
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Microstructure-Property Relationship in Advanced Ni-Based Superalloys
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1. Introduction Ni based superalloys have been developed more or less empirically over the past 60 years
from a simple Ni-Cr matrix to the present multi element and phase systems[1], having a
fully austenitic face centred cubic (fcc) structure which maintains a superior tensile, fatigue
and creep properties at high temperature to a body centred cubic (bcc) alloy[1]. One of the
major applications of Ni superalloys is gas turbine engines. They comprise over 50% of the
weight of advanced aircraft engines and include wrought and cast turbine blades and
powder metallurgy (P/M) route turbine discs [1]. One of the most important goals of engine design is increasing turbine entry temperature
(TET): the temperature of the hot gases entering the turbine arrangement [2]. This implies
that the resistance against the environmental attack, i.e. high temperature, under a severe
mechanical force is the priority challenge and indeed Ni based superalloys are used in the
hottest as well as the highest tensile pressure of the gas turbine engine component as shown
in the schematic diagram in Fig. 1. Nowadays, for the advanced cast single crystal
superalloys in the turbine blades, the alloy capability exceeds 1,000ºC [2]. In this chapter, the
polycrystalline Ni superalloys, which have slightly less temperature capability up to 800°C,
applied in the turbine discs and the adjoined shafts, will be introduced focusing on their
microstructures correlating with the mechanical properties. http://dx.doi.org/10.5772/52011 http://dx.doi.org/10.5772/52011 © 2012 Kitaguchi, licensee InTech. This is an open access chapter distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Microstructure-Property Relationship in
Advanced Ni-Based Superalloys Hiroto Kitaguchi Additional information is available at the end of the chapter 2.1. Hardening precipitates On the other hand, the precipitates arise at lower temperature such as during cooling after
heat treatment stay small (Fig. 4 (left hand side of the image)). γ´ has the perfect coherency
with the γ matrix, hence their morphologies are mostly sphere, whereas γ´´ has a tall crystal
unit tetragonal structure where a axis has the identical lattice parameter with the γ matrix but
c axis has nearly double the length of the γ, hence γ´´ always precipitate with the perfect
coherency on the basal plane with γ and grow along the longitudinal direction (Fig. 5). 2.1. Hardening precipitates From the point of view of microstructure, Ni superalloys are complex [4]. The fcc matrix,
known as γ, mainly consists of nickel, cobalt, iron, chromium and molybdenum. The strength
of superalloys are conferred by the hardening precipitates known as γ´ (Ni3Al based L12 Metallurgy – Advances in Materials and Processes
20 Figure 1. (a) Schematic diagram of a turbine engine Ref. [3]. (b) Schematic diagram of the temperature
and pressure gradients throughout the engine component correlating with the diagram (a) Ref. [4] Figure 1. (a) Schematic diagram of a turbine engine Ref. [3]. (b) Schematic diagram of the temperature
and pressure gradients throughout the engine component correlating with the diagram (a) Ref. [4] structure) (Fig. 2). In some nickel – iron superalloys such as IN718 and IN706, which contain
niobium, they are hardened by γ´´ (Ni3Nb based D022 structure) (Fig. 3) [2]. Homogeneously
distributed coherent hardening precipitates confer excellent tensile and fatigue life properties
at high temperatures. Their volume fraction is controlled by the nominal chemical
composition. The size and the morphology are controlled by the process and their
crystallographic relations with γ matrix. The precipitates arise close to the solvus temperature
grow larger which subsequently restrict the grain growth pinning grain boundaries (Fig. 4). On the other hand, the precipitates arise at lower temperature such as during cooling after
heat treatment stay small (Fig. 4 (left hand side of the image)). γ´ has the perfect coherency
with the γ matrix, hence their morphologies are mostly sphere, whereas γ´´ has a tall crystal
unit tetragonal structure where a axis has the identical lattice parameter with the γ matrix but
c axis has nearly double the length of the γ, hence γ´´ always precipitate with the perfect
coherency on the basal plane with γ and grow along the longitudinal direction (Fig. 5). structure) (Fig. 2). In some nickel – iron superalloys such as IN718 and IN706, which contain
niobium, they are hardened by γ´´ (Ni3Nb based D022 structure) (Fig. 3) [2]. Homogeneously
distributed coherent hardening precipitates confer excellent tensile and fatigue life properties
at high temperatures. Their volume fraction is controlled by the nominal chemical
composition. The size and the morphology are controlled by the process and their
crystallographic relations with γ matrix. The precipitates arise close to the solvus temperature
grow larger which subsequently restrict the grain growth pinning grain boundaries (Fig. 4). 2.2. Carbides and borides Carbon and boron are added as a grain boundary strengthener by segregating in the grain
boundaries and forming carbides and borides. They are believed to be formed during
solidification, aging treatment which strengthen grain boundaries at elevated temperatures Microstructure-Property Relationship in Advanced Ni-Based Superalloys 21 but the ones arising during service must be controlled carefully since they can impair
properties [4]. p
p
[ ]
Figure 2. γ΄ L12 structure. Ni atoms are blue and Al purple
Figure 3. γ΄΄ D022 structure. Ni atoms are blue and Nb, Al and Ti purple
Carbides are traditionally classified by their chemical composition, mainly MC, M6C and
M23C6, where M stands for metal elements such as Ti, Cr, Nb, Mo, Hf and Ta [4]. MC carbides are usually coarse (Fig. 6), having a fcc densely packed structure [4]. Ti, Nb, Hf
and Ta are the main metal elements. They are very strong and are normally considered to be
some of the most stable compounds in nature, justified by their high precipitation and Figure 2. γ΄ L12 structure. Ni atoms are blue and Al purple Figure 2. γ΄ L12 structure. Ni atoms are blue and Al purple Figure 2. γ΄ L12 structure. Ni atoms are blue and Al purple Figure 2. γ΄ L12 structure. Ni atoms are blue and Al purple Figure 2. γ΄ L12 structure. Ni atoms are blue and Al purple Figure 3. γ΄΄ D022 structure. Ni atoms are blue and Nb, Al and Ti purple Figure 3. γ΄΄ D022 structure. Ni atoms are blue and Nb, Al and Ti purple Figure 3. γ΄΄ D022 structure. Ni atoms are blue and Nb, Al and Ti purple Carbides are traditionally classified by their chemical composition, mainly MC, M6C and
M23C6, where M stands for metal elements such as Ti, Cr, Nb, Mo, Hf and Ta [4]. MC carbides are usually coarse (Fig. 6), having a fcc densely packed structure [4]. Ti, Nb, Hf
and Ta are the main metal elements. They are very strong and are normally considered to be
some of the most stable compounds in nature, justified by their high precipitation and
melting temperature: they are believed to precipitate during processing shortly after
solidification of the superalloy [4]. They usually have little or no orientation relationship
with the alloy matrix [4]. 2.2. Carbides and borides M6C carbides have a complex cubic structure and they precipitate when the alloy contains
highly refractory elements, for example Mo and W. These carbides are believed to be the
product of MC carbide decomposition during service or relatively high heat treatment
between 815 and 980ºC [4]. The examples of the micrographs of M6C can be found in Ref. [5, 6]. Metallurgy – Advances in Materials and Processes
22 Figure 4. TEM dark field (DF) image. γ΄ pinning grain boundary, shown by the white dashed line. The
small spherically shaped precipitates inside the grain are also γ´. (g = 01 1 B = [111]) Figure 4. TEM dark field (DF) image. γ΄ pinning grain boundary, shown by the white dashed line. The
small spherically shaped precipitates inside the grain are also γ´. (g = 01 1 B = [111]) Figure 5. TEM DF image of the γ΄΄ in IN718. The growth direction is c axis parallel to the a axis of γ (
(gγ´´ = 0 0 2) B = [100]). Figure 5. TEM DF image of the γ΄΄ in IN718. The growth direction is c axis parallel to the a axis of γ (
(gγ´´ = 0 0 2) B = [100]). Microstructure-Property Relationship in Advanced Ni-Based Superalloys 23 Figure 6. Coarse Nb and Ti based carbide in IN718 Figure 6. Coarse Nb and Ti based carbide in IN718 Figure 6. Coarse Nb and Ti based carbide in IN718 M23C6 carbides (Fig. 7) form mainly along grain boundaries at a relatively low temperature
for carbides: between 760 and 980ºC. The crystal structure is complex cubic structure. The
lattice parameter is exactly three times larger than γ matrix, hence they precipitate with cube-
cube orientation with the matrix (Fig. 8). They are believed to form either by the
decomposition of MC or M6C or they nucleate directly on the grain boundaries. They are
known as having a high content of Cr. M23C6 carbides have a significant effect on Ni based
superalloy properties [4] since they are profuse in alloys with moderate to high Cr content [4] Figure 7. Fine M23C6 type carbides precipitate along the grain boundary running diagonally. Figure 7. Fine M23C6 type carbides precipitate along the grain boundary running diagonally. Metallurgy – Advances in Materials and Processes 24 and are controversial carbides. 2.2. Carbides and borides Firstly, this is because their different morphologies: the
blocky shaped ones at grain boundaries have a beneficial effect on rupture strength; on the
contrary the film ones are regarded as promoting early rupture failure [4]. Secondly, this is
because that they make a Cr depleted zone (Fig. 9) around the precipitate. In this area, it is
difficult to form a protective oxide, namely Cr2O3, due to lack of Cr. Figure 8. M23C6 and γ matrix perfect coherent diffraction pattern (left) and the bright field image from
another beam direction to make M23C6 outstanding (right) Figure 8. M23C6 and γ matrix perfect coherent diffraction pattern (left) and the bright field image from
another beam direction to make M23C6 outstanding (right) Figure 9. Left: STEM EDX line scan results across M23C6 revealed the Cr depletion as indicated by the
arrows (Cr nominal composition is 15 wt%). Right: STEM bright field image of the M23C6 (the thin arrow
shows the length and the direction of the EDX line scan) Figure 9. Left: STEM EDX line scan results across M23C6 revealed the Cr depletion as indicated by the
arrows (Cr nominal composition is 15 wt%). Right: STEM bright field image of the M23C6 (the thin arrow
shows the length and the direction of the EDX line scan) 25 Microstructure-Property Relationship in Advanced Ni-Based Superalloys It is broadly acknowledged that boron segregation along the grain boundary increases the
cohesive strength of the grain boundaries. The role of borides is, however, still under
open discussion. Those so far identified have a base centred tetragonal (BCT), M3B2 [4] or
M5B3 [7] formula, where M is typically a refractory element, namely Mo or Cr. They
appear as various shapes such as blocky to half-moon [4]. The examples shown in Fig. 10
were found in an advanced polycrystalline Ni superalloy after a thermal exposure at
980°C for 1 hour. Figure 10. Some examples of M5B3 type boride appeared in TEM bright field (left) and in SEM (right) Figure 10. Some examples of M5B3 type boride appeared in TEM bright field (left) and in SEM (right) 2.3. Other phases Adding excess quantity of refractory elements, such as Mo, W and Re, promotes the
precipitation of hard intermetallic phases [2], so called TCP phase, which are believed to
deteriorate the alloy ductility [4] and the creep life [8]. In the ternary phase diagrams for
superalloy elements, such as Ni-Cr-Mo, there are two phase spaces: one is austenite (γ) fcc
and the other is bcc [4]. Between these two major fields, a band of numerous small phase
volumes can be identified such as σ, μ, R and so on [4], which are characterized firstly as
having a high and uniform packing density of atoms[2] and secondly as having complex
crystal structures [2], either hcp, body centred tetragonal or rhombohedral. With the careful
control of these refractory elements, TCP phases occur after a long time service or a
prolonged heat treatment [9]. Some are believed to be the products of transformation from
another beneficial phase: for example η(Ni3X) results from γ΄ [4] and σ has the same crystal
structure as that of M23C6, but without the carbon atoms. The example of σ phase shown in
Fig. 11 was found to be Cr, Mo and Co based chemistry after a thermal exposure at 720°C
for 1,100 hours in a newly developed advanced Ni superalloy. The second phases
introduced above and some other important second phases for the Ni superalloy
microstructure are summarized in Table 1. Metallurgy – Advances in Materials and Processes
26 Figure 11. Sigma (σ) phase precipitates on the grain boundary running diagonally from top left to
bottom right Figure 11. Sigma (σ) phase precipitates on the grain boundary running diagonally from top left to
bottom right Figure 11. Table 1. Summary of second phases in the polycrystalline Ni based superalloys [10] The lattice
parameter may vary (less than 5%) by changing chemical composition. rameter may vary (less than 5%) by changing chemical composition. 2.3. Other phases Sigma (σ) phase precipitates on the grain boundary running diagonally from top left to
bottom right Phase Prototype Pearson
symbol
Strukturbericht
symbol
Lattice [nm]
Chem
Comp Phase Prototype Pearson
symbol
Strukturbericht
symbol
Lattice [nm]
Chemical
Composition (Appx)
γ'
Cu3Au
cP4
L12
a 0.36
(Ni Co)3(Al Ti)
γ''
Al3Ti
tI8
D022
a 0.36 c 0.74
(Ni Fe)3(Nb Al Ti)
MC
NaCl
cF8
B1
a 0.44
(Ti Ta)C or TiC, TaC,
NbC, WC
M6C
Fe3W3C
cF112
E93
a 1.11
(Mo Cr W)6C
M7C3
Cr7C3
oP40
D101
a 0.45 b 0.70 c 1.21 Cr7C3
M23C6
Cr23C6
cF116
D84
a 1.07
Cr21Mo2C6
M5B3
Cr5B3
tI32
D8l
a 0.55 c 1.06
(Cr Mo)5B3
M3B2
Si2U3
tP10
D5a
a 0.60 c 0.32
(Mo Cr)3B2
σ
CrNi
tP30
D8b
a 0.88 c 0.46
Cr Mo Co based
δ
Cu3Ti (β)
oP8
D08
a 0.51 b 0.43 c 0.46 Ni3Nb
η
Ni3Ti
hP16
D024
a 0.51 c 0.83
Ni3(Ti Ta)
μ
Fe7W6
hR13
D85
a 0.48 c 2.5
Mo Co based
Table 1. Summary of second phases in the polycrystalline Ni based superalloys [10] The lattice
parameter may vary (less than 5%) by changing chemical composition. Phase Prototype Pearson
symbol
Strukturbericht
symbol
L Table 1. Summary of second phases in the polycrystalline Ni based superalloys [10] The lattice
parameter may vary (less than 5%) by changing chemical composition. Microstructure-Property Relationship in Advanced Ni-Based Superalloys 27 27 3. Microstructures and mechanical properties It is worth noting the microstructure related mechanical properties in detail. We will discuss
briefly how microstructure affects various mechanical properties in polycrystalline Ni
superalloys. Altering grain sizes results in various effects with regard to the different mechanical
properties. Tensile and fatigue life properties are optimized by a fine grain microstructure,
on the other hand, good creep and fatigue crack growth properties at elevated temperature
are favoured by a coarse grain microstructure [2]. The former is a result of grain orientation
and stress concentration by dislocation movement along the slip plane [2]. The latter is
about intergranular crack propagation susceptibility. For example, Bain et al [11] showed the
significance of the grain size for the crack growth rate using UDIMET720. Testing at 650°C,
the crack growth rate reduced by more than two orders of magnitude by changing the size
from 20 to 350 μm in diameter. (Fig. 12). Figure 12. UDIMET 720 fatigue crack growth rate for different grain sizes (ASTM grain size between 0
and 8.5: 360μm and 19μm in diameter) tested at 650ºC [11]
650ºC Figure 12. UDIMET 720 fatigue crack growth rate for different grain sizes (ASTM grain size between 0
and 8.5: 360μm and 19μm in diameter) tested at 650ºC [11] The size of the hardening precipitates significantly affects the yield strength of the material
via the interaction between the precipitate and the dislocation. If the precipitates are large,
dislocation bowing around the precipitates becomes dominant; for small sized precipitates,
dislocation cutting becomes dominant. For bowing For bowing
߬ൌܩ∗ܾ
ܮെ2ݎ
(1)
and for cutting
߬ൌݎ∗ߛ∗ߨ
ܾ∗ܮ
(2) ߬ൌܩ∗ܾ
ܮെ2ݎ
(1) (1) and for cutting and for cutting ߬ൌݎ∗ߛ∗ߨ
ܾ∗ܮ
(2) (2) τ is the strength of the material, G is the shear modulus, b is the magnitude of the Burgers
vector, L is the distance between the hardening precipitates, r is the radius of the precipitates
and γ is the surface energy. In general in Ni-Al binary system, the optimum size to τ is the strength of the material, G is the shear modulus, b is the magnitude of the Burgers
vector, L is the distance between the hardening precipitates, r is the radius of the precipitates
and γ is the surface energy. In general in Ni-Al binary system, the optimum size to Metallurgy – Advances in Materials and Processes 28 maximize the strength is found to be around 5 - 30 nm in diameter (Fig. 3. Microstructures and mechanical properties 13). The size of the
precipitates also affects the creep strain as shown in Fig. 14. In their study [12], the size of
the precipitate was changed by changing the heat treatment temperature and time and
found that the smaller the precipitate the slower the creep strain rate is, which is achieved
via the smaller γ´ - γ΄ channel width [12, 13]. Figure 13. γ´ particle diameter against the critical shear stress in Ni-Al system [28] Figure 13. γ´ particle diameter against the critical shear stress in Ni-Al system [28]
Figure 14. Creep strain tested at 700ºC for different heat treatments (HT1, HT2 and HT3) The size of γ´:
HT2>HT1>HT3 [12]. It is well known fact that in general both the yield strength and the creep rupture strength
increases by increasing the hardening precipitate volume fraction [2] Historically low cycle Figure 13. γ´ particle diameter against the critical shear stress in Ni-Al system [28] Figure 14. Creep strain tested at 700ºC for different heat treatments (HT1, HT2 and HT3) The size of γ´:
HT2>HT1>HT3 [12]. Figure 14. Creep strain tested at 700ºC for different heat treatments (HT1, HT2 and HT3) The size of γ´:
HT2>HT1>HT3 [12]. It is well known fact that in general both the yield strength and the creep rupture strength
increases by increasing the hardening precipitate volume fraction [2]. Historically, low cycle
fatigue life was the main concern for turbine disc alloys, but fatigue crack growth rate and
damage tolerant design have attracted more attention over the last two decades [11, 14]. 29 Microstructure-Property Relationship in Advanced Ni-Based Superalloys They can be strongly influenced not only by the size of the grains as introduced above, but
also by the size of the precipitates; the striking results were shown in Ref. [15, 16]. The
results show that the larger the hardening precipitates the better the crack growth property. However, this conflicts with the creep life property as mentioned above. Research on
damage tolerant design originally started to investigate the grain boundary chemistry since
fast crack growth (FCG) is always observed with intergranular cracks and tends to
disappear at low temperature. Additionally, transgranular ductile cracking replaces
intergranular crack when the tests carried out in the reduced oxygen partial pressure [17, 18]
(Fig. 15) Thus, FCG embrittlement has been attributed to oxidation [11, 19]. Grain boundary
engineering has been explored by changing the morphology of the grain boundary. 3. Microstructures and mechanical properties Optical microscopy image of serrated grain boundaries. The arrows indicate the serrated
boundaries [15] 3. Microstructures and mechanical properties For
example, Ref. [15, 20] reported a complex grain boundary geometry, so called ‘serrated’ (Fig. 16), by slow cooling after solution treatment. The result showed slower intergranular crack
growth rate than with a normal grain boundary [15]. However, the improvement above did
not account for the property change by the different size of the hardening precipitate
mentioned above. The fast intergranular crack growth at high temperature in superalloys
added a new dimension after intensive studies with regard to the correlation between the
hardening precipitate distribution and the crack growth rate. Ref. [15, 16, 21] claimed that
the prevention of stress relaxation of the crack tip by the hardening precipitates can increase
the crack growth rate. Some experimental work support the idea, for example Andieu et al
[22] carried out a unique dwell fatigue crack propagation test where oxygen was introduced
in different phases of the low cycle fatigue crack growth test and found that it is potent for
the fast crack growth when oxygen is introduced at the beginning of the loading rather than
introducing in the later part of the loading. This may imply that the oxidation at the crack
tip happens during the stress concentrated at the crack tip. Molins et al [23, 24] concluded
that the local microstructure at the crack tip, which can be controlled by an appropriate heat
treatment against the stress accumulation, can significantly affect the crack propagation
behaviour in Ni superalloys. This conclusion recalls an arguable grain boundary
microstructure feature, namely the precipitate free zone (PFZ). One suggested that the PFZ
would promote plastic deformation and fracture [25, 26]. Another suggested that the PFZ in
some nickel alloys is beneficial for crack tip stress relaxation [27]. Figure 15. Typical intergranular (left) and transgranular (right) fracture surfaces. Alloy 718 tested at
650ºC in air (left) and vacuum (right) [18] Figure 15. Typical intergranular (left) and transgranular (right) fracture surfaces. Alloy 718 tested at
650ºC in air (left) and vacuum (right) [18] Metallurgy – Advances in Materials and Processes 30 These findings above suggest that not only the macroscopic structure such as the grain size
and the distribution of the hardening precipitates, but also the microscopic structure, such
as the grain boundary shape and the relationship with the hardening precipitates, can
significantly affect the mechanical properties. Figure 16. Optical microscopy image of serrated grain boundaries. The arrows indicate the serrated
boundaries [15] Figure 16. 4. Polycrystalline superalloy grain boundary structure The details of the Ni superalloy grain boundary microstructure will be demonstrated in this
section. Particular attention will be paid to the relationship between the hardening
precipitates and the high grain boundaries. Fig. 17 shows the STEM bright field image of the
grain boundary and the hardening precipitate morphology in an advanced polycrystalline
superalloy. The grain boundary running top left to bottom right cuts through γ´. This was
confirmed by the conventional TEM image analysis combining with the crystallographic
analysis that the either side of the γ´ keeps the coherency with the matrix (Fig. 18). With
respect to the morphologies of γ´ on the grain boundaries, it is the same as those inside the
grains. It has, however, two different crystallographic orientations keeping the coherency
with the either side of the matrix. This morphology is believed to form during the process
with the high boundary mobility [29]. There are at least four different possibilities of
interactions between the migrating grain boundaries and the precipitates, which are
illustrated in Fig. 19. Following Fig. 19, a. the boundary migrates with no effect on the precipitates; the precipitates thus become
incoherent after the migrating grain boundary passes through them. a. the boundary migrates with no effect on the precipitates; the precipitates thus become
incoherent after the migrating grain boundary passes through them. Microstructure-Property Relationship in Advanced Ni-Based Superalloys 31 31 b. the precipitates dissolve in contact with migrating boundary and reprecipitate
coherently within the new grain. b. the precipitates dissolve in contact with migrating boundary and reprecipitate
coherently within the new grain. c. the grain boundary is held by the coherent precipitates which then coarsen, leading to
complete halting of the boundary movement. c. the grain boundary is held by the coherent precipitates which then coarsen, leading to
complete halting of the boundary movement. d. the grain boundary can pass through the coherent precipitate which undergoes the
same orientation change as the grain surrounding it and thereby retains the coherent
low-energy interface between the precipitate and the matrix. d. the grain boundary can pass through the coherent precipitate which undergoes the
same orientation change as the grain surrounding it and thereby retains the coherent
low-energy interface between the precipitate and the matrix. Figure 17. General aspect of the high angle grain boundary and γ´ (dark spheres). The grain boundary
is running diagonally from top left to bottom right
100nm Figure 17. 4. Polycrystalline superalloy grain boundary structure General aspect of the high angle grain boundary and γ´ (dark spheres). The grain boundary
is running diagonally from top left to bottom right Figure 18. A crystallographic analysis of the cutting γ´. The dark field images of the lower and the
upper grain are in (I) and (II), respectively. The white arrows indicate the grain boundary Figure 18. A crystallographic analysis of the cutting γ´. The dark field images of the lower and the
upper grain are in (I) and (II), respectively. The white arrows indicate the grain boundary Metallurgy – Advances in Materials and Processes 32 With regard to theory a), this is often observed with high solvus temperature precipitates,
such as carbides and oxides. b) is not applicable in this study. This can be, however, the case
for less γ΄ volume fraction superalloys with small amount of nominal Al content such as
Nimonic PE16 or the case in high temperature very close to the γ´ solvus. With regard to c),
it can be applicable in the case of the larger γ´ such as the one in Fig. 4. d) is relevant to
explain the results of Fig. 17 and Fig. 18. As the grain boundary impinges on the γ΄, the
grain boundary apparently cuts off γ΄. Firstly, the interface free energy between γ´ and the
grain boundary increases. This results in dissolving the γ´ at the interface but due to the
supersaturation of γ´ formers, such as Al and Ti, γ´ immediately re - nucleates in the next
grain coherent with the next grain discontinuously [30, 31]. Thus, this phenomenon can be
concluded the result of γ΄ dissolution and subsequent (discontinuous) precipitation. Figure 19. The possible interactions between the migrating grain boundary and the precipitate [29] gure 19. The possible interactions between the migrating grain boundary and the precipitate [29] Another example is forming a precipitate free zone (PFZ) as shown in Fig. 20 in IN718. γ´
and γ´´ coexist in IN718, however, γ´´ denude along the grain boundary and form a γ´´ PFZ. On the contrary, the minor hardening precipitate in IN718: γ´ exist along the grain
boundary. Vacancy deficiency is one of the causes of the PFZ along grain boundaries due to
lack of the nucleation sites as grain boundary acts as a good vacancy sink [32]. 4. Polycrystalline superalloy grain boundary structure One of the
important factors to create the PFZ in the superalloys can be the difference of the interfacial
free energy, i.e. the free energy between γ´-matrix and γ´´-matrix. The γ´´-γ´´ nucleus
channel distance along the grain boundary can be larger than the critical distance to
aggregate two γ´´ nuclei. On the other hand, the critical distance for the γ´ is smaller than
that of γ´´ or γ´ can nucleate their precipitate independently as γ´ has smaller interfacial
energy. Thus, the γ´ nuclei can grow and form precipitates along the grain boundary but not
for γ´´ and the γ´´ PFZ arises. Microstructure-Property Relationship in Advanced Ni-Based Superalloys 33 Figure 20. γ´´ PFZ appeared in IN718, although γ´ still exist in the γ´´PFZ adjacent to the grain boundary Figure 20. γ´´ PFZ appeared in IN718, although γ´ still exist in the γ´´PFZ adjacent to the grain b Figure 20. γ´´ PFZ appeared in IN718, although γ´ still exist in the γ´´PFZ adjacent to the grain boundary 5. High temperature oxidation along grain boundaries The context of ‘High temperature’ in this section is the temperature range of 600-700°C
which is the high temperature regime of the disc in the turbine engine application. It is more than a half century ago, the investigation of the oxidation assisted fast crack growth
started. Cr is believed to be an important element for the oxidation assisted crack growth. For
example, as shown in Fig. 21, crack growth tests were conducted under various oxygen partial
pressure on Ni-Cr binary alloys with 5, 20 and 30 wt% of Cr. It was found that the higher the
Cr content the higher the transition oxygen partial pressure from transgranular to intergranular
cracking is. The highest Cr content alloy: Ni-30wt%Cr did not show a transition pressure. Oxidation process on a freshly exposed alloy surface had been characterised intensively and
well understood. As illustrated in Fig. 22, both Ni and Cr oxide formation takes place at the
beginning of the oxidation [33]. This is particularly important for alloys on the borderline
between protective and non-protective behaviour [34]. But in the early stage, the fast kinetics
Ni oxide grows quickly and dominates the oxide. In general, there are two types of oxidation:
the cation diffusion type and the anion diffusion type [34] . The difference between the two is
the movement of the ions; the former involves cation (metal ion) transport, the latter anion
(oxygen ion) transport. For the cation diffusion type the oxides form between the oxide and
the free surface, but, for the anion diffusion, the oxides form between the oxide and the metal Metallurgy – Advances in Materials and Processes 34 interface. During the transient stage which corresponds to the middle of the illustrations in
Fig. 22, Cr2O3 particles are embedded inside the NiO layer. As NiO grows and the oxidation
rate becomes slower, Cr2O3 soon establishes its own layer underneath the NiO layer, which
implies that Cr2O3 can be the anion diffusion. Eventually the Cr2O3 layer is completed, where
the layer prevents further diffusion of oxygen into the alloy, called passivation [34]. Figure 21. Transition oxygen partial pressure from the transgranular to intergranular cracking against
the Cr concentration in Ni alloys after crack propagation tests at 650°C [24] Figure 21. Transition oxygen partial pressure from the transgranular to intergranular cracking against
the Cr concentration in Ni alloys after crack propagation tests at 650°C [24] Figure 22. 5. High temperature oxidation along grain boundaries Schematic diagram of the oxidation process (from top to bottom) of the freshly exposed Ni
alloy [33] e 22. Schematic diagram of the oxidation process (from top to bottom) of the freshly exposed Ni
[33] Figure 22. Schematic diagram of the oxidation process (from top to bottom) of the freshly exposed Ni
alloy [33] 35 Microstructure-Property Relationship in Advanced Ni-Based Superalloys 35 Nevertheless, the oxidation process at (ahead) of the crack tip has remained under debate. This is due to the technical difficulties of studying the microstructure of such small regions,
which is predictable taking consideration of the size of the crack tip: less than a micron and
even smaller for the oxides adjacent. To overcome the problem, so called ‘site specific
specimen preparation’ has been developed since the late 90’s using focused ion beam (FIB)
technique [35, 36]. The use of gallium ions in a focused ion beam accelerated in a FIB
apparatus to energies up to 30 keV enables us to mill specimens selectively to reveal
structural features and to deposit films at selected locations. An example carried out in the
University of Birmingham UK (2009) is shown in Fig. 23. The specimen is an advanced Ni
based superalloy after an interrupted crack propagation test at 650°C in air. A plan-view
crack tip TEM specimen was prepared [37]. Using the two different modes: the tungsten
deposition to protect the region of interest (Fig. 23 a)) and the milling (trenching) (Fig. 23 b)),
the crack tip was transported to a TEM copper grid (c) and d)). The size of the focused Ga
ion beam can be achieved as small as a few nanometres in radius. It is possible to prepare
the site specific TEM specimen foil as thin as 50 nm in thickness. Fig. 24 shows TEM bright field images; they are from the same material: polycrystalline
advanced superalloy, but they are after different testing conditions. Fig. 24 (a) is from a
specimen after interrupting a crack propagation test at 650°C; the intergranular crack
propagation was identified. Fig. 24 (b) is from a specimen after interrupting the same crack
propagation test mentioned above except for the crack growth rate: 0 μm/s (~0.9 Kth: just
below the crack growth threshold) held for 5 hours. The oxides ahead of the crack tip are
also along grain boundaries. 5. High temperature oxidation along grain boundaries The dashed line boxes indicate the area analysed by EDX
shown later in this section. Comparing the two bright field images in Fig. 24, it is apparent
that the oxides penetration in the metal ahead of the crack tip is approximately 5 times
longer for the static crack specimen (b). The EDX mapping and the EDX line scan of the oxides close to the crack tip for the moving
crack specimen from the region I in Fig. 24 are shown in Fig. 25. It is apparent that the grain
boundary is completely filled with oxides (oxygen map). There is a Co and Ni rich oxide in
the middle. There are Cr rich oxide areas on both sides of the oxide. Cr thus forms a thin layer
between the Ni (Co) oxides and the alloy. EDX line scans across the oxide revealed that Ti, Al
are also segregating in this region. Crystallographic analysis using selected aperture
diffraction confirmed that the middle oxide is cubic (Nix Co1-x)O and the rim oxide is hcp (Cr
Al Ti)2O3. The higher oxygen partial pressure region in the middle of the oxide is consisted by
the Ni and Co oxide. The rim of the oxide between the Ni (Co) oxide and the matrix are
consisted by the passive Cr, Al and Ti layer. This is correlating with the freshly exposed Ni
alloy oxidation process described above. Fig. 26 shows the oxide chemistry at the tip of the
oxide corresponding to the region II in Fig. 24. According to the EDX mapping, the oxides
formation manner looks similar to that of the region I; Ni(Co) oxide in the middle and the Cr,
Al and Ti oxide in the rim. It is questionable to argue the stoichiometry of the oxide from the
results of the EDX cross section line scan due to the x-ray emission from the matrix, however
it revealed from the line scan in Fig. 26 that the oxide is Cr based; Ni, Co, Al and Ti deplete in
the very tip of the oxide. Fig. 27 shows the chemistry of the oxides in the middle of the oxide
ahead of the crack tip for the static crack corresponding to the region x in Fig. 24. EDX
mapping revealed that the Ni and Co are depleted even in the middle of the oxide and the Metallurgy – Advances in Materials and Processes
36 Figure 23. 5. High temperature oxidation along grain boundaries SEM secondary electron images of the process of the site specific TEM sample preparation e 23. SEM secondary electron images of the process of the site specific TEM sample preparation Microstructure-Property Relationship in Advanced Ni-Based Superalloys 37 Figure 24. TEM bright field images of the two specimens. The crack tips locate just next to the specimen
and the crack propagate from the left to right. Figure 24. TEM bright field images of the two specimens. The crack tips locate just next to the specimen
and the crack propagate from the left to right. Figure 25. STEM dark field image from the region I in Fig. 24 (top left); the thick white arrow indicates
the crack growth direction and the thin black arrow indicates the area and the direction of the EDX line
scan. The EDX line scan across the oxide (top right) and the EDX mapping results of the oxide (bottom). Figure 25. STEM dark field image from the region I in Fig. 24 (top left); the thick white arrow indicates
the crack growth direction and the thin black arrow indicates the area and the direction of the EDX line
scan. The EDX line scan across the oxide (top right) and the EDX mapping results of the oxide (bottom). Metallurgy – Advances in Materials and Processes
38 Figure 26. STEM dark field image from the region II in Fig. 24 (top left). The EDX line scan across the
oxide (top right) and the EDX mapping results of the oxide (bottom). Figure 26. STEM dark field image from the region II in Fig. 24 (top left). The EDX line scan across the
oxide (top right) and the EDX mapping results of the oxide (bottom). Figure 27. STEM dark field image from the region x in Fig. 24 (top left). The EDX line scan across the
oxide (top right) and the EDX mapping results of the oxide (bottom). Figure 27. STEM dark field image from the region x in Fig. 24 (top left). The EDX line scan across the
oxide (top right) and the EDX mapping results of the oxide (bottom). Microstructure-Property Relationship in Advanced Ni-Based Superalloys 39 line scan revealed that the oxide is consisted mainly by Cr, Al. Fig. 28 shows the tip of the
oxide for the static crack corresponding to the region z in Fig. 24. 6. Summary One of the challenges of the advanced Ni based superalloys is in the damage tolerance
properties without reducing their superior strength at high temperature. The microstructure,
particularly the grain boundaries, was found to be controlled by the two factors in this study. Firstly, it is the nominal chemical composition, especially the hardening precipitate
participants. Secondly, heat treatment has a profound influence of microstructure. The damage tolerance properties are also concerned with the environmentally assisted crack
propagation along grain boundaries, which is essentially the oxidation assisted crack
propagation in this study. In general, chromium oxide (Cr2O3) has been regarded as a
healing agent of the oxidation process in Ni alloys. The state-of-the-art technique enabled us
to observe the crack tip oxidation. In this study, it was successfully presented that the
oxidation sequence is following the free energies for the oxides to form. Thus, for example,
Cr2O3 is one of the earliest oxides to form at the crack tip. Understanding the environmentally assisted crack propagation is one of the crucial fields of
research to increase the turbine entry temperature (TET), which is indeed one of the most
significant attainments in the propulsion industries. Author details
Hiroto Kitaguchi
Department of Materials, University of Oxford, OX1 3PH, Oxford, UK 5. High temperature oxidation along grain boundaries Cr still exists in the oxide
in the middle, but particularly at the very tip of the oxide approximately 100 nm or so, Cr is
depleted and only Al and Ti enriched at the tip of the oxide (see also the cross section line
scan). It is also difficult in this case due to the thickness effect to discuss the stoichiometry of
the oxide, however, in this region, Al and Ti based oxide formation takes place. Figure 28. STEM dark field image from the region z in Fig. 24 (top left). The EDX line scan across the
oxide(top right) and the EDX mapping results of the oxide (bottom). Table 2 shows the oxygen dissociation pressure to form the oxide and the free energy for the
elements forming oxides introduced above. It is clear that the formation of the oxide at the Figure 28. STEM dark field image from the region z in Fig. 24 (top left). The EDX line scan across the
oxide(top right) and the EDX mapping results of the oxide (bottom). Table 2 shows the oxygen dissociation pressure to form the oxide and the free energy for the
elements forming oxides introduced above. It is clear that the formation of the oxide at the
crack tip follows the thermodynamics suggesting the oxygen partial pressure gradient along
the oxide tip ahead of the crack tip. Metallurgy – Advances in Materials and Processes
40 Oxide formation
Free energy
Oxygen dissociation pressure in log10
[kJ/mole]
[bar]
Al
Al2O3
-1425
-48
Ti
Ti2O3
-1295
-44
TiO2
-798
-40
Cr
Cr2O3
-877
-31
Co
CoO
-166
-18
Ni
NiO
-149
-16
Table 2. Oxide formation free energy and the dissociation pressure at 727°C for the elements
introduced in this section [38] Table 2. Oxide formation free energy and the dissociation pressure at 727°C for the elements
introduced in this section [38] Author details Hiroto Kitaguchi Department of Materials, University of Oxford, OX1 3PH, Oxford, UK Acknowledgement The author would thank to Profs. I.P. Jones and P. Bowen at The University of Birmingham
and Dr. M.C. Hardy at Rolls-Royce plc for their advice and the guidance. The joint support
of the UK Engineering and Physical Sciences Research Council (EPSRC) and Rolls-Royce plc
is also gratefully acknowledged. Many thanks are also due to Dr H.Y. Li, Research Fellow at
the School of Metallurgy and Materials, The University of Birmingham and Dr Y.Y. Tse,
former Research Fellow at the School of Metallurgy and Materials, The University of Microstructure-Property Relationship in Advanced Ni-Based Superalloys 41 Birmingham, currently Lecturer at Loughborough University, for their assistance with the
mechanical tests and the FIB TEM sample preparation. 7. References [1] G.W. Meetham, The Development of Gas Turbine Materials 1st ed., Applied Science,
London, 1981. [2] R.C. Reed, The Superalloys, Cambridge University Press, Cambridge, 2006. [3] H. Cohen, G.F.C. Rodgers, H.I.H. Saravanamuttoo, Gas turbine theory. Third edition,
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Engineering A (Structural Materials: Properties, Microstructure and Processing), A361
(2003) 191-197. ] X.Z. Qin, J.T. Guo, C. Yuan, J.S. Hou, H.Q. Ye, Materials Letters, 62 (2008) 2275-2278 [7] S.T. Wlodek, M. Kelly, D.A. Alden, in, TMS, Warrendale, PA, USA, 1996, pp. 129-136. [8] T. Sugui, W. Minggang, L. Tang, Q. Benjiang, X. Jun, Materials Science and Engineering
A, 527 (2010) 5444-5451. [9] R.J. Mitchell, C.M.F. Rae, S. Tin, Materials Science and Technology, 21 (2005) 125-132. [10] P.Villars, ASM International, 1997. [10] P.Villars, ASM International, 1997. [11] K.R. Bain, M.L. Gambone, J.M. Hyzak, M.C. Thomas, Proceedings of the International
Symposium on Superalloys, (1988) 13-22. [12] D. Locq, P. Caron, S. Raujol, F. Pettinari-Sturmel, A. Coujou, N. Clement, Superalloys
2004. Proceedings of the Tenth International Symposium on Superalloys (2004) 179-187. [13] P.R. Bhowal, E.R. Wright, E.L. Raymond, Metallurgical Transactions A (Physical
Metallurgy and Materials Science), 21A (1990) 1709-1717. [14] N.J. Hide, M.B. Henderson, P.A.S. Reed, SUPERALLOYS 2000. Proceedings of the
Ninth International Symposium on Superalloys, (2000) 495-503. [15] J. Telesman, P. Kantzos, J. Gayda, P.J. Bonacuse, A. Prescenzi, Superalloys 2004. Proceedings of the Tenth International Symposium on Superalloys (2004) 215-224. [16] J. Telesman, T.P. Gabb, A. Garg, P. Bonacuse, J. Gayda, Superalloys 2008, (2008) 8 [16] J. Telesman, T.P. Gabb, A. Garg, P. Bonacuse, J. Gayda, Superalloys 2008, (2008) 807-816. [17] E. Andrieu, R. Molins, H. Ghonem, A. Pineau, Materials Science and Engineering A,
A154 (1992) 21-28. [16] J. Telesman, T.P. Gabb, A. Garg, P. Bonacuse, J. Gayda, Superalloys 2008, (2008) 807-816. [17] E Andrieu R Molins H Ghonem A Pineau Materials Science and Engineering A [17] E. Andrieu, R. Molins, H. Ghonem, A. Pineau, Materials Science and Engineering A,
A154 (1992) 21-28. [18] H. Ghonem, D. Zheng, Materials Science and Engineering A (Structural Materials:
Properties, Microstructure and Processing), A150 (1992) 151-160. 9] M. Gao, D.J. Dwyer, R.P. Wei, Scripta Metallurgica et Materialia, 32 (1995) 1169-1174 [20] A.K. Koul, R. Thamburaj, Metallurgical Transactions A (Physical Metallurgy and
Materials Science), 16A (1985) 17-26. 7. References )
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[21] D. Turan, D. Hunt, D.M. Knowles, Materials Science and Technology, 23 (2007) 183-188. [22] E. Andrieu, A. Pineau, Journal de Physique IV (Proceedings), 9 (1999) 3-11. D. Turan, D. Hunt, D.M. Knowles, Materials Science and Technology, 23 (2007) 183-18 [21] D. Turan, D. Hunt, D.M. Knowles, Materials Science and Technology, 23 (2007) 183-188. [22] E Andrieu A Pineau Journal de Physique IV (Proceedings) 9 (1999) 3 11 [22] E. Andrieu, A. Pineau, Journal de Physique IV (Proceedings), 9 (1999) 3-11. [23] R. Molins, J.C. Chassaigne, E. Andrieu, Materials Science Forum, 251-254 (1997) 4 [23] R. Molins, J.C. Chassaigne, E. Andrieu, Materials Science Forum, 251-254 (1997) 445-452. [24] R. Molins, G. Hochstetter, J.C. Chassaigne, E. Andrieu, Acta Materialia, 45 (1997) 663-
674 [24] R. Molins, G. Hochstetter, J.C. Chassaigne, E. Andrieu, Acta Materialia, 45 (1997) 663-
674. Metallurgy – Advances in Materials and Processes
42 [25] R.G. Baker, J. Nutting, Iron and Steel, 32 (1959) 606-612. [25] R.G. Baker, J. Nutting, Iron and Steel, 32 (1959) 606-612. [26] G. Thomas, J. Nutting, Institute of Metals -- Journal, 86 (1957) 7-14. [27] The Journal of the institute of metals; Discussion, 91 (1963-64) 153. [28] H. Gleiter, E. Hornbogen, Materials science and engineering, 2 (1968) 285-302. [29] E. Grant, A. Porter, B. Ralph, Journal of Materials Science, 19 (1984) 3554-3573. [30] A. Porter, B. Ralph, Journal of Materials Science, 16 (1981) 707-713. [31] R.D. Doherty, Metal Science, 16 (1982) 1-13. [32] G.W. Lorimer, R.B. Hicholson, Acta Metallurgica, 14 (1966) 1636. [33] G.C. Wood, F.H. Stott, Materials Science and Technology, 3 (1986) 519-530. [34] L.L. Shreir and R.A. Jarman and G.T. Burstein, Corrosion, Butterworth-Heinemann Ltd,
Oxford, 1963. [35] S. Lozano-Perez, Y. Huang, R. Langford, J.M. Titchmarsh, Electron Microscopy and
Analysis 2001. Proceedings, 5-7 Sept. 2001, IOP Publishing, Bristol, UK, 2001, pp. 191-
194. [36] D.M. Longo, J.M. Howe, W.C. Johnson, Ultramicroscopy, 80 (1999) 69-84. [37] F.A. Stevie, R.B. Irwin, T.L. Shofner, S.R. Brown, J.L. Drown, L.A. Giannuzzi,
Characterization and Metrology for ULSI Technology. 1998 International Conference,
23-27 March 1998, AIP, USA, 1998, pp. 868-872. [38] G.V. Samsonov, The oxide handbook, Plenim Publishing Company, Ltd, London, 1973,
pp. 23.
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Dynamic and transient processes in warm dense matter
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this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Dynamic and transient processes in warm dense matter White, T. G., Dai, J., & Riley, D. (2023). Dynamic and transient processes in warm dense matter. Philosophical
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Introduction
Cite this article: White TG, Dai J, Riley D. 2023
Dynamic and transient processes in warm
dense matter. Phil.Trans.R.Soc.A 381:
20220223. https://doi.org/10.1098/rsta.2022.0223
Received: 5 May 2023
Accepted: 22 May 2023
One contribution of 11 to a theme issue
‘Dynamic and transient processes in warm
dense matter’. Subject Areas:
astrophysics, high-pressure physics, plasma
physics
Keywords:
warm dense matter, review, plasmas,
experimental techniques, simulation
Author for correspondence:
David Riley
e-mail: d.riley@qub.ac.uk Subject Areas: This article is part of the theme issue ‘Dynamic and
transient processes in warm dense matter’. astrophysics, high-pressure physics, plasma
physics Introduction 1Department of Physics, University of Nevada, Reno, NV 89557, USA
2College of Science, National University of Defense Technology,
Changsha 410073, People’s Republic of China
3School of Mathematics and Physics, Queen’s University Belfast,
Belfast BT7 1NN, UK 1Department of Physics, University of Nevada, Reno, NV 89557, USA
2College of Science, National University of Defense Technology,
Changsha 410073, People’s Republic of China
3School of Mathematics and Physics, Queen’s University Belfast,
Belfast BT7 1NN, UK Cite this article: White TG, Dai J, Riley D. 2023
Dynamic and transient processes in warm
dense matter. Phil.Trans.R.Soc.A 381:
20220223. TGW, 0000-0002-3865-4240; DR, 0000-0002-6212-3212 https://doi.org/10.1098/rsta.2022.0223 In this paper, we discuss some of the key challenges
in the study of time-dependent processes and non-
equilibrium behaviour in warm dense matter. We
outline some of the basic physics concepts that have
underpinned the definition of warm dense matter as
a subject area in its own right and then cover, in
a selective, non-comprehensive manner, some of the
current challenges, pointing along the way to topics
covered by the papers presented in this volume. One contribution of 11 to a theme issue
‘Dynamic and transient processes in warm
dense matter’. 1. Introduction warm dense matter, review, plasmas,
experimental techniques, simulation The term warm dense matter (WDM) was coined over
20 years ago, e.g. [1–3] to describe matter that is at
high pressures, generally over 100 GPa, but at the same
time, at high temperatures of order 104–106 K. This
range of conditions leads to partially degenerate and
partially ionized matter, with strong correlations between
charged particles and both populated excited and bound
states playing a role in the microscopic structure and
particle dynamics [4]. It is a transitional state between
solid and plasma and exhibits properties of both. Its inherent complexities make perturbative techniques
unreliable, leading to significant differences in theoretical
and computational predictions of important quantities
[5]. Moreover, due to the difficulty of creating and 2. Background Much of the physics relevant to WDM was studied in the fields of dense plasma physics [7,8] and
liquid metal physics [9] long before the term was coined. The remit of these two fields includes
a wider range of conditions than is usually included within the definition of WDM but they do
share some issues, such as the effect of degeneracy and particle correlation on basic properties
such as thermal and electrical conductivity. A principal and oft-cited motivation for the study of WDM is that the relevant conditions are
applicable to many planetary interiors, especially for large planets such as Jupiter and Saturn. For the former, the expected conditions at the core, depending on the equation of states used,
include a pressure of order 4 TPa and temperature approximately 104 K. A key point emerging
from work such as that by Nettelmann and colleagues [10] is that, by using different equation of
state models for the hydrogen and helium within the planetary core, various models of the interior
layers and conditions can be constructed that still agree with observations of surface temperature
and gravitational moments observed by missions such as Voyager and Cassini, e.g. [11–13]. The
significantly improved gravity data from the Juno mission (see [14–16] and references therein)
means that more detailed models of the interior of Jupiter can be considered and compared. For example, the more accurate measurements of the J4 and J6 moments have suggested a more
diluted core with heavy and light element mixing. This means that more exact information on
the equation of state for hydrogen and helium has more impact than ever and efforts to improve
measurements in the laboratory are more important than ever. These considerations highlight the
necessity of laboratory experiments to determine equations of states for the WDM regime. The
discovery over recent decades of thousands of exoplanets [17,18] only gives increased motivation
in this respect. Interest is not limited to planetary interiors. For any matter taken on a path from a cold solid to
hot plasma, such as components of an inertial confinement fusion (ICF) capsule, the sample must
pass through the WDM regime, and thus the properties of WDM may affect the trajectory. The
design of ICF implosions depends on the thermal conductivity of both the ablator materials and
the fuel mixture [19–21]. 2
royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . diagnosing WDM in the laboratory [6], there is a shortage of experimental results with which to
benchmark predictions. diagnosing WDM in the laboratory [6], there is a shortage of experimental results with which to
benchmark predictions. 2 Author for correspondence:
David Riley 2023 The Authors. Published by the Royal Society under the terms of the
Creative Commons Attribution License http://creativecommons.org/licenses/
by/4.0/, which permits unrestricted use, provided the original author and
source are credited. 2. Background [27,28] can only
probe a peripheral part of the WDM regime. (Adapted from [29].) y
yp
g g j
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
1000
DAC Fe (Dubrovinsky)
Ramp compression Fe (Wang)
Uranus core
'Super' Earth
explosive
compression
Saturn core
Jupiter
Brown Dwarf
Fe Hugoniot
Earth core
DAC Fe
(Yoo)
Pressure (GPa)
0.1
1
temperature (eV)
10
10 000
Γ = 50 for Fe
H Hugoniot
EF/KT = 1 Fe 3 temperature (eV) 1 Figure1. Mappingofthewarmdensematterregion.Theboundariesareabroadguide.Wecanseethewayinwhichconditions
in Jupiter as a function of depth (dashed blue line) pass through the WDM regime, as do conditions in a Brown Dwarf (dashed
orange line). We note that static methods of compression and heating, such as diamond anvil cells (DAC), e.g. [27,28] can only
probe a peripheral part of the WDM regime. (Adapted from [29].) can see that the regime extends across a wide range of pressures and temperatures from a few eV
to several 10s of eV. The significance of planetary interiors as a reason to study WDM means that the conditions
we are motivated to study are often in a steady or quasi-steady state with a very long timescale of
evolution. For static methods of producing WDM states, Anzellini & Boccato [30] have recently
reviewed the accessible range of DACs for both laser and resistive heating. Temperatures of up to
6000 K are reachable for pressures in the region of more than 100 GPa. Tateno et al. [31] reported
measurements of iron up to 5700 K and 377 GPa, while Dubrovinsky et al. [32] report pressures
above 750 GPa with a double-stage DAC. Although these extreme conditions are relevant, for
example, to the structure of the Earth’s core, as we can see from figure 1, the temperatures reached
are at the lower border of the WDM regime and the interiors of larger planets may be out of reach
for DAC and other static methods. 2. Background At the same time, the efficiency of the process can be reduced by fuel
degradation through mechanisms like diffusion [22–24]. The Rayleigh–Taylor instability, which
can affect the interface between the ablator and fuel within the fusion capsule, is particularly
sensitive to the diffusion coefficient [25]. The recent successes at the National Ignition Facility, e.g. [26] can only serve to further increase interest in WDM research within the ICF community. In figure 1, we see the WDM regime mapped out in pressure–temperature space. We can see
how the shock Hugoniots for two elements of astrophysical interest, hydrogen and iron, pass
through the WDM regime. The label, Γ , against the grey dashed curve refers to the ion–ion
coupling parameter, which measures the ratio of Coulombic potential energy to thermal energy: Γ = Z2e2
akBT
(2.1) (2.1) where Z is the average charge on the ions, and a is a characteristic distance between ions (Wigner–
Seitz radius) given by a =
3
4πni
1/3
(2.2) (2.2) where ni is the average density of the ions. For strong shocks, we typically see values of Γ ≲50,
and the thermal energy stops being a small perturbation to the Coulomb interaction. The yellow
line denotes where the Fermi energy (EF) for Fe is equal to the temperature, in eV, indicated in the
figure. In this regime, we cannot treat the free electrons in the sample as fully degenerate and we where ni is the average density of the ions. For strong shocks, we typically see values of Γ ≲50,
and the thermal energy stops being a small perturbation to the Coulomb interaction. The yellow
line denotes where the Fermi energy (EF) for Fe is equal to the temperature, in eV, indicated in the
figure. In this regime, we cannot treat the free electrons in the sample as fully degenerate and we 100
1000
DAC Fe (Dubrovinsky)
Ramp compression Fe (Wang)
Uranus core
'Super' Earth
explosive
compression
Saturn core
Jupiter
Brown Dwarf
Fe Hugoniot
Earth core
DAC Fe
(Yoo)
Pressure (GPa)
0.1
1
temperature (eV)
10
10 000
Γ = 50 for Fe
H Hugoniot
EF/KT = 1 Fe
Figure1. Mappingofthewarmdensematterregion.Theboundariesareabroadguide.Wecanseethewayinwhichconditions
in Jupiter as a function of depth (dashed blue line) pass through the WDM regime, as do conditions in a Brown Dwarf (dashed
orange line). We note that static methods of compression and heating, such as diamond anvil cells (DAC), e.g. 2. Background This presents a challenge for experimental study, and it is for
this reason we often look to dynamic methods of production, such as shocks and other rapid
heating mechanisms, as discussed in the next section. 3. Timescale considerations for dynamic production of warm dense matter However, answering this question is not a simple matter, as samples expand upon
heating, and the hydrodynamic timescale is dependent on both the pressure and sample size. For
example, if we take a sound speed given by cs ≈
P
ρ ,
(3.1) (3.1) for a sample at pressure 100 GPa and density of 103 kgm−3, we can see that the sound speed is
of order 104 m s−1. For a millimetre-sized sample, the estimated timescale for decompression is
t ∼d/cs ∼100 ns, where d is the size of the sample. In this sense, we can say that experiments
carried out with nanosecond creation and probing can be applicable to steady-state conditions. p
g
pp
y
However, it is important to note that hydrodynamic expansion timescales are not the only
timescale of interest. Not all species in a sample (e.g. ions and electrons) are initially heated in
the same way when creating WDM. For example, shock compression heats ions, e.g. [43,44],
which transfer energy to electrons through collisions. By contrast, X-ray heating, e.g. [45] transfers
energy to electrons via photo-ionization and these transfer energy to ions via collisional processes
and electron–phonon coupling. The rate at which energy is transferred between ions and
electrons, and indeed the rate at which processes, such as melting, occur, is an important factor to
consider when studying the formation of WDM and forms an important topic in this collection
addressed by several papers. Not only will this rate determine if we have conditions relevant
to equilibrium situations, but macroscopic quantities such as electrical and thermal conduction
depend on this rate. These latter quantities are of vital importance in planetary interiors. Interest in the issue of electron–ion equilibration is further motivated by the fact that short-
pulse laser systems have also been used to extend WDM experiments to the other end of the
temporal scale, for example, Lecherbourg et al. [46] will present results on the evolution of
solid copper into a warm dense matter state, in this case, after excitation with a femtosecond
laser pulse. More recently, and of particular significance to the work in this issue, the advent of
X-ray free-electron lasers has made it possible to achieve volumetric heating on sub-picosecond
timescales [45,47]. Using this property, Ren et al. 3. Timescale considerations for dynamic production of warm dense matter As noted in the previous section, when attempting to access conditions relevant to either
planetary interiors or inertial fusion efforts, the appropriate conditions can be well outside the
range accessible to static methods. Therefore, to reach conditions within the range of interest, we must turn to dynamic heating and compression methods, which can take several forms. For
example, strong shocks (more than 100 GPa) can generate matter at several times solid density
and temperatures well in excess of 104 K [33,34]. Shocks strong enough to heat a solid beyond
this temperature can be generated in a variety of ways; for example, intense lasers of nanosecond
duration have commonly been used to generate pressures ranging from 0.1 to 1 TPa [34]. Intense
lasers [35,36] or Z-pinch machines [37] can also be used to generate in the range of 100s kJ of
X-ray radiation in the sub-keV to keV photon range that can, on timescales of several
nanoseconds, volumetrically heat a sample into the WDM region. In a similar vein, intense
short-pulse lasers can be used to generate high-intensity electron [38] or proton beams [39] to
heat solid targets volumetrically on sub-nanosecond timescales. There are also longer timescale
experiments; for example, Gardelle et al. used beams of intense electrons from pulsed power
generators to generate WDM on 100 ns timescales [40]. Finally, the longest-lived experiments
utilize flyer plates driven by explosives in gas-gun experiments that create shocks on microsecond
timescales that can reach pressures in excess of 600 GPa [41]. Z-pinches can, in addition to being
used to drive X-rays, also be used to accelerate flyer plates to over 40 km s−1 using the enormous
JXB forces possible, see [42] for a review of Z-pinch HED science. 4 royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Considering the timescale of experiments raises concerns regarding the validity of
extrapolating laboratory experiments to conditions of interest, such as those encountered in a
planetary core. 4. Experimental approaches It is essential to validate our theoretical knowledge of warm dense matter through experiments,
and significant effort has been invested in producing benchmark-quality experimental results. A significant portion of the experimental effort has been dedicated to measuring transport
coefficients, including attempts to measure electrical conductivity via the reflectivity of incident
electromagnetic radiation from a solid target [50–53]. An early example was the use of a
nanosecond optical probe as a diagnostic looking at the time resolved reflectivity history of
the rear surface of a shock-compressed foil, e.g. [54] with approximately 10 ps resolution. With the development of chirped pulse amplification (CPA) laser technology in the late 1980s,
Strickland & Mourou [55] allowed for much shorter, sub-picosecond pulses. This made it possible
to conduct experiments where the reflectivity of the target is measured before any substantial
decompression from the well-defined solid density is possible. This can be done for a target
heated by the probe itself, e.g. [56] or with a pump-probe arrangement in a relatively simple
reflectivity measurement, e.g. [57]. For a more complex arrangement, such as Fourier domain
interferometry (FDI), e.g. in [58–60], the motion of the heated surface can be followed and factored
into calculations and experimental conclusions. An important issue closely related to conductivity measurements is the experimental
investigation into electron–ion equilibration in the WDM regime. However, hindered by the
opacity of WDM, there are limited experimental measurements of the electron–ion equilibration
rate in this regime, with only a few model-dependent techniques in use. Historically, the response
of the electron subsystem was measured in pump–probe experiments that used optical [57]
reflectivity, surface optical emission [44,61] or X-ray absorption spectroscopy [62,63]. Likewise,
the bulk ion temperature has only been inferred from the atomic structure, measured through
ultrafast electron [64] and X-ray diffraction [38,39,65,66]. For the case of X-ray diagnostics of
WDM, the opacity is an important quantity, and Hansen [67] presents a method of calculating
non-equilibrium opacity using a DFT-based average atom approach that can be applied across a
wide range of conditions including rapidly heated, non-equilibrium samples. g
g
p
y
q
p
The experiments of Chen et al. [57] on warm dense gold revealed an electron–ion coupling
parameter that was notably lower than predicted by classical Spitzer theory or the Fermi
Golden Rule approach, which allows for electron–ion energy exchange through normal modes
of the electron fluid interacting with normal modes of the ions. 5
royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 3. Timescale considerations for dynamic production of warm dense matter [48] discuss the application of inner shell
photoionization to creation of a solid density X-ray laser using volumetric pumping with a
free electron laser, with particular attention paid to collisional effects of photo-ejected electrons,
an issue of relevance to volumetric X-ray heating of WDM. In another paper, Hu et al. [49]
discuss electron-lattice equilibration within the context of ultra-fast X-ray diagnostics, but
again, with physics encompassing processes relevant to WDM production. The development
of the field of WDM physics means experimental and modelling techniques progress together, often hand-in-hand. However, for convenience, in the next two sections we present their
discussion separately. 6
royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . terahertz (THz) radiation, derived from intense ultra-short pulse optical interaction, e.g. [71]. The
advantage is that, for a Drude-type model of the conductivity, we can relate the ac conductivity
to the dc value via terahertz (THz) radiation, derived from intense ultra-short pulse optical interaction, e.g. [71]. The
advantage is that, for a Drude-type model of the conductivity, we can relate the ac conductivity
to the dc value via 6 royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . σ(ω) =
σDC
1 −iωτ . (4.1) (4.1) In reality, of course, there may be complicating factors such as band structure at higher
density and lower temperatures. Indeed, non-Drude like behaviour has been reported with DFT
calculations of the dynamic conductivity of warm dense Al for energy transfers large enough to
cause excitations, e.g. [72]. The significantly lower frequencies in the THz regime mean the initial measurements are closer
to the dc values pursued. Despite being complex and difficult to implement, one key advantage
of terahertz radiation is that it can penetrate into solid-density samples and, thus, is capable of
probing bulk properties of highly excited solid foil samples, e.g. [73]. A more common approach to probing bulk properties of a WDM sample has been to
use laser-produced X-rays. Some important early experiments used X-ray Thomson scattering
to extract thermodynamic quantities such as temperature and density [74]. However, careful
measurement of the plasmon spectrum encodes dynamic information on particle collisions as
well as detailed balance [75]. The use of X-ray Thomson scattering to extract thermodynamic
properties from experimental data is complex and depends on making approximations in
the various theoretical models used. Dornheim et al. [76] address this by discussing a new
approach based in use of imaginary-time correlation functions. More recently, high-resolution
X-ray radiography experiments are being developed with the aim of directly observing the effects
of transport properties, such as the thermal conductivity and particle diffusion, across interfaces
in WDM [77–80]. 4. Experimental approaches Instead, it was concluded that
coupled modes of the electrons and ions likely play an important role. The observed lower
coupling parameter is in line with earlier results from Ng et al. [61] and Celliers et al. [44],
who used temporally resolved measurement of the optical emission from the rear surface of
shock-compressed WDM to infer electron-equilibration rates. These studies also indicated that the
timescale for electron–ion coupling in strongly coupled plasma was over an order of magnitude
longer than expected from equilibrium models such as Spitzer [68] and Lee & More [69], including
degeneracy corrections [70], suggesting that equilibration could take hundreds of picoseconds. This has significant implications for shock measurements [44], which commonly use the Rankine–
Hugoniot equations to connect shock and particle speeds to density, pressure, and internal energy
density in order to assess the equation of state for WDM. These equations rely on accurate
temperature measurements to connect to internal energy, which can be affected by equilibration
effects and are often obtained using techniques such as streaked optical pyrometry as used by Ng
et al. [61] and Celliers et al. [44]. Many experiments, such as those cited above, are naturally carried out in the optical region
where ultra-short pulse pump-probe experiments down to 10 s of femtoseconds duration are
available. However, the reflectivity measured then gives access to the ac conductivity. There
is much interest in dc conductivity as this is more relevant to understanding WDM in many
situations of interest. To this end, there has been a strong development of probing capability using 5. Modelling approaches This volume includes papers that discuss various numerical simulation techniques used in
the field of WDM, and their recent advancements. As noted in the Introduction, sometimes
approaches from liquid metal theory have been used. An example used to investigate the ion–ion
structure is the use of the Ornstein–Zernicke equation and hypernetted chain closure relations
(e.g. [90]), where a given interionic potential is used to develop a self-consistent solution to
the pair-distribution function, g(r). This given potential might, for example, take the form of
a screened Coulombic potential introduced by Wünsch et al. [91]. This potential included the
addition of a stiff repulsive term to account for the overlap of bound shells as ions approach
closely, V(r) = A
r4 + Z2 e4
r
e(−r/λ),
(5.1) (5.1) where the strength of the first term is determined by the parameter A, and there is a screening
length, λ, which is typically set somewhere between the Debye and Thomas–Fermi screening
lengths, depending on the degeneracy [92]. The HNC approach is practical and fast for an
assumed steady state with a fixed, equilibrated temperature, a known interaction potential and
moderate coupling [93]. However, this is not the case for many of the cases of interest here. For
example, solids that are rapidly heated, such as those produced by FEL sources, may experience
rapid changes in temperature as the energy exchange between electrons and ions takes place. This can result in both thermal melting, in which the ions gain enough energy to reach the
melting point at the given pressure, and non-thermal melting, in which the ejection of electrons
through photo-ionization and subsequent electron–electron collisions leads to a potential surface
where the ions are no longer bound to each other. For more strongly coupled systems, it may
be necessary to use other techniques, such as ab initio molecular dynamics, to understand the
material behaviour and evolution. Molecular dynamics is an important class of simulation techniques that can model a system
of many particles with differing levels of physics assumptions made. A classical approach can be
taken, with a predetermined pairwise potential to govern the forces between ions, in a similar
way to the HNC approach above. Historically, a Coulomb [94] or screened Coulomb potential
[95] was used. However, recent more advanced potentials have been developed, including short-
range repulsion effects [91,96] and thermally damped Friedel oscillations [97]. 6
royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [89] will discuss the related issue of diffusion of ions in warm dense matter. This is important in the field of fusion
pellets, where fuel mixing is a key factor in capsule performance. 7 royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The development of new facilities such as free-electron lasers (FELs) in the extreme ultraviolet
radiation (XUV) to hard X-ray range has revitalized research on WDM, allowing for the heating
and probing of samples on ultrafast timescales of 1–100 fs, which is shorter than the inverse
phonon frequency of solids. This opens up the possibility of conducting experiments that,
rather than trying to achieve an equilibrated system, intentionally create a non-equilibrium state
with hot electrons and cold ions in order to study non-equilibrium effects [81,82]. However,
the novel sources provided by FELs not only provide an ability to isochorically heat systems
to well-defined conditions, but are also an extremely bright source for scattering, imaging,
and spectroscopic probing [83]. A particular improvement in diagnostic capability provided
by FELs is in X-ray Thomson scattering, with some key advantages over the use of X-rays
from laser plasmas. The enhanced collimation and monochromatic nature of the probe are
extremely well suited to this diagnostic and this has allowed many advances. For example,
XUV Thomson scattering experiments have been used to test electron–ion equilibration models
on ultra-short timescales [84]. At the same time, X-ray scattering experiments have been able
to probe the structure and dynamics of warm dense matter with unparalleled resolution [85],
with measurements of the damped electron plasmon wave by Sperling et al. being used to show
that the dynamic conductivity exhibits non-Drude-like behaviour that must be considered when
determining the optical properties of WDM, rather than relying on the widely used random phase
approximation [86]. In addition to transport and equilibration, another important aspect of the electronic properties
of warm dense matter is the stopping power of charged particles, which is relevant to fields
such as planetary science, geophysics and fusion research, where the stopping of alpha particles
can have a significant impact on capsule performance. Experiments on stopping power in warm
dense matter, such as those conducted by Malko et al. [87], have found deviations from classical
models for protons in warm dense carbon but have shown better agreement with first-principles
calculations using time-dependent DFT, as demonstrated by Ding et al. when applied to warm
dense Be experiments performed by Zylstra et al. [88]. This time-dependent DFT approach
showed a significant deviation from classical models when applied to alpha-particle stopping
in warm dense DT plasmas relevant to fusion capsules. Angermeier et al. 8
royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . matter at high temperatures due to the need to include a large number of excited state orbitals in
the simulation, which scales cubically with the number of orbitals. Using DFT, Mazevet et al. [106]
have studied the time evolution of a sample of gold assuming an initial, essentially instantaneous,
input of energy to the electrons. Using a combination of classical molecular dynamics with ab
initio methods, where the forces acting are derived in a time-dependent way, governed by the
configuration of the ions at a particular point in the simulation, they were able to follow the
melting of the lattice and equilibration of the ion and electron temperatures. 8 g
q
p
These ab initio methods are well suited to the study of time-dependent problems [107],
in which the electronic states and orbitals are calculated from the atomic structure, which
then determines the forces that move the particles—essentially, using DFT to calculate the
potential for MD on-the-fly. With increased computing power, such simulations have become
possible over the last decade, with DFT-MD being used to simulate the dynamic properties
of WDM with increasing levels of complexity [108–110]. Of course, there have also been
significant developments in the method. White & Collins [111] developed algorithms that
allow temperatures in the 50 eV range to be handled with acceptable computing times while
considerable effort is now being spent on the development of high-temperature exchange and
correlation functionals [112]. With the increased interest in machine learning, the accurate
interatomic potentials from DFT data can be produced for WDM, so that we can extend the study
of WDM to millions of atoms within ab initio accuracy [113,114]. Incorporating electronic quantum effects into first-principles simulations is inherently
computationally demanding, limiting their applicability to larger spatiotemporal scales and non-
equilibrium systems; one way around this is to use reduced models. Quantum hydrodynamics,
which has recently gained renewed interest [115], is able to include the important role of quantum
effects in cases where the quantum correlations are not too prominent [116] and consider the
role of quantum non-locality through the inclusion of the Bohm potential, which is neglected
in classical hydrodynamics. It is noteworthy that in certain scenarios, the Bohm force arising
from this approach can be comparable in magnitude to the forces generated by exchange
and correlation pressures, highlighting the relevance of quantum hydrodynamics in capturing
essential quantum phenomena [117]. 5. Modelling approaches These classical
simulations have been used to investigate many WDM properties such as density fluctuations
[96,98], transport coefficients [99,100] and electron–ion equilibration [101]. Molecular dynamics is
able to capture the strongly coupled nature of warm dense matter, but the quantum effects are
hidden in the details of the interatomic potential that ultimately defines the accuracy of these
simulations. In many cases, the parameters and even the form of the potential are unknown. In
such cases, we must use more advanced techniques, such as density functional theory (DFT), to
calculate the potential. As an example of the power of the molecular dynamics approach, Ling
et al. [102] used molecular dynamics to simulate the formation of warm dense Cu using the shock
compression method, and find the complicated evolution of the thermodynamic paths during the
compressing processes. p
g p
DFT is a popular simulation technique in the WDM field, with DFT molecular dynamics
(DFT-MD) a popular method of choice. The theory is based on the concept that the total energy is
a function of the electron density distribution, and by minimizing this energy, we can determine
the actual electron distribution. The Thomas–Fermi model is often seen as a precursor to DFT, but
it was Hohenberg & Kohn’s [103] and Kohn & Sham’s [104] work that truly advanced the field. For more information about DFT, see Kohanoff’s work [105]. One challenge for DFT is properly
formulating expressions for exchange and correlation energies in non-uniform systems. While
DFT is commonly used in solid-state physics, it can be computationally intensive for warm dense 9
royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . heat, momentum and mass, respectively, are a necessary input for continuum models such as
magnetohydrodynamics, and all three coefficients play a vital role in our understanding of
planetary interiors and the design and analysis of inertial confinement fusion (ICF) implosions. As highlighted at a recent workshop [126], there still exist large order-of-magnitude differences
between theoretical predictions. 9 Several authors have carried out extensive theoretical discussions of electron-ion relaxation
times for dense plasmas and warm dense matter (e.g. [127]). Dharma-Wardana & Perrot [128] used
a Fermi Golden Rule (FGR) approach to calculate the interaction between the normal modes of
independent ion and electron subsystems. Depending on whether they assumed the subsystems
to have independent spectra or a spectrum of modes generated by the interaction between the
subsystems, they found for solid density Al at temperatures in the range 3–40 eV, the coupling rate
was 4 or 5 orders of magnitude lower than calculated with the classical Spitzer–Brysk method. For systems where the ion acoustic or phonon modes interact strongly with the electrons, coupled
modes may form, further reducing the equilibration rate and forming a relaxation bottleneck
[39,128]. Gericke et al. [129] investigated the validity of ad-hoc approximations to the Coulomb
logarithm that are designed to extend a classical Landau–Spitzer approach to strongly coupled
systems where linear approximations to the electron trajectory are not valid and both close and
weak collisions are included. In highly non-equilibrium systems undergoing rapid changes, such as in laser-excited solids,
using FGR is thought to overestimate the transition rates on short time scales [130,131]. Models
based on the extension of FGR, such as those based on the Eliashberg spectral function formalism
[132], are thought to overestimate the coupling strength compared to experimental data. The most
successful descriptions take into account the varying coupling between electrons and different
phonon modes [133], with a hybrid approach based on tight-binding molecular dynamics code
showing good agreement with the limited experimental data [134]. In this volume, the issue of
electron–ion equilibration is addressed further in the paper of Ziaja et al. [135]. 8
royalsocietypublishing.org/journal/rsta Phil. Trans. R.Soc. A 381: 20220223
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A further important development has been wave packet molecular dynamics (WPMD). This time-dependent quantum mechanical method simultaneously simulates the classical point
particle motion of ions and the quantum-mechanical behaviour of electrons [118]. It has become
popular in recent years as a computationally fast way to study dynamic processes in warm
dense matter that go beyond the Born–Oppenheimer approximation [119,120]. Such non-adiabatic
techniques are necessary for describing states that are not in local thermal equilibrium (Te ̸= Ti),
in which energy can be exchanged between electrons and ions through collisions [121]. However,
recent research has indicated that these techniques may also be important for studying systems
in equilibrium [122,123]. Angermeier et al. [89] investigate the role non-adiabatic effects have in
particle diffusion in warm dense hydrogen, while Svensson et al. [124] present an extension of
the wave packet description of quantum plasmas, in which non-isotropic wave packets can be
elongated in arbitrary directions. As we have noted, molecular dynamics methods, particularly those based on DFT, have
been de-rigeur in warm dense matter modelling. However, we should not ignore the potential
contributions of other methods. For example, Filinov et al. [125] present ab initio calculations
of helium based on path integral Monte-Carlo methods that could be applied across a range of
conditions, including WDM. The evolution of the microscopic structure of warm dense matter in rapidly evolving systems
is clearly a key interest for this volume. However, the calculation of transport coefficients,
which describe the rate at which key properties are transferred throughout the plasmas,
is another important category of dynamic processes. Transport properties such as thermal
conductivity, viscosity and diffusion are some most fundamental dynamical parameters that
reflect the nature of the interatomic potential and characterize the thermodynamics of the
system. The corresponding transport coefficients, which encapsulate the rate of transfer of 6. Conclusion It is clear that warm dense matter is a field that is of wide interest for applications in fusion
sciences, geophysics and planetary sciences. It is also a vibrant field with many advances, both
in experimental techniques, computational simulation and underlying theory. These advances
are exemplified by the papers presented and the works that they build upon and reference. The
literature on warm dense matter is wide enough and varied enough that even within the scope of
this special edition, concentrating on issues of time dependence and dynamic processes, we can
see a huge variety of techniques and topics addressed. Data accessibility. This article has no additional data. Data accessibility. This article has no additional data. Authors’ contributions. D.R.: conceptualization, project administration, writing—original draft, writing—review
and editing; D.J.: writing—original draft, writing—review and editing; T.G.W.: writing—original draft,
writing—review and editing. All authors gave final approval for publication and agreed to be held accountable for the work performed
therein.l Conflict of interest declaration. This theme issue was put together by the Guest Editor team under supervision
from the journal’s Editorial staff, following the Royal Society’s ethical codes and best-practice guidelines. The Guest Editor team invited contributions and handled the review process. Individual Guest Editors were
not involved in assessing papers where they had a personal, professional or financial conflict of interest with
the authors or the research described. Independent reviewers assessed all papers. Invitation to contribute did
not guarantee inclusion. Funding. This work was supported by the National Science Foundation (grant no. PHY-2045718). This work
was funded in part by the US Department of Energy, National Nuclear Security Administration (NNSA)
(Award no. DE-NA0004039). D.J.Y. thanks the support of the Science and Technology Innovation Program
of Hunan Province China (grant no. 2021RC4026). D.R. thanks the UK Engineering and Physical Sciences
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English
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Implications of providing social support to close network members on the social well-being of older people in Kitui County, Kenya
|
Frontiers in aging
| 2,022
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cc-by
| 10,426
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TYPE Original Research
PUBLISHED 16 November 2022
DOI 10.3389/fragi.2022.897508 TYPE Original Research
PUBLISHED 16 November 2022
DOI 10.3389/fragi.2022.897508 TYPE Original Research
PUBLISHED 16 November 2022
DOI 10.3389/fragi.2022.897508 KEYWORDS social
well-being,
social
support,
instrumental
support,
emotional
support,
informational support, close network members Implications of providing social
support to close network
members on the social
well-being of older people in
Kitui County, Kenya OPEN ACCESS
EDITED BY
Joyce S. Pang,
Nanyang Technological University,
Singapore
REVIEWED BY
Pauline Thuku,
Karatina University, Kenya
Jan Oyebode,
University of Bradford, United Kingdom
*CORRESPONDENCE
Kezia Mbuthia,
mbuthiakezia@gmail.com
SPECIALTY SECTION
This article was submitted to
Interventions in Aging,
a section of the journal
Frontiers in Aging
RECEIVED 16 March 2022
ACCEPTED 24 October 2022
PUBLISHED 16 November 2022
CITATION
Mbuthia K, Mwangi S and Owino G
(2022), Implications of providing social
support to close network members on
the social well-being of older people in
Kitui County, Kenya. Front. Aging 3:897508. doi: 10 3389/fragi 2022 897508 OPEN ACCESS EDITED BY
Joyce S. Pang,
Nanyang Technological University,
Singapore REVIEWED BY
Pauline Thuku,
Karatina University, Kenya
Jan Oyebode,
University of Bradford, United Kingdom Kezia Mbuthia*, Samuel Mwangi and George Owino Department of Sociology, Gender and Development Studies, Kenyatta University, Nairobi, Kenya Social support is a strong predictor of social well-being. Older people are key
providers of social support to others, but an overemphasis on received social
support in gerontological literature portrays them as mere recipients. We
examined the association between social support provided by older people
and its influence on their social well-being. Data were collected from
369 respondents residing in rural dwellings in Kitui County using mixed
methods and were analyzed for association using chi-square statistics. Instrumental, emotional, and information support provision was determined
by asking four questions in each category about whether the respondents
provided social support to close network members. The subjective experience
of support provision using a satisfaction question for each domain of social
support was used to determine the influence of providing social support on the
social well-being of older people. Provision of social support across the three
domains was found to be significantly associated with social well-being. The
level of statistical significance was highest for emotional and information
support compared to instrumental support. Older people are important
providers of social support. The majority of those who provided social
support
reported
being
satisfied. Therefore,
offering
social
support,
especially emotional and information support, is an important contributor to
satisfaction with these aspects of social well-being. COPYRIGHT
© 2022 Mbuthia, Mwangi and Owino. 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. social
well-being,
social
support,
instrumental
support,
emotional
support,
informational support, close network members (Elliott, 2017). In addition, studies on social support infrequently examine
whether helping others has any benefits. According to Brown
et al. (2003), the effects are frequently attributed to receiving
support or occasionally to reciprocal support. Even if some social
support measures actually appear to examine giving, they do so
possibly inadvertently. Therefore, the study aims to investigate
the
effects
of
providing
instrumental,
emotional,
and
informational social support to others on the social well-being
of the providers (i.e., older people). According to Dunaeva (2018), social support is a significant
element of social well-being. It relates to social exchanges that are
regarded
as
beneficial
(Dykstra,
2015),
such
as
advice,
encouragement, and love (Thoits, 2011). These supportive
qualities sustain social relationships (Umberson & Montez,
2010). From this definition, similar to Schwarzer, Knoll, and
Rieckmann (2004), social support is investigated in several types
such as emotional (i.e., love), informational (i.e., giving advice),
and instrumental (i.e., assistance with a problem). It serves as a
potential resource to individuals and is associated with well-being
benefits, especially among older adults (Mohd et al., 2019). 2 Materials and methods Social support is described as either perceived or received
support. Received social support refers to the actual reported
exchanges, while perceived social support refers to potential
access to support by an individual (Haber et al., 2007;
Uchino, 2009). The multidimensionality of social support has
led to some of its aspects being given more attention than others,
which may be attributed to the lack of a standardized method of
measuring and reporting the multiple dimensions (Mohd et al.,
2019). Specifically, perceived social support has attracted wider
scholarly attention than received social support (Mohd et al.,
2019). 1 Introduction Social well-being can be defined as a person’s evaluation of their situation, social
relations, and how well they can function in a community (Dunaeva, 2018). This
construct has been classified into two dimensions: subjective experiences and objective
circumstances (Boreham, Povey, & Tomaszewski, 2013). Objective circumstances relate to
support that is apparent, tangible, and direct (Yu et al., 2020). Frontiers in Aging 01 frontiersin.org Mbuthia et al. 10.3389/fragi.2022.897508 Mbuthia et al. Subjective experiences reflect a person’s internal thoughts
and feelings that are not visible to outside observers (Lucas,
2018). A singular objective life situation or circumstance can be
evaluated
differently
by
different
people. The
subjective
experiences are evaluations of life in terms of satisfaction
(Keyes,
Shmotkin,
&
Ryff,
2002)
based
on
objective
circumstances. According to Sen, Prybutok, and Prybutok
(2021) social well-being involves both external and internal
factors, such as the presence of exchanges or connections and
satisfaction with the quality of those exchanges or connections. Therefore, in the context of this study, social well-being refers to
satisfaction with the quality of social support provided. However,
although the social well-being of older people is a topic that has
drawn scholarly consideration in high-income nations, it has
received little attention in low- and middle-income countries
(Elliott, 2017). people who did not. Other studies however, found that providing
social support elicits feelings of burden and frustration (Thomas,
2010; Morelli et al., 2015) and is overwhelming (Krause & Shaw,
2002) for older people. The mixed and contradictory findings call
for more research to understand the influence of providing social
support on well-being. Numerous crucial aspects of social
support provision and its effects on social well-being have not
been systematically documented (Thomas, 2010). This is because
prior works focus on two categories of social support,
instrumental, and emotional, with each category affecting
support providers differently (Morelli et al., 2015). Therefore,
this study focused on three categories of social support,
instrumental, emotional, and information, to assess their effect
on support providers because the findings in prior studies are
mixed and contradictory. people who did not. Other studies however, found that providing
social support elicits feelings of burden and frustration (Thomas,
2010; Morelli et al., 2015) and is overwhelming (Krause & Shaw,
2002) for older people. The mixed and contradictory findings call
for more research to understand the influence of providing social
support on well-being. 1 Introduction Numerous crucial aspects of social
support provision and its effects on social well-being have not
been systematically documented (Thomas, 2010). This is because
prior works focus on two categories of social support,
instrumental, and emotional, with each category affecting
support providers differently (Morelli et al., 2015). Therefore,
this study focused on three categories of social support,
instrumental, emotional, and information, to assess their effect
on support providers because the findings in prior studies are
mixed and contradictory. 2.1 Participants A cross-sectional survey design was employed to collect
primary
data
in
June
and
July
2021. The
total
study
population of older people in four sampled sub-counties of
Kitui County was 32,839, according to the Kenya National
Bureau of Statistics (KNBS, 2019). Using Yamane’s (1967)
formula at a 95% confidence level and p = 0.05, a sample of
396 older people was determined. Multistage cluster sampling
was employed, and participants within each of the clusters were
selected using a simple random sampling technique. Received social support is said to be provided and received in
social relationships. Received social support has attracted wide
theoretical and empirical attention, unlike provided social
support (Song, Son, and Lin, 2011). This is especially the case
when investigating social support among older people. Received
social support has been found useful in providing protection
against threats to well-being and in helping people to deal with
setbacks that are detrimental to social well-being (Dykstra, 2015). The overemphasis in gerontological literature of social support
received by older people portrays them as mere recipients and
disregards their role as its providers (Albertini, Kohli, & Vogel,
2007; Dykstra, 2015). Some studies found that provided social
support was beneficial to providers. For instance, Bai et al. (2020)
observed that providing social support enhanced well-being in
older people who took care of their grandchildren compared to In the first stage, administrative locations in the four sub-
counties were identified. In each sub-county, three locations were
selected using the fish bowl draw method (Kumar, 2011). In the
second stage, two sub-locations were purposively identified from
the selected locations, and a sampling frame (i.e., list of older
people living within the sub-locations) was obtained from the
area administrative chiefs and village elders. These steps were
necessary
because
the
population
was
geographically
heterogeneous (Sedgwick, 2015), and the sampling units for
the different stages were different (Acharya et al., 2013). The
names of older people provided by the area chiefs and village
elders were entered into Microsoft Excel 2016. Older people in
the sampled sub-locations were randomly selected using the
Rand function. Frontiers in Aging frontiersin.org 02 10.3389/fragi.2022.897508 10.3389/fragi.2022.897508 Mbuthia et al. Mbuthia et al. 2.4 Data analysis Statistical Package for the Social Sciences (SPSS) Version
21.0 was used to analyze the data. The chi-square test of
association was used to establish the association between the
independent and the dependent variables. For each item in
the provision of social support questionnaire, the relationship
to overall satisfaction in that domain of social support was
assessed to establish whether it was significant. Because the
sample sizes were small, Fisher’s exact test was preferred
because it does not depend on any large-sample asymptotic
distribution assumptions (Field & Miles, 2010). The results
were reported with a 95% confidence interval (CI) and a p-
value of 0.05. Descriptive statistics (frequencies, percentages)
were also calculated and are presented in tables. Thematic
analysis was used to analyze qualitative data. Thematic
analysis is a technique for assessing qualitative data that
involves looking through data to find, examine, and report
recurring themes (Viji & Benedict, 2014). Kiger and Varpio
(2020) contend that rather than choosing an easy-to-use
method of analysis, deciding to utilize thematic analysis
should be based on the objectives of the study itself. In 2.2.2 Dependent variable: Social well-being The study measured social well-being by assessing how the
respondents appraised the social support that they provided
using a satisfaction question for each domain. The satisfaction
question was indexed by responses (from 1 = extremely
dissatisfied, 2 = dissatisfied, 3 = satisfied, and 4 = extremely
satisfied) to the following statement: In the last 12 months, how
satisfied are you with the social support that you provided to
CNMs? The responses were highly skewed because very few
respondents
selected
extremely
dissatisfied
and
extremely
satisfied in all the three domains of social support. At the
analysis stage, responses for 1 and 2 were collapsed from the
original scale to 1 = dissatisfied and those for 3 and 4 were
collapsed to 2 = satisfied, creating a dichotomized scale. The
research instrument was pre-tested with 30 respondents from a
subsample of older people, and a post-test was conducted 3 weeks
later. The test–retest reliability was reasonably measured by the
correlation coefficient between the two scores, which was 0.718. 2.2.1 Independent variable: Social support 2.2.1 Independent variable: Social support Participants were asked to report whether they provided
social support to close network members (CNMs), including
spouse, children, neighbors, relatives, and friends, in the
12 months prior to the study. The study measured three
domains of social support with four items in each: (a)
instrumental social support—lend or give money, help with
chores/errands, provided care in sickness, lent items or tools;
(b) emotional support—cheered up or helped CNM feel better,
showed interest in their personal life, did or said things that were
kind or considerate, trusted CNM to solve their problems; (c)
informational support—gave helpful advice when needed to
make important decision, agreed with CNM’s actions or
thoughts, gave information to understand an issue, and gave
CNM feedback on an action they wanted to take. These items in
the three domains of social support were drawn from the
previous studies (Morelli, Lee, Arnn & Zaki, 2015; Schwarzer
et al., 2004; Thoits, 2011; Newsom et al., 2005). Participant’s
response categories were: Yes [1] or No [2]. 2.2 Measures With the assistance of local leaders, a distinct group of
older people from those who filled out the questionnaire were
purposively sampled. This was done in order to include
individuals who could stimulate one another to explore
their opinions and experiences in light of the discussion
topics
(Moser
&
Korstjens,
2018). The
focus
group
discussion themes were developed using the quantitative
data
from
the
questionnaire. For
instance,
the
questionnaire was divided into five sections: demographic
characteristics of the respondents, instrumental, emotional,
and
informational
social
support,
and
each
domain’s
satisfaction
questions. These
social
support
domain
questions served as the basis for the focus group template
and were the starting point for addressing the crucial
concerns of “why” and “how” providing social support
influenced satisfaction with doing so. This was done in
order to understand the reasoning underlying participants’
feelings and actions. The discussants were welcomed to the
FGD, and the purpose of the study was explained to them. The researcher moderated the focus group discussions as the
research assistants allocated each participant’s initials and
transcribed the discussion of each participant verbatim. The
socio-demographic questions for the focus group participants
included
gender,
marital
status,
level
of
education
attainment, and their source of livelihood (multi-response
question). Because demographic characteristics can influence
sharing within the group discussion, homogeneity with
sufficient variation among members to support opposing
viewpoints was established. Frontiers in Aging 2.3 Procedure A questionnaire was developed for this study that covered the
three domains of social support and the social well-being question, as
well as socio-demographic questions, including gender, participant’s
marital status, level of education attainment, source of livelihood, and
average monthly income. These were administered by the researcher
and the research assistants at the respondent’s homes. Four focus
group discussions comprising older men and women were also
conducted,
and
the
data
were
used
to
complement
the
quantitative data from the questionnaire. Frontiers in Aging frontiersin.org 03 Mbuthia et al. 10.3389/fragi.2022.897508 TABLE 1 Demographic profiles of FGD participants. Variable
Categories
n = 369
(%)
n = 32
(%)
Gender
Male
162
40.9
14
47.3
Female
234
59.1
18
56.3
Age
60–69
200
50.5
18
56.3
70–79
110
27.8
12
37.5
80–89
63
15.9
1
3.1
90–99
17
4.3
1
3.1
100+
6
1.5
0
0
Marital Status
Single
13
3.3
2
6.3
Married
228
57.6
15
46.9
Divorced
3
0.8
1
3.1
Separated
9
2.3
1
3.1
Widowed
143
36.1
13
40.6
Level of education
No formal education
142
35.9
6
18.7
Primary
165
41.7
14
43.8
Secondary
74
18.7
7
21.9
Tertiary
15
3.8
5
15.6
Source of livelihood
Farming
284
57.8%
25
43.9
Older persons cash transfer (OPCT)
94
19.1%
6
10.5
Pension
31
6.3%
0
0
Business
31
6.3%
0
0
Casual labor
25
5.1%
2
8.8
Support by children and other kin
18
3.7%
22
38.6
Employment
8
1.6%
2
8.8
Average monthly Ksh
Below 1,000
99
25.0
1,001–5,000
227
57.3
5,001–10,000
25
6.3
Over 10,000
45
11.4 qualitative studies and, in this case, demonstrated that the
emerging themes were trustworthy and plausible. this case, the focus group template of the key themes that were
developed from the questionnaire study was checked against
the focus group transcripts. Verbatim quotations from each
of the domains were chosen to complement the quantitative
data that represented the main themes that emerged from the
discussions. To ascertain the trustworthiness of FGDs, the
researcher
and
two
research
assistants
engaged
in
transcribing and categorizing the themes. Final agreement
on the themes for each domain of social support was reached
by comparing the three researchers’ independently drawn
theme categories. The data were triangulated across the four
sub-counties by conducting one FGD in each. 2.3 Procedure Verbatim
quotations that were selected also reflected the main
themes that emerged from the discussions, and there was
prolonged engagement with the transcriptions as well as
persistent observation of the emerging themes. According
to Guion et al. (2011) triangulation establishes the validity of 3.1 Socio-demographic characteristics Most
respondents
were
women
(234,
59.1%)
aged
between 60 and 69 years. Of which, 200 (50.5%) were
married, 228 (57.6%) had attained a primary level of
education,
165
(41.7%)
earned
their
livelihood
from
farming, and 224 (57.8) reported on average a monthly
income of KES 1,001–5,000 (equivalent to USD 10–50). Results are shown in Table 1. I left my watchman job to attend to my sick brother because
he is my brother and needed me (AG, 67-year-old male FGD
participant). I help in cleaning utensils because I can’t help in the farms and
am happy am useful (JK, 84-year-old female). I feel good when I help the people close to me with chairs and
sufurias (cooking pot) because I know that they have sought
my assistance since they don’t have the items (FK, 75-year-old
female participant). 3 Results My son was having a very hard time paying rent in Nairobi
because of COVID-19 lockdown, I gave my son money to rent
a cheaper house and felt good helping him (JM, 68-year-old
male FGD participant). My son was having a very hard time paying rent in Nairobi
because of COVID-19 lockdown, I gave my son money to rent
a cheaper house and felt good helping him (JM, 68-year-old
male FGD participant). I left my watchman job to attend to my sick brother because
he is my brother and needed me (AG, 67-year-old male FGD
participant). 3.2.1 Instrumental social support The results show that most respondents provided social
support within each item of instrumental social support and
were satisfied with the social support they provided to CNMs. The calculated chi-square statistic using Fisher’s exact test
demonstrated
a
highly
statistically
significant
association between the instrumental social support variables
and satisfaction with provided social support [satisfaction with
lending money (χ2 = 6.05, p < 0.014)], satisfaction with providing
care to CNM in sickness (χ2 = 13.26, p < 0.000), satisfaction with
providing help with chores/errands (χ2 = 4.48, p < 0.034),
satisfaction with lending household tools/items (χ2 = 8.90 p <
0.017)]. Results are shown in Table 2. 2.5 Ethical approval Study participants were informed about the study by the
researcher and research assistants. They were notified about the
procedure to be followed and that participation was voluntary. They were made aware that they could withdraw from the study
at any time and that the information they provided would be used
for academic purposes only. The approval to conduct the study
was granted by the Kenyatta University Graduate School and
permitted by the Kenyatta University Ethical Review Committee
(KU-ERC), approval number PKU/2235/11379, and the National
Commission
for
Science,
Technology,
and
Innovation
(NACOSTI) [Permit number NACOSTI/P/21/11012. Frontiers in Aging frontiersin.org 04 10.3389/fragi.2022.897508 Mbuthia et al. TABLE 2 Cross-tabulation of social support provided and satisfaction with provided instrumental support. Support provided
Responses
Satisfaction with provided instrumental
support
χ2
df
p-value
Satisfied %
Dissatisfied %
Lend money
Yes
281 (97.2%)
74.1%
8 (2.8%)
47.1%
6.05
1
0.014 e*
No
98 (91.6%)
25.9%
9 (8.4%)
52.9%
Care in sickness
Yes
325 (97.3%)
85.8%
9 (2.7%)
52.9%
13.26
1
0.000 e*
No
54 (87.1%)
14.7%
8 (12.9%)
47.1%
Help with household chores
Yes
287 (97.0%)
74.7%
9 (3.0%)
52.9%
4.475
1
0.034 e*
No
92 (92.0%)
25.3%
8 (8.0%)
47.1%
Lending household items/tools
Yes
358 (96.5%)
94.5%
13 (3.5%)
76.5%
8.901
1
0.017 e*
No
21 (84.0%)
5.5%
4 (16.0%)
23.5%
*Fisher’s exact test. 3.2 Social support and social well-being These narratives demonstrate that older people felt useful
when they were able to meet the needs of their CNM. Therefore,
providing instrumental support was satisfying for older people
because it elicited a feeling of being useful to others when they
met the needs of CNMs. 3.2.2 Emotional social support The results show that most respondents provided social
support within each item of emotional social support and
were satisfied with the social support that they provided to
CNMs. The
smallest
expected
frequency
in
the
cross-
tabulation Table 3 did not exceed 5, and the assumption of
chi-square was not met. Fisher’s exact test (Fisher, 1922) was
used to compute the exact probability of the chi-square statistic. The association between emotional support provided and
satisfaction with providing emotional support was highly
significant [satisfied cheering up or helping CNM feel better
(χ2 = 32.54, p < 0.000), satisfaction with showing interest in their
personal life (χ2 = 40.44, p < 0.000), satisfied doing or saying
things that were kind or considerate (χ2 = 67.41, p < 0.000),
satisfied helping CNM to solve their problems (χ2 = 41.83, p <
0.000)]. The results are shown in Table 3. Participants in the focus group discussions agreed with
the study findings that providing instrumental social support
is pervasive among older people. They further alluded that
providing for others is a good thing to do as long as it is within
a
person’s
ability
to
provide. The
narrative
below
demonstrates
that
providing
instrumental
social
support elicited positive effects on older peoples’ social
well-being: Frontiers in Aging frontiersin.org 05 10.3389/fragi.2022.897508 Mbuthia et al. TABLE 3 Cross-tabulation of social support provided and satisfaction with provided emotional support. Support provided
Responses
Satisfaction with provided emotional
support
χ2
df
p-value
Satisfied %
Dissatisfied %
Cheered CNM up
Yes
351 (97.8%)
92.4
8 (2.2%)
50.0
32.538
1
0.000*
No
29 (78.4%)
76
8 (21.6%)
50.0
Showed interest in the personal life of CNM
Yes
350 (98.0%)
92.1
7 (2.0%)
43.8
40.435
1
0.000*
No
30 (76.9%)
7.9
9 (23.1%)
56.3
Did or said things that were kind or considerate
Yes
374 (97.4%)
98.4
10 (2.6%)
62.5
67.419
1
0.000*
No
6 (50.0%)
1.6
6 (50.0%)
37.5
Trusted CNM to solve your problems
Yes
351 (98.0%)
92.4
7 (2.0%)
43.8
41.834
1
0.000*
No
29 (96.0%)
7.6
9 (4.0%)
56.3
*Fisher’s exact test. These findings were congruent with the voices of the FGD
participants, who reported that they provide emotional support to
CNMs and found fulfillment in this activity. This support is meant to
help CNMs when they face difficulties, as explained in the excerpts
below: they succeed at it, their own social well-being is enhanced. 3.2.2 Emotional social support Providing the emotional support was satisfying to older
people
by
increasing
their
social
connection
and
engagement with CNMs. 3.2.3 Informational social support Anytime they
are unhappy, it affects me too (MK, a 70-year-old male
participant). The voices of FGD participants echo the findings from the
quantitative analysis. I am always happy when my children are happy. I celebrate
when they celebrate and thank God for them. Anytime they
are unhappy, it affects me too (MK, a 70-year-old male
participant). My neighbor has been quarreling with his neighbor about a land
boundary and wanted to take the case to court. I suggested to him
that the case could be solved at the clan level instead of going to
court. He heard my advice, and the issue has been resolved (AW,
72-year-old male participant). I am the treasurer of the community self-help group. My son
needed some money and borrowed it from me. I took some
from the kitty and trusted my son to refund on time before our
next community meeting. He refunded the money on time,
and I was very proud of him for being worthy of my trust (AN,
a 68-year-old male participant). y
My neighbor had kept a man (co-habit), and the neighbors
thought her actions were not right and the man was not good
for her, but I saw him as a good man and agreed with her
action to stay with him. I told her that she made a good
decision to think about herself for once since her husband
died ten years ago (AM, 72-year-old female participant). My grandchildren have been raised in Nairobi and do not
understand much about our Kamba culture. I explained to
them when they visited about our clan, which is called
Anzauni, about how hardworking we are. It felt good My neighbor had kept a man (co-habit), and the neighbors
thought her actions were not right and the man was not good
for her, but I saw him as a good man and agreed with her
action to stay with him. I told her that she made a good
decision to think about herself for once since her husband
died ten years ago (AM, 72-year-old female participant). These voices from the FGD participants imply that older
people go to great lengths to provide help to their CNMs. This
is because when CNMs are facing challenges, the older people
are also affected and thus put effort in helping the CNM return
to a state that can potentially elicit positive emotions. 3.2.3 Informational social support Out of my four children, three of them lost their jobs due to the
COVID-19 pandemic. I cheered them up by reminding them of
where we have come from and also sent them maize and beans
from the village and encouraged them not to give up despite the
hard times they were going through (DK, a 73-year-old FGD male
participant). The association between information support and satisfaction
with providing information support was computed. The expected
frequencies in the distribution were not greater than 5, and so the chi-
square assumption was not met. Fisher’s exact test, a precise test that is
suitable when samples sizes are small (Kim, 2017), was used, and it
demonstrated significance between the variables giving helpful advice
when needed to make important decision and satisfaction providing
information support (χ2 = 73.02, p < 0.000), satisfaction agreeing with
CNM’s actions or thoughts (χ2 = 41.55, p < 0.000), gave information
to understand an issue (χ2 = 49.62, p < 0.000), gave CNM feedback on
an action they wanted to take (χ2 = 39.01, p < 0.000). Results are
shown in Table 4. My son had a fight with the wife and chased her away. She
went to town and got a house for herself and the kids. I knew
my son was in the wrong, and he was not listening to my
scolding. I left my son in the village and joined his estranged
wife in town and stayed with her until my son accepted her
back. She is a good wife and exactly the person my son needs. I
feel good they reconciled even if it compelled me to intrude in
their personal lives (SK, a 69-year-old female participant). My son had a fight with the wife and chased her away. She
went to town and got a house for herself and the kids. I knew
my son was in the wrong, and he was not listening to my
scolding. I left my son in the village and joined his estranged
wife in town and stayed with her until my son accepted her
back. She is a good wife and exactly the person my son needs. I
feel good they reconciled even if it compelled me to intrude in
their personal lives (SK, a 69-year-old female participant). I am always happy when my children are happy. I celebrate
when they celebrate and thank God for them. Frontiers in Aging 3.2.3 Informational social support My brother came to me because he had problems paying
school fees for his son who was to join university. He wanted
to sell a portion of land that our parents left me to oversee. I
did not have a response at the time and told him I would think
about it. A few days later, I went to him and gave him reasons
why we cannot sell that portion of land. I suggested ways we
could raise the money without having to dispose our only
prime land. My brother’s patience and willingness to look for
alternative ways showed how much he respects me (SM, a 68-
year-old male participant). My brother came to me because he had problems paying
school fees for his son who was to join university. He wanted
to sell a portion of land that our parents left me to oversee. I
did not have a response at the time and told him I would think
about it. A few days later, I went to him and gave him reasons
why we cannot sell that portion of land. I suggested ways we
could raise the money without having to dispose our only
prime land. My brother’s patience and willingness to look for
alternative ways showed how much he respects me (SM, a 68-
year-old male participant). Generally,
the
significant
finding
that
providing
instrumental social support was associated with social
well-being has been observed in the literature. Providing
instrumental social support was associated with higher
levels of life satisfaction (Brown et al., 2003) and greater
life satisfaction among rural Taiwanese older adults (Ku et al.,
2013). However, in a study on contributory behaviors and life
satisfaction among older Chinese adults, Liu et al. (2019)
found that providing instrumental social support and
satisfaction with life were unrelated. From these voices, it is evident that an effect is generated that has
the potential to influence social well-being. Older people weigh in on
whether the information support they provide will benefit the CNM. In addition, they feel knowledgeable and needed by the CNM, which
benefits their social well-being. Actively offering information support
to others was satisfying to older people by making them part and
parcel of the decision-making process. Additionally, it increased
communication between them and their CNM and gave them a
sense of resourcefulness. 3.2.3 Informational social support In
the
first
instrumental
item
[lending
money
and
satisfaction with lending money (χ2 = 6.05, p < 0.014)],
satisfaction was attributed to the feelings of being useful to
CNMs and the assurance of support during their own time of
need. Older people have support expectations from children and
grandchildren that they assist, in the present or past, in their time
of need (Schatz & Ogunmefun, 2007; Chitaka, 2017). These
findings resonate with a longitudinal study in Ireland that
found that older people who provide financial support to
CNMs have the highest quality of life compared to those who
only receive support (Ward & McGarrigle, 2017). Similarly,
spending money on others was found to produce positive
effect in a study of 136 higher- and lower-income countries
around the world (Aknin et al., 2013). The results are reinforced In summary, the study shows that each dimension of social
support provided by older people was significantly associated
with social well-being, as measured by participants’ reports of
their satisfaction with that dimension. However, emotional and
informational
support
had
a
stronger
association
than
instrumental support. 3.2.3 Informational social support When My grandchildren have been raised in Nairobi and do not
understand much about our Kamba culture. I explained to
them when they visited about our clan, which is called
Anzauni, about how hardworking we are. It felt good Frontiers in Aging frontiersin.org 06 10.3389/fragi.2022.897508 Mbuthia et al. TABLE 4 Cross-tabulation of social support provided and satisfaction with provided informational support. Support provided
Responses
Satisfaction with provided
informational support
χ2
df
p-value
Satisfied %
Dissatisfied %
Offered helpful advice
Yes
358 (98.9%)
93.7
4 (1.1%)
28.6
73.025
1
0.000*
No
24 (70.6%)
6.3
10 (29.4)
71.4
Agreed with CNM’s thoughts/actions
Yes
347 (98.6%)
90.8
5 (1.4%)
35.7
41.549
1
0.000*
No
35 (79.5%)
9.2
9 (20.5%)
64.3
Gave CNM information to understand an issue
Yes
337 (99.1%)
88.2
3 (0.9%)
21.4
49.620
1
0.000*
No
45 (80.4%)
11.8
11 (19.6%)
78.6
Gave CNM feedback on an action they wanted to take
Yes
326 (99.1%)
85.6
3 (0.9%)
21.4
39.013
1
0.000*
No
56 (83.6%)
14.4
11 (16.4%)
78.6
*Fisher’s exact test. sharing about our clan with the children (AP 78-year-old male
participant). sharing about our clan with the children (AP 78-year-old male
participant). instrumental,
informational,
and
emotional. Instrumental
support includes doing things for others (DeHoff et al., 2016)
and offering goods and services (Shakespeare-Finch & Obst,
2011;
Southwick
et
al.,
2016)
that
help
solve
practical
problems. Generally, a statistically significant (p < 0.05)
association
was
reported
between
providing
instrumental
social support and social well-being. The high significance
recorded between the two variables could be attributed to the
expectation that such support will be availed to older people at a
later date. According to Chitaka (2017), the expectations of older
people that the support they offered can be reciprocated when
they need it is satisfying. instrumental,
informational,
and
emotional. Instrumental
support includes doing things for others (DeHoff et al., 2016)
and offering goods and services (Shakespeare-Finch & Obst,
2011;
Southwick
et
al.,
2016)
that
help
solve
practical
problems. Generally, a statistically significant (p < 0.05)
association
was
reported
between
providing
instrumental
social support and social well-being. The high significance
recorded between the two variables could be attributed to the
expectation that such support will be availed to older people at a
later date. According to Chitaka (2017), the expectations of older
people that the support they offered can be reciprocated when
they need it is satisfying. 4.1 Instrumental social support Social support is a multi-dimensional construct that Schaefer,
Coyne, and Lazarus (1981) classified into three types: tangible/ Frontiers in Aging frontiersin.org 07 10.3389/fragi.2022.897508 Mbuthia et al. emotional support (χ2 = 32.54, p < 0.000)] was statistically
significant. This was associated to the bond shared between
the older person and a CNM as well as the opportunity to
share knowledge and experience gained over the years. This
finding agrees with Carstensen, Freedman, and Larson
(2016), who noted that older people are in a unique
position to serve as supporters and guides to CNMs, which
allows the older people to experience fulfillment and purpose
in their own lives. Similarly, in Kenya, Kimamo and Kariuki
(2018) noted that older people often cheer their CNM,
especially during major public holidays, by slaughtering
chicken, sheep, or goats and joyfully sharing a meal with
CNMs, while at the same time enquiring how each is doing in
their personal lives. It is satisfying for older people when their
CNMs are all cheerful in their home. emotional support (χ2 = 32.54, p < 0.000)] was statistically
significant. This was associated to the bond shared between
the older person and a CNM as well as the opportunity to
share knowledge and experience gained over the years. This
finding agrees with Carstensen, Freedman, and Larson
(2016), who noted that older people are in a unique
position to serve as supporters and guides to CNMs, which
allows the older people to experience fulfillment and purpose
in their own lives. Similarly, in Kenya, Kimamo and Kariuki
(2018) noted that older people often cheer their CNM,
especially during major public holidays, by slaughtering
chicken, sheep, or goats and joyfully sharing a meal with
CNMs, while at the same time enquiring how each is doing in
their personal lives. It is satisfying for older people when their
CNMs are all cheerful in their home. by the Blau (1968) tenet, which stresses that social actors engage
in activities that entail some costs in order to obtain desired goals. The second instrumental item [providing care in sickness
and satisfaction with providing instrumental support (χ2 =
13.26, p < 0.000)] was also significantly associated with
satisfaction
with
providing
instrumental
support. This
significant finding was attributed to the unintentionality of
being/getting sick as well as how providing care perpetuates
life and survival. 4.1 Instrumental social support According to Inagaki and Orehek (2017),
caring for others is not only a good thing to do but also
necessary for the survival of our species. However, these
findings are not in agreement with Milne et al. (2014), who
found that the likelihood of older people providing care to sick
CNMs is high and has the potential to affect their own health
problems, which is not satisfying. The significant results reported on the third item of the
instrumental social support investigated [helping with household
chores and satisfaction with providing instrumental support (χ2 =
4.48, p < 0.034)] could be attributed to this activity’s effect of
keeping older people busy and active. According to Koblinsky
et al. (2021), household chores may include activities such as
cleaning, tidying, dusting, and home maintenance work like yard
work and home repairs. These household chores keep older
people active and serve as a low-risk form of beneficial
exercise (Ward & McGarrigle, 2017; Koblinsky et al., 2021). The findings of this study corroborate those of Adjei and
Brand (2018), which showed that older people were satisfied
with their household chores because those chores were beneficial
to their health and well-being. Similarly, the study by Crisp and
Robinson (2010) also revealed that household chores enhanced
the social well-being of older people and their relationships
with CNMs. Showing interest in the personal life of a CNM was also
significantly
associated
with
satisfaction
with
providing
emotional social support (χ2 = 40.44, p < 0.000). This
significant association was attributed to CNMs and their
personal concerns being important to older people. According
to Vaillant (2012), older people are motivated to get involved in
the lives of others. This interest in others is not limited to some
aspects of life but to their great desire to lavish time, affection,
information, concern, and resources on others (Erikson, 1994). These results resonate with Amati et al.’s (2018) findings that
older people provide CNMs with emotional support by showing
interest in their personal or family matters, serving as a resource
pool to help the CNMs solve their problems. The older people
find it satisfying to be part of the solution. 4.1 Instrumental social support The findings of this
study, however, do not concur with those of Juma, Okeyo, and
Kidenda (2004), which showed that showing interest in the life of
CNMs, especially those under their care like orphans, cause older
people to worry about who will continue providing care when
they die. The significant (p < 0.05) association between lending
household
tools/items
and
satisfaction
with
providing
instrumental support (χ2 = 8.90 p < 0.017) was attributed to
the feelings of being helpful to others and meeting a need. Lending household tools/items is a form of assistance in social
relationships that serves as a resource to meet needs (Amurwon
et al., 2017). This study’s results conform with Wang et al.’s
(2019) findings that social reciprocity takes place in close
relationships like those of CNMs and older people, which
eases access to what one has and the other does not in a time
of need and is thus beneficial to social well-being. The third emotional item was doing or saying things that
were kind or considerate and was significantly associated with
satisfaction with providing emotional social support (χ2 = 67.41,
p < 0.000). This significance was attributed to the effect
generated when something kind was said to a CNM. When
the CNM experienced pleasant emotions because of something
the older person said or did, it resultantly produced a positive
effect in that older person. Oerlemans, Bakker, and Veenhoven
(2011) stated that people experience happiness as a pleasant and
somewhat stimulated emotional state in their daily lives. The
kind and considerate words or deeds from older people to
CNMs
stimulated
pleasant
emotions,
which
elicited
satisfaction. These results are in agreement with a South
African study by Makiwane (2010)thatshowed that older
people are great encouragers who are happy about the
success of the CNM and also offer support when things are
not working well. Because the older people have amassed much
experience, they can offer hope and wisdom to calm or
cheer CNMs. 4.2 Emotional social support Emotional
social
support
includes
being
there
for
someone (DeHoff et al., 2016) and behavior that fosters
and expresses feelings of being loved, respected, and cared
for (Shakespeare-Finch & Obst, 2011; Southwick et al., 2016). The first item of emotional social support [cheering up or
helping a CNM feel better and satisfaction with providing Frontiers in Aging frontiersin.org 08 Mbuthia et al. Mbuthia et al. 10.3389/fragi.2022.897508 10.3389/fragi.2022.897508 Lastly,
trusting
CNMs
to
solve
their
problems
was
significantly associated with satisfaction with providing social
support (χ2 = 41.83, p < 0.000). This was attributed to the
presence of a trustworthy person and the relief obtained when
a viable solution to an issue was provided by a CNM. The
presence of trustworthy CNMs whom older people can
confide in provides access to social support that addresses
older
people’s
problems
(Storchi,
2017). These
findings
contradict those of the Clough et al. (2007) study, which
reported that older people expressed concerns about not
knowing whom to trust to solve their problems. He argued
that older people fear for their safety and lives when they do
not know or have someone they can trust to solve their problems. They face challenges and lack trustworthy people, which creates
worry that affects their social well-being. desire to give back. These findings are in tandem with the
Michel et al. (2019) study on the roles of a grandmother in
African societies. They concluded that older people provide
invaluable and unbiased advice to every family member and are
satisfied taking the role of an adviser. Similarly, Crisp and
Robinson (2010) found that older people offer useful advice to
CNMs, especially on navigating life’s challenges, which makes
them feel good about themselves. desire to give back. These findings are in tandem with the
Michel et al. (2019) study on the roles of a grandmother in
African societies. They concluded that older people provide
invaluable and unbiased advice to every family member and are
satisfied taking the role of an adviser. Similarly, Crisp and
Robinson (2010) found that older people offer useful advice to
CNMs, especially on navigating life’s challenges, which makes
them feel good about themselves. In the second item, agreeing with a CNM’s actions or
thoughts was significantly associated with satisfaction with
providing emotional support (χ2 = 41.55, p < 0.000). The
significant result was associated with the CNM’s decision to
act according to the older person’s expectations. 4.2 Emotional social support Otherwise, the
older people could not accept the CNM’s decision to act contrary
to what they had strongly suggested and advised. According to
Noftle and Fleeson (2010), older adults are significantly more
agreeable than younger adults. This is because they possess
higher emotional control (Charles & Carstensen, 2007) to
weigh the costs and benefits of the thoughts or actions
presented to them, which corroborates this study. Generally, the highly significant association observed in the
four items of emotional support and social well-being was
attributed to the presence of CNMs who have a close
relationship with the older person. According to Thomas, Liu,
and Umberson (2017), closeness in family ties, particularly to
adult children who give social support to their elderly parents,
can have a major impact on well-being. Providing emotional
support has been reported in prior studies as significantly
associated with reduced stress (Shakespeare-Finch, & Obst,
2011), beneficial for the life satisfaction of aging parents
(Silverstein, Cong, and Li, 2006) and has the potential to
enhance satisfaction with life for older people regardless of
gender (Liu et al., 2019). Similarly, Morelli et al. (2015) also
observed that emotionally supportive individuals also report
higher levels of satisfaction. Older people gave their CNM information to understand an
issue, which was significantly associated with satisfaction with
providing informational support (χ2 = 49.62, p < 0.000). This was
attributed to the positive effects elicited when older people gave a
CNM information to understand an issue, which was satisfying
to them. Annear et al. (2017) indicated that older people have
wisdom, values, and skills that can benefit the younger
generation. They
possess
a
wide
array
of
information,
knowledge, and expertise that they pass along to CNMs
(Michel et al., 2019). These findings are in agreement with
those of Chadha (1999), who found that CNMs consult older
people on a majority of life’s major issues, which makes older
people feel useful and valued. Amurwon, J., Hajdu, F., Yiga, D B., and Seeley, J. (2017). ‘Helping my neighbour is
like giving a loan’–the role of social relations in chronic illness in rural Uganda.
BMC Health Serv. Res. 17 (1), 1–12. 4.3 Informational social support inally, giving feedback to a CNM on an action that they wanted
to take was significantly related to satisfaction with providing
information support (χ2 = 39.913, p < 0.05). This was attributed
to the feelings of being valued arising from being consulted on a
matter and being given time to think things through and offer
feedback. According to Storchi (2017), when contacted about a
subject, older people contribute feedback to a CNM because they
feel valued and significant. Therefore, they take time to find the best
possible response in a situation because they are strategic
communicators who want to make a difference in the lives of
others (Carstensen et al., 2016). The findings of this study are in
agreement with Carstensen et al. (2016), who reported that before
giving feedback on any issue, older people tend to rely on their past
experiences, which are directly linked to their emotions, short- and
long-term goals, and desire to give back. Being in a position to
provide feedback when requested contributes to their satisfaction
with life. Informational social support is sharing knowledge and
resources (DeHoff et al., 2016), provision of advice and
guidance (Lu et al., 2016; Southwick et al., 2016), or advice
regarding the environment (Shakespeare-Finch & Obst, 2011),
which is intended to help individuals accomplish a task or cope
with a difficult life situation. The results observed in the four
items studied are significantly associated with satisfaction with
providing information support. In the first item, providing helpful advice when needed to
make an important decision was significantly associated with
the older person’s satisfaction with providing information
support (χ2 = 73.02, p < 0.000). This was attributed to the
feeling of being knowledgeable on a matter and contributing to
a CNM’s ability to make sound decisions. According to
Carstensen et al. (2016), older people possess an almost
mystical ability to assist CNMs to develop capabilities to
deal with daily concerns. The authors argued that older
people tend to rely on past experiences, which are directly
linked to their emotions, temporary and permanent goals, and Overall, providing information support, as evidenced in the
four items, significantly influenced satisfaction with providing
information support. Having gone through different challenges Frontiers in Aging frontiersin.org 09 10.3389/fragi.2022.897508 Mbuthia et al. Conflict of interest The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Data availability statement All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations
or those of the publisher, the editors, and the reviewers. Any product
that may be evaluated in this article, or claim that may be made by its
manufacturer, is not guaranteed or endorsed by the publisher. The original contributions presented in the study are
included
in
the
article/Supplementary
Material;
further
inquiries can be directed to the corresponding author. Ethics statement and experiences, older people are able to understand the
informational needs of a CNM and offer useful information
(Heaney & Israel, 2008). This finding concurs with other studies
that found that providing information support to others is linked
to better health outcomes and well-being (Brown et al., 2003). Generally, providing social support in the three dimensions
requires both cognitive judgments of satisfaction with social
support and affective appraisals of moods and emotions about
that support (Tov & Diener, 2013). The older people had to
consider the help they offered to CNMs and how doing so
affected their moods and emotions. The studies involving human participants were reviewed and
approved by the Kenyatta University Institutional Ethical Review
Committee, Kenya, approval number PKU/2235/11379, and the
National Commission for Science, Technology and Innovation
(NACOSTI) Kenya, approval number NACOSTI/P/21/11012. The
patients/participants provided their written informed consent to
participate in this study. Author contributions The findings of this study contribute significantly to the limited
research evidence on the relationship between social support in the
three categories and social well-being among older individuals in
Kenya. The strengths of this study lie in its use of a randomly selected
sample of older people, a high response rate, and detailed information
on provided social support. The use of mixed methods—quantitative
data from the questionnaire and qualitative data from focus group
discussions—allowed the study to examine the association between
social support and social well-being
and obtain a deeper
understanding through narratives, revealing subjective appraisals
of providing support. The potential in the data lies in its scope
and detail of the social support variables provided across the three
domains. KM conceived the idea for the study, collected data, analyzed,
and developed the manuscript. SM and GO revised the draft
manuscript critically for important intellectual content. All
authors approved the final manuscript to be published. Acknowledgments The study acknowledges the study participants who provided
important data for this study. The main shortcoming of this study was that the data are cross-
sectional, and thus, the causal ordering of providing social support
and social well-being is precluded. In future studies, data from
longitudinal studies would be necessary to provide a temporal
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s12888-020-02826-3 12 Frontiers in Aging 12 frontiersin.org
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Joint genome-wide association study of progressive supranuclear palsy identifies novel susceptibility loci and genetic correlation to neurodegenerative diseases
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Molecular neurodegeneration
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Permalink https://escholarship.org/uc/item/79n4d1xn ISSN
1750-1326
Authors
Chen, Jason A
Chen, Zhongbo
Won, Hyejung
et al. Publication Date
2018-08-01
DOI
10.1186/s13024-018-0270-8
Peer reviewed UCLA
UCLA Previously Published Works UCLA
UCLA Previously Published Works UCLA Previously Published Works Title
Joint genome-wide association study of progressive supranuclear palsy identifies novel
susceptibility loci and genetic correlation to neurodegenerative diseases. Joint genome-wide association study of progressive supranuclear palsy identifies novel
susceptibility loci and genetic correlation to neurodegenerative diseases. © 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. Abstract Background: Progressive supranuclear palsy (PSP) is a rare neurodegenerative disease for which the genetic
contribution is incompletely understood. Methods: We conducted a joint analysis of 5,523,934 imputed SNPs in two newly-genotyped progressive
supranuclear palsy cohorts, primarily derived from two clinical trials (Allon davunetide and NNIPPS riluzole trials in
PSP) and a previously published genome-wide association study (GWAS), in total comprising 1646 cases and 10,662
controls of European ancestry. Results: We identified 5 associated loci at a genome-wide significance threshold P < 5 × 10−8, including replication
of 3 loci from previous studies and 2 novel loci at 6p21.1 and 12p12.1 (near RUNX2 and SLCO1A2, respectively). At
the 17q21.31 locus, stepwise regression analysis confirmed the presence of multiple independent loci (localized
near MAPT and KANSL1). An additional 4 loci were highly suggestive of association (P < 1 × 10−6). We analyzed the
genetic correlation with multiple neurodegenerative diseases, and found that PSP had shared polygenic heritability
with Parkinson’s disease and amyotrophic lateral sclerosis. Conclusions: In total, we identified 6 additional significant or suggestive SNP associations with PSP, and discovered
genetic overlap with other neurodegenerative diseases. These findings clarify the pathogenesis and genetic
architecture of PSP. Keywords: Genome-wide association study, Progressive supranuclear palsy, Neurodegeneration dementia, characterized by pathological tau aggregation and
a clinical syndrome of postural instability, falls, and supra-
nuclear ophthalmoplegia [1]. It shares symptomatic and
neuropathologic overlap with a large group of diseases, that
are collectivity known as “tauopathies” due to characteristic
tau deposits; however, compared to these diseases, PSP
appears to be more clinically, neuropathologically, and genet-
ically homogenous [2–4]. Notably, the clinical syndrome has
high correlation with the neuropathology [5]. These charac-
teristics have thrust PSP into a central role for studying Powered by the California Digital Library
University of California eScholarship.org Chen et al. Molecular Neurodegeneration (2018) 13:41
https://doi.org/10.1186/s13024-018-0270-8 RESEARCH ARTICLE
Open Access
Joint genome-wide association study of
progressive supranuclear palsy identifies
novel susceptibility loci and genetic
correlation to neurodegenerative diseases
Jason A. Chen1, Zhongbo Chen2, Hyejung Won3, Alden Y. Huang1, Jennifer K. Lowe3, Kevin Wojta4,
Jennifer S. Yokoyama5, Gilbert Bensimon6,7,8, P. Nigel Leigh9, Christine Payan6,7, Aleksey Shatunov2,
Ashley R. Jones2, Cathryn M. Lewis10, Panagiotis Deloukas11, Philippe Amouyel12, Christophe Tzourio13,
Jean-Francois Dartigues13, Albert Ludolph14, Adam L. Boxer5, Jeff M. Bronstein3, Ammar Al-Chalabi2,
Daniel H. Geschwind1,3 and Giovanni Coppola1,3,4,15* Open Access Joint genome-wide association study of
progressive supranuclear palsy identifies
novel susceptibility loci and genetic
correlation to neurodegenerative diseases Jason A. Chen1, Zhongbo Chen2, Hyejung Won3, Alden Y. Huang1, Jennifer K. Lowe3, Kevin Wojta4,
Jennifer S. Yokoyama5, Gilbert Bensimon6,7,8, P. Nigel Leigh9, Christine Payan6,7, Aleksey Shatunov2,
Ashley R. Jones2, Cathryn M. Lewis10, Panagiotis Deloukas11, Philippe Amouyel12, Christophe Tzourio13,
Jean-Francois Dartigues13, Albert Ludolph14, Adam L. Boxer5, Jeff M. Bronstein3, Ammar Al-Chalabi2,
Daniel H. Geschwind1,3 and Giovanni Coppola1,3,4,15* * Correspondence: gcoppola@ucla.edu
1Interdepartmental Program in Bioinformatics, University of California, Los
Angeles, CA 90095, USA
3Program in Neurogenetics, Department of Neurology and Department of Human
Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA
Full list of author information is available at the end of the article Background Tau pathology is a prominent hallmark of neurodegenerative
diseases, including Alzheimer’s disease (AD) and frontotem-
poral dementia (FTD). Progressive supranuclear palsy (PSP)
is a relatively pure tauopathy associated with parkinsonism - * Correspondence: gcoppola@ucla.edu
1Interdepartmental Program in Bioinformatics, University of California, Los
Angeles, CA 90095, USA
3Program in Neurogenetics, Department of Neurology and Department of Human
Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA
Full list of author information is available at the end of the article Page 2 of 11 Chen et al. Molecular Neurodegeneration (2018) 13:41 subjects. For the Illumina HumanQuad 660 W Array
(Hoglinger et al. study), we used phs000103.v1.p1 “Genome--
Wide Association Studies of Prematurity and Its Complica-
tions”, phs000289.v1.p1 “National Human Genome Research
Institute (NHGRI) GENEVA Genome-Wide Association
Study of Venous Thrombosis”, phs000188.v1.p1 “Vanderbilt
Genome-Electronic Records (VGER) Project: QRS Dur-
ation”, phs000203.v1.p1 “A Genome-Wide Association Study
of Peripheral Arterial Disease”, phs000237.v1.p1 “Northwest-
ern NUgene Project: Type 2 Diabetes”, phs000234.v1.p1
“Group Health/UW Aging and Dementia eMERGE study”,
and phs000170.v1.p1 “A Genome-Wide Association Study
on Cataract and HDL in the Personalized Medicine Research
Project Cohort”. For the Illumina HumanOmni 2.5 Array
(UCLA – this study, and NNIPPS study), we used
phs000371.v1.p1 “Genetic Modifiers of Huntington’s Dis-
ease”, phs000429.v1.p1 “NEI Age-Related Eye Disease Study
(AREDS) - Genetic Variation in Refractive Error Substudy”,
and phs000421.v1.p1 “A Genome-Wide Association Study
of Fuchs’ Endothelial Corneal Dystrophy (FECD)”. For the
Illumina HumanCore Array (UCLA – this study), we used
the WTCCC2 cohort, which was typed on the related Illu-
mina OmniExpress Array. Subjects with an ascertained
phenotype (e.g., disease) were removed. More detailed in-
formation regarding these datasets is available in Add-
itional file 1: Table S1. neurodegeneration, enabling clinical trials of a relatively
homogenous patient population with potentially more uni-
form response to treatment. Therefore, PSP has become a
target of intense clinical research [6, 7]. While the disease
shares neuropathological overlap with other tauopathies, the
polygenic genetic correlation with other neurodegenerative
diseases remains to be clarified. The major known genetic risk factor is an extended
H1 haplotype on chromosome 17q21.31, which includes
MAPT (the gene encoding the tau protein), and is
homozygous in almost all PSP patients [2]. Other risk
factors identified include genome wide significant associ-
ations at loci near MAPT, MOBP, STX6, and EIF2AK3,
suggesting a strong contribution of common variation in
its genetic architecture [8]. Data preprocessing p
p
g
Genotypes for all datasets were converted to the forward
strand, and converted into coordinates based on the hg19
reference sequence using UCSC liftOver [10]. The genotypes
were then merged and pre-processed according to platform. Determination of cryptic relatedness (pairwise proportion
IBD, PI-HAT > 0.2), sample missingness (> 0.05), genotype
missingness (> 0.05), Hardy-Weinberg equilibrium p-value
(< 10−5), and sex-matching was performed in PLINK
v1.90b3.28 [11] and used to quality-control (QC) samples
using standard parameters [12]. Ancestry was predicted by
multidimensional scaling based on raw Hamming distances,
implemented
in
PLINK. Only
samples
of
presumed
European ancestry that clustered with known Europeans
from the HapMap3 cohort [13] were included. Preprocessing
steps are further elaborated in Additional file 2: Figure S1. Imputation Imputation was performed separately for each geno-
typing platform using the IMPUTE v2.3.2 algorithm
[14]. Prephasing of chromosomes using the Segmented
HAPlotype Estimation & Imputation Tool (SHAPEIT)
v2.r837 was performed as previously described [15, 16]. IMPUTE2 was run on the prephased haplotypes using the
1000 Genomes Project Phase 3 reference in non-overlapping
5 megabase chunks with a 250 kilobase buffer and an effect-
ive population size of 20,000. Imputed variants with an Genotyping Genotyping in the UCLA study cohort was performed as
a prelude to whole-genome sequencing, and was per-
formed by Illumina (using the Illumina HumanOmni 2.5
Array) and the New York Genome Center (using the
Illumina HumanCore Array). Genotyping calls were
made using the Illumina GenomeStudio software. Methods
Cohort Three cohorts of primarily European ancestry were in-
cluded in the study – “UCLA”, a combination of 349
PSP patients and 130 controls from the UCSF Memory
and Aging Center [2, 9] and the Allon Therapeutics
Davunetide trial [6]; “NNIPPS”, a group of 341 PSP pa-
tients from the Neuroprotection and Natural History in
Parkinson Plus Syndromes (NNIPPS) trial [7] and the
Blood
Brain
Barrier
in
Parkinson
Plus
syndromes
(BBBIPPS) study; and “Hoglinger”, 1112 PSP patients
from a previously published GWAS [8]. The UCLA co-
hort was divided into two, because of differences in
genotyping platform: “UCLA Omni 2.5” and “UCLA
HumanCore”. Further details are available in the Supple-
mentary Methods. Background We reasoned that the inclu-
sion of additional cases and controls could increase the
statistical power for genome-wide association, potentially
yielding novel loci that could provide insight into the
molecular mechanisms of PSP and other more common
tauopathies. Credible set of causal variants at PSP GWAS loci Credible set of causal variants at PSP GWAS loci
A credible set (potential causal variants) was identified
at each of total of seven genome-wide significant loci
identified in this study using the CAusal Variants Identi-
fication in Associated Regions (CAVIAR) software pack-
age [28]. Because of the extended linkage disequilibrium
patterns in the chromosome 17q21.31 haplotype region,
causal variants were not identified at this associated
locus. Within each of the selected loci, the SNP with the
minimum joint association p-value was chosen as the
index SNP, and variants with p-value < 10−5 and in LD
(r2 > 0.6) with the index SNP were input into CAVIAR. The CAVIAR-identified credible set contains potential
causal variants (with a confidence level of 95% under the
statistical model) that could explain the association at
each locus. Prediction of gene expression differences associated with
PSP-associated SNPs Genetic associations with PSP may be due to genetic
control of gene expression. We used TWAS to pre-
dict differential gene expression in PSP from the joint
analysis summary statistics, integrating paired geno-
typing and gene expression data from the GTEx
Consortium [27]. Correcting for approximately 5000
effective independent tests per brain region (taking
into account 5483 genes with significantly heritable
weights and the interdependence of gene expression,
particularly across tissues), the significance threshold
was set at P < 1 × 10−5. Public datasets Genotypes from the Hoglinger et al. GWAS [8] (cases
only – no controls) were obtained from the NIAGADS
database. Out-of-sample
controls
were
obtained
from
dbGAP Authorized Access to match each genotyping
platform. In total, for the HumanOmni 2.5 M platform, we
obtained 2364 subjects; for the OmniExpress platform, 870
subjects; and for the HumanQuad 660 W platform, 8756 Chen et al. Molecular Neurodegeneration (2018) 13:41 Chen et al. Molecular Neurodegeneration (2018) 13:41 Page 3 of 11 calculated and used as covariates for restricted max-
imum likelihood (REML) analysis. imputation genotype probability < 0.9, missingness > 0.05, or
minor allele frequency < 0.01 were removed, and genotypes
across platforms were merged. Cryptic relatedness across co-
horts was assessed, and related/duplicated samples were
removed. Association Association was performed using a linear mixed model to
correct for population structure, using BOLT-LMM [17]. The genotyping platform was used as a categorical covariate. The standard infinitesimal model p-values were chosen for
downstream anaylsis. Odds ratios were calculated as exp. (beta). Because some of the individual cohort sizes violate
the large sample size assumptions of BOLT-LMM, odds ra-
tios for association (for individual cohorts) were computed
using a logistic regression model in PLINK, using the first 5
eigenvectors, derived from Principal Component Analysis
(PCA), as covariates. Power calculations were performed
using the Genetic Power Calculator [18], assuming a variant
with risk allele frequency of 0.5 and relative risk of 1.3 in an
additive genetic model, a disease with a prevalence of 10 in
100,000, and a p-value threshold of 5 × 10−8, using a geno-
typic, 2 df case-control test. QQ and Manhattan plots were
constructed using the R package “qqman” [19]. Forest plots
were constructed using the R package “metafor” [20]. The
genomic inflation factor λ was computed with PLINK. Correction of the genomic inflation factor to an equivalent
sample size of 1000 cases and 1000 controls was performed
as previously described [21]. To control for the extended
haplotype on chr17q21 and to identify independent associ-
ation signals, we performed association as before, but includ-
ing the haplotype (tagged by the SNP rs1560310) [22] as a
covariate. Proportion variance in liability explained p
y
p
The explained variance in liability at each of the
genome-wide significant loci was calculated according to
the method of So et al., which requires the frequency of
the risk allele, the relative risk of the heterozygous geno-
type, the relative risk of the homozygous risk genotype,
and the prevalence of the disease in the population [23]. The allele frequencies were calculated from the control
population of the joint genotyping cohort. Relative risks
were approximated with the corresponding odds ratios,
which converges to relative risk when the prevalence of
disease is rare. Genotypic odds ratios were estimated by
assuming an additive model. The prevalence of PSP was
estimated at 6.5 per 100,000 in accordance with prior
epidemiological studies [24, 25]. The genome-wide poly-
genic variance in liability explained was calculated using
GCTA v1.24.7 [26]. The genetic relationship matrix was
calculated
chromosome-by-chromosome
and
then
re-combined. The first 5 principal components were Genetic variation can result in changes to the coding se-
quence of a gene (e.g., nonsense and missense variants)
or can regulate the gene’s expression (e.g., by affecting
transcription factor binding in promoter or enhancer
regions). We first identified credible SNPs as “func-
tional” (stopgain variant, frameshift variant, splice donor
variant, nonsense-mediated decay transcript variant, or
missense variant). Of the remaining credible SNPs, we
identified those in the promoter region of a gene, de-
fined as the range 2 kb upstream to 1 kb downstream
relative to the transcription start site (TSS). Finally, the
remaining credible SNPs were considered possible regu-
latory variants and tested for short- or long- range inter-
action with other regions of chromatin to identify
potential downstream target genes. The interactions
were determined by Hi-C experiments in IMR90 and
embryonic stem cells from public data [29, 30], and fetal
brain germinal zone (ventricular and subventricular Chen et al. Molecular Neurodegeneration (2018) 13:41 Page 4 of 11 Chen et al. Molecular Neurodegeneration (2018) 13:41 zone) and cortical plate (intermediate zone and marginal
zone) from our group [31]. zone) and cortical plate (intermediate zone and marginal
zone) from our group [31]. To increase statistical power, we combined all three
cohorts in a joint analysis framework. We estimated that
this combined cohort had 90% power to detect associ-
ation of a variant with allele frequency of 0.5 and relative
risk of 1.3. Results We analyzed subjects from three GWAS cohorts, including
1) a multi-center cohort [2, 6] in whom we performed
genotyping using the Illumina HumanOmni2.5 BeadChip
and the Illumina HumanCore BeadChip (“UCLA”); 2) pa-
tients from centers in France, Germany, and the United
Kingdom as part of the Neuroprotection and Natural
History in Parkinson Plus Syndromes (NNIPPS) study, a
double-blind randomized placebo-controlled clinical trial of
riluzole [7], genotyped with the Illumina HumanOmni2.5
BeadChip (“NNIPPS”), and 3) a cohort of autopsy-proven
cases from a previously published [8] GWAS (“Hoglinger”). A more detailed description of the cohorts is provided in
Additional file 1: Table S1. We combined each cohort with
platform-matched, out-of-sample controls from dbGAP
(Additional file 1: Table S1). Stringent QC – excluding
SNPs that had low genotype call rates (< 0.95) or did not
follow Hardy-Weinberg equilibrium, and excluding subjects
with low sample call rate (< 0.95), non-European ancestry,
incompatible sex, cryptic relatedness, or duplication across
cohorts (Additional file 2: Figure S1) – was applied to each
cohort (including platform-matched controls). We then im-
puted variants implementing IMPUTE2 [14] using the
1000 Genomes Phase 3 Reference Panel to estimate geno-
types at more than 77,000,000 SNPs. Imputed variants with
imputation quality scores (r2 < 0.9) or low minor allele
frequency (< 0.01) were filtered, and genotypes across all
cohorts were combined in a joint analysis. In total, we ex-
amined 6,419,662 SNPs in 1646 PSP cases and 10,662
controls. The results of the joint analysis genome-wide association
are shown in Fig. 1 and Additional file 2: Figure S4. SNPs at
5 loci, in cytobands 17q21.31 (in an extended haplotype con-
taining MAPT, lead SNP rs71920662, odds ratio OR = 0.19,
p = 3.9 × 10−113), 3p22.1 (within MOBP, rs10675541, OR =
0.71, p = 7.2 × 10−19), 1q25.3 (within STX6, rs57113693, OR
= 1.3, p = 8.7 × 10−16), 6p21.1 (within RUNX2, rs35740963,
OR = 0.77, p = 1.8 × 10−8), and 12p12.1 (within SLCO1A2,
rs7966334, OR = 1.5, p = 3.2 × 10−8), reached genome-wide
significance (p < 5 × 10−8) (Additional file 1: Table S2,
Additional file 2: Figure S5). An additional SNP reported in a
previous GWAS [8], rs7571971, was also analyzed. Although
this SNP did not reach genome-wide significance in the joint
analysis (OR = 1.18, p = 2.7 × 10−5), the direction of the asso-
ciation was consistent with the previous association in each
cohort. Proportion variance in liability explained For a cohort of the sample size of that in a
previous PSP GWAS from Hoglinger et al., the power to
detect association was only 33%. In the primary analysis,
we assessed the genome-wide association between the
genotype at each SNP and case-control status using a
linear mixed model to correct for population stratifica-
tion. The genomic inflation factor λ for the joint analysis
was 1.05; for the UCLA-Omni2.5, UCLA-HumanCore,
NNIPPS, and Hoglinger cohorts, λ was 1.03, 1.02, 1.11,
and 1.11, respectively (Fig. 1, Additional file 2: Figure S2
and S3). We considered the joint inflation factor to be
acceptable in the setting of a relatively large joint ana-
lysis sample size [33]. Scaled for sample size, the ad-
justed genomic inflation factor λ1000 was 1.02. Data availability Full and imputed genotyping results from the UCLA
and NNIPPS cohorts will be made available on the
NIAGADS database. Genetic correlation with neurodegenerative diseases g
Genetic correlation was assessed from GWAS summary sta-
tistics using the Linkage Disequilibrium Score Regression
method (LDSC) [32]. Summary statistics were filtered by
only considering SNPs that overlap with the HapMap3 refer-
ence panel. Refer to the Supplementary Methods for further
details. Results 1 Genome-wide SNP association in the joint analysis. a Manhattan plot indicating the SNP association P values. The vertical axis displays the
strength of association (−log10 P value) as a function of genomic position, with alternating colors for sequential chromosomes. Genome-wide
significant and suggestive loci are labeled with the nearest gene symbol. The thresholds for significant (P < 5 × 10−8, red horizontal line) and
suggestive (P < 1 × 10−6, blue horizontal line) associations are shown. b-d Quantile-quantile plots for: b all SNPs, including the strongly associated
extended haplotype on chromosome 17; c SNPs excluding chromosome 17; and d SNPs excluding genome-wide significant and suggestive loci. The 95% confidence interval for the expected distribution of p-values is shaded To further understand how variation at each of the loci
contributes to disease risk, we assessed the functional con-
sequences of significant SNPs. We first identified a set of
potential causal SNPs using the CAVIAR method, which
identifies a “credible set” of SNPs that encompasses those
likely to be causal [28]. The 17q21.31 locus was excluded
from the analysis because of its unusual, long-range link-
age disequilibrium pattern. In some loci, potentially causal
coding variants were identified (in genome-wide signifi-
cant loci, at 6p21.1, in RUNX2, and at 12p12.1, in
SLCO1A2; and in suggestive loci, at 8q24.21, in ASAP1,
and at 12q13.13, in AMHR2; Additional file 1: Table S5). Other SNPs in the credible set fell within regulatory re-
gions; we identified the gene associated with each SNP
using data from Hi-C experiments, mapping chromosome
conformation patterns on a genome-wide scale from four
human cell types (IMR-90 fetal lung fibroblasts, embry-
onic stem cells, fetal brain, and fetal brain germinal zone). Each potential regulatory SNP in the credible set was then
associated with genes in close proximity by chromosomal
conformation, yielding potential downstream causal genes
(Additional file 1: Table S5). entire set of genotyped SNPs explains 9.4 ± 0.8% (estimate
±standard error) of the variance on the liability scale, sug-
gesting that many loci are yet to be found. The association between PSP and the chr17q21 haplo-
type (H1/H2) has been widely characterized, but inde-
pendent
SNPs
in
the
chr17q21
region
may
also
contribute to disease susceptibility. To test this, we per-
formed linear regression, taking haplotype as a covariate. Results In order to decrease the likelihood that the results
were influenced by population stratification, we assessed
association at the loci in each of the study cohorts (Fig. 2). Associations at the lead SNPs in each of the regions were
consistent across the three most well-powered study
cohorts (Hoglinger, NNIPPS, and UCLA Omni2.5)
while in general, the HumanCore subset of the UCLA
cohort was underpowered to detect association. An add-
itional 4 loci demonstrated suggestive association (1 × 10−6
< P < 5 × 10−8),
in
1q41
(intergenic,
near
DUSP10,
rs12125383, OR = 1.28, p = 5.3 × 10−8), 12q13.13 (within
SP1,
rs147124286,
OR = 0.74,
p = 4.1 × 10−7),
8q24.21
(within ASAP1, rs2045091, OR = 1.25, p = 4.7 × 10−7), and
1p22.3 (near WDR63 and MIR4423, rs114573015, OR = 2.1,
p = 5.9 × 10−7) (Additional file 1: Table S3). Overall, the
genome-wide significant loci explained a combined 5.9% of
the variance in heritable liability of PSP (Additional file 1:
Table S4). The locus tagging the chr17q21 haplotype sur-
rounding MAPT contributed the majority (5.0%), while
new loci contributed an additional 0.2% of the total liability. Using a polygenic model implemented in GCTA [26], the Association was initially performed using the 616 cases
represented from the UCLA and NNIPPS cohorts. Genome-wide significant association was detected at loci
near MAPT and largely corresponded to the haplotype
region (lead SNP rs79730878, p = 5.4 × 10−45; Additional
file 1: Table S2). Other top associations were found at
loci near MOBP, STX6, SEMA4D, DDX27, and SP1,
though these did not reach genome-wide significance. Chen et al. Molecular Neurodegeneration (2018) 13:41 Page 5 of 11 Fig. 1 Genome-wide SNP association in the joint analysis. a Manhattan plot indicating the SNP association P values. The vertical axis displays the
strength of association (−log10 P value) as a function of genomic position, with alternating colors for sequential chromosomes. Genome-wide
significant and suggestive loci are labeled with the nearest gene symbol. The thresholds for significant (P < 5 × 10−8, red horizontal line) and
suggestive (P < 1 × 10−6, blue horizontal line) associations are shown. b-d Quantile-quantile plots for: b all SNPs, including the strongly associated
extended haplotype on chromosome 17; c SNPs excluding chromosome 17; and d SNPs excluding genome-wide significant and suggestive loci. The 95% confidence interval for the expected distribution of p-values is shaded Fig. Results At a threshold of P < 1 × 10−5, we identified a
number of genes that were called as differentially
expressed (Additional file 1: Table S6). As expected due
to the length and lack of recombination in the region,
most of these genes (17) clustered around the chromo-
some 17 haplotype. Notably, MAPT (within the associ-
ated 17q21.31 locus) was among the genes predicted to
be differentially expressed, as well as STX6 (within the
associated
1q24
locus),
SP1
(within
the
suggestive
12q13.13 locus), SKIV2L (within 6p21.33, nearby the as-
sociated 6p21.1 locus), and RPSA (within the associated
3p22.1 locus). Other genes that were pinpointed outside
of association regions were CEP57 (in 11q21) and
RPS6KL1 (in 14q24.3). many common neurodegenerative diseases. Altogether,
from a joint analysis of three disease cohorts, we have
found 2 novel genome-wide significant susceptibility loci in
PSP and replicated 3 previously reported loci. Of the loci
identified in this study, three (within MAPT, MOBP, and
STX6) were reported significant in a previous GWAS [8]. In the MAPT region, a third independent association
was identified, also in MAPT intron 1, speculatively sug-
gesting important regulatory functions in this region;
however, no effects in differential expression have been
uncovered. Overall, the mechanisms of the MAPT asso-
ciations have been unclear. The larger H1/H2 haplotype
appears to affect splicing at MAPT exon 3 but not over-
all tau expression [34]; while other MAPT variants, such
as rs242557, may affect tau expression in some tissues
[35], the effect is not robust in brain tissue. The
additional association identified here may provide an or-
thogonal point of investigation into this curious region. An additional locus near the EIF2AK3 gene (encoding
PERK, a key component of the unfolded protein re-
sponse) was also previously identified; however, the re-
ported SNP did not reach genome-wide significance in
this joint analysis or in the new “Hoglinger” cohort
(using different controls). The strong neuropathological overlap of PSP with
other tauopathies suggests that genetic overlap may
exist. Using the LDSC software [32], we assessed genetic
overlap of PSP with other neurodegenerative diseases,
including AD, behavioral variant FTD (bvFTD), Parkin-
son’s disease (PD), and amyotrophic lateral sclerosis
(ALS), by using GWAS summary statistics. Results Additionally, we identified subjects that were homozy-
gous for the risk allele (H1/H1), and performed associ-
ation
in
this
subset
of
patients. Both
approaches
identified similar independent associations from the H1
haplotype in the 17q21.31 region, with the most signifi-
cant SNPs at rs8078967 (P = 1.9 × 10−14) and rs9904290
(P = 8.9 × 10−12) in the haplotype-regressed and H1/H1
only datasets, respectively (Additional file 2: Figure S5). These SNPs did not appear to be in strong linkage dis-
equilibrium with a previously reported SNP association,
rs242557 [8] (r2 = 0.008 and 0.007 in the 1000 Genomes
Project data – EUR super-population, respectively) that
was filtered from this dataset in variant QC; however,
they were highly correlated with each other (r2 = 0.996). Additionally, both variants and rs242557 are within the
first intron of the MAPT gene. To supplement the mapping information from Hi-C,
we also identified the functional consequences of GWAS Chen et al. Molecular Neurodegeneration (2018) 13:41 Page 6 of 11 Fig. 2 (See legend on next page.) Fig. 2 (See legend on next page.) Chen et al. Molecular Neurodegeneration (2018) 13:41 Chen et al. Molecular Neurodegeneration (2018) 13:41 Page 7 of 11 (See figure on previous page.)
Fig. 2 Forest plots showing association across each individual cohort for selected SNPs. A total of six genome-wide significant loci were
identified, with representative SNPs: a rs71920662 in 17q21.31, near MAPT; b rs57113693 in 1q25.3, near STX6; c rs10675541 in 3p22.1, near MOBP;
d rs35740963 in 6p21.1, near RUNX2; and e rs7966334 in 12p12.1, near SLCO1A2. An additional four suggestive loci were also identified: f
rs12125383 in 1q41, near DUSP10 in an intergenic region; g rs147124286 in 12q13.13, near SP1; h rs2045091 in 8q24.21, near ASAP1; and i
rs114573015 in 1p22.3, near WDR63. j Additionally, a previously reported GWAS SNP rs7571971 in 2p11.2, near EIF2AK3, was not identified as
genome-wide significant in the joint analysis hits using the TWAS method to predict genes that may
be affected by risk alleles [27]. TWAS estimates gene ex-
pression values using paired reference transcriptome/
genotyping datasets (e.g., for expression quantitative trait
loci - eQTL studies) and genotype information from
summary statistics, and predicts differential expression
between cases and controls. Using reference data from
the GTEx Consortium, TWAS predicted the effect of
gene expression from the risk haplotypes in multiple
tissues. Results As controls,
we included summary statistics from GWAS for heritable,
non-neurodegenerative diseases of brain (schizophrenia
and bipolar disorder), a quantitative trait (height), and a
non-brain disease (type 2 diabetes) (for further details,
refer to the Supplementary Methods). Each of these traits
was shown to be heritable. Statistically significant genetic
correlations were identified for PD (P = 9.7 × 10−5) and
ALS (P = 1.8 × 10−3), but not for non-neurodegenerative
disease control GWAS (Fig. 3). We also identified 2 novel genome-wide significant
susceptibility loci at 6p21.1 and 12p12.1 (near RUNX2
and SLCO1A2, respectively). At 6p21.1, we identified a
lead SNP as well as several coding SNPs in the credible
set within RUNX2, a gene thought to be a transcriptional
factor involved in regulation of osteoblastic differentiation
[36]. While seemingly unrelated to PSP, a curious number
of neurodegeneration-related genes are also involved in
bone diseases (e.g. TREM2, which has been linked to AD
and Nasu-Hakola disease [37, 38], and VCP, linked to
amyotrophic lateral sclerosis and Paget’s disease of bone
[39, 40]). At 12p12.1, we identified a lead SNP and credible
set coding SNPs within SLCO1A2, a transporter present
(among other places) at the blood-brain barrier, where it
regulates solute trafficking [41]. An additional four loci
(near the genes DUSP10, SP1, ASAP1, and WDR63/
MIR4423) were suggestive of association, but did not reach
genome-wide significance. While this study raises the possi-
bility of involvement of these genes in PSP pathogenesis,
further fine-mapping and functional studies are needed to
confirm their possible roles. Discussion Phenotypes that
share a common polygenic background are positively correlated 1
0.5
0
0.5
1
PSP_Chen
ALS_ProjectMinE
AD_IGAP
bvFTD_Ferrari
PD_23&Me_meta
SCZ_PGC
BIP_PGC
Height_GIANT
T2D_DIAGRAM
PSP_Chen
ALS_ProjectMinE
AD_IGAP
bvFTD_Ferrari
PD_23&Me_meta
SCZ_PGC
BIP_PGC
Height_GIANT
T2D_DIAGRAM
1
(0)
0.52
(0.002)
0.12
(0.4)
0.6
(0.2)
0.4
(1e 04)
0.18
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0.06
(0.6)
0.02
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0.52
(0.002)
1
(0)
0.15
(0.5)
0.45
(0.5)
0.11
(0.5)
0.32
(0.007)
0.083
(0.5)
0.29
(0.2)
0.003
(1)
0.12
(0.4)
0.15
(0.5)
1
(0)
0.29
(0.5)
0.077
(0.5)
0.039
(0.6)
0.023
(0.8)
0.055
(0.8)
0.016
(0.9)
0.6
(0.2)
0.45
(0.5)
0.29
(0.5)
1
(0)
0.31
(0.4)
0.054
(0.8)
0.16
(0.6)
0.11
(0.8)
0.034
(0.9)
0.4
(1e 04)
0.11
(0.5)
0.077
(0.5)
0.31
(0.4)
1
(0)
0.079
(0.3)
0.12
(0.1)
0.16
(0.3)
0.034
(0.6)
0.18
(0.05)
0.32
(0.007)
0.039
(0.6)
0.054
(0.8)
0.079
(0.3)
1
(0)
0.66
(2e 27)
0.062
(0.6)
0.089
(0.2)
0.06
(0.6)
0.083
(0.5)
0.023
(0.8)
0.16
(0.6)
0.12
(0.1)
0.66
(2e 27)
1
(0)
0.009
(1)
0.12
(0.1)
0.02
(0.9)
0.29
(0.2)
0.055
(0.8)
0.11
(0.8)
0.16
(0.3)
0.062
(0.6)
0.009
(1)
1
(0)
0.1
(0.4)
0.053
(0.6)
0.003
(1)
0.016
(0.9)
0.034
(0.9)
0.034
(0.6)
0.089
(0.2)
0.12
(0.1)
0.1
(0.4)
1
(0)
Fig. 3 Heatmap of genetic correlation between GWAS summary statistics for neurodegenerative diseases (PSP – progressive supranuclear palsy,
ALS – amyotrophic lateral sclerosis, AD – Alzheimer’s disease, bvFTD – behavioral variant frontotemporal dementia, PD – Parkinson’s disease),
calculated by LDSC. GWAS for non-neurodegenerative phenotypes (SCZ – schizophrenia, BIP – bipolar disorder, height, and T2D– type 2 diabetes
mellitus) are also included for comparison. In each cell, the genetic correlation coefficient (and P value in parentheses) is shown. Phenotypes that
share a common polygenic background are positively correlated Fig. 3 Heatmap of genetic correlation between GWAS summary statistics for neurodegenerative diseases (PSP – progressive supranuclear palsy,
ALS – amyotrophic lateral sclerosis, AD – Alzheimer’s disease, bvFTD – behavioral variant frontotemporal dementia, PD – Parkinson’s disease),
calculated by LDSC. GWAS for non-neurodegenerative phenotypes (SCZ – schizophrenia, BIP – bipolar disorder, height, and T2D– type 2 diabetes
mellitus) are also included for comparison. In each cell, the genetic correlation coefficient (and P value in parentheses) is shown. Phenotypes that
share a common polygenic background are positively correlated Fig. Discussion As a prototypical tauopathy, insight into PSP susceptibility
alleles can help to illuminate the downstream molecular ef-
fects of tau pathology, which is a major component of Chen et al. Molecular Neurodegeneration (2018) 13:41 Page 8 of 11 1
0.5
0
0.5
1
PSP_Chen
ALS_ProjectMinE
AD_IGAP
bvFTD_Ferrari
PD_23&Me_meta
SCZ_PGC
BIP_PGC
Height_GIANT
T2D_DIAGRAM
PSP_Chen
ALS_ProjectMinE
AD_IGAP
bvFTD_Ferrari
PD_23&Me_meta
SCZ_PGC
BIP_PGC
Height_GIANT
T2D_DIAGRAM
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1
(0)
Fig. 3 Heatmap of genetic correlation between GWAS summary statistics for neurodegenerative diseases (PSP – progressive supranuclear palsy,
ALS – amyotrophic lateral sclerosis, AD – Alzheimer’s disease, bvFTD – behavioral variant frontotemporal dementia, PD – Parkinson’s disease),
calculated by LDSC. GWAS for non-neurodegenerative phenotypes (SCZ – schizophrenia, BIP – bipolar disorder, height, and T2D– type 2 diabetes
mellitus) are also included for comparison. In each cell, the genetic correlation coefficient (and P value in parentheses) is shown. Discussion 3 Heatmap of genetic correlation between GWAS summary statistics for neurodegenerative diseases (PSP – progressive supranuclear palsy,
ALS – amyotrophic lateral sclerosis, AD – Alzheimer’s disease, bvFTD – behavioral variant frontotemporal dementia, PD – Parkinson’s disease),
calculated by LDSC. GWAS for non-neurodegenerative phenotypes (SCZ – schizophrenia, BIP – bipolar disorder, height, and T2D– type 2 diabetes
mellitus) are also included for comparison. In each cell, the genetic correlation coefficient (and P value in parentheses) is shown. Phenotypes that
share a common polygenic background are positively correlated Aside from identifying additional associated loci and
highlighting potential PSP susceptibility genes, we ana-
lyzed the polygenic overlap between neurodegenerative
diseases, identifying shared heritability with PD and ALS. Curiously, these diseases do not have predominant tau
neuropathology, as PSP and other tauopathies do. Typic-
ally, PD is associated with aggregation of α-synuclein, and
ALS with aggregation of TDP43 and other proteins, while
tau pathology is prominent in AD. However, there are
known shared genetic risk factors among these diseases. The 17q21.31 haplotype is highly associated with PD, in
the same direction as in PSP [44], and SNPs near the
MOBP gene have been recently associated with ALS [45]. Our results indicate the existence of common neurode-
generative disease pathways even across traditional protein
aggregate-based subdivisions, and could potentially lead to
effective treatment strategies. Our results also implicate possible alternative causal
genes in previously reported genome-wide significant
loci. At 3p22.1, the gene closest to the GWAS lead SNP
was reported as MOBP. This locus has previously been
implicated in differential expression of the SLC25A38/
appoptosin gene, which may regulate tau cleavage [42]. Using Hi-C, we have identified chromatin interactions
with MYRIP and EIF1B that could also explain this asso-
ciation. Similarly, at 1q25.3, the gene closest to the
GWAS lead SNP was STX6; by Hi-C, we have also iden-
tified XPR1 as a possible candidate gene. Interestingly,
our group has previously demonstrated XPR1 mutations
in primary familial brain calcification [43], though any
mechanistic overlap with PSP is unclear. Analysis of eQTL
datasets (in GTEx) suggests that RPSA at 3p22.1 and
SKIV2L near 6p21.1 may also be the causal genes but the
tissue-relevant datasets were relatively underpowered. Chen et al. Molecular Neurodegeneration (2018) 13:41 Page 9 of 11 Chen et al. Molecular Neurodegeneration (2018) 13:41 Page 9 of 11 A limitation of the study includes the case-control
matching design. URLs CAVIAR, http://genetics.cs.ucla.edu/caviar/index.html;
dbGAP, http://www.ncbi.nlm.nih.gov/gap;
GCTA, http://cnsgenomics.com/software/gcta/index.html;
Genetic Power Calculator, http://zzz.bwh.harvard.edu/gpc/;
HapMap, ftp://ftp.ncbi.nlm.nih.gov/hapmap//;
IMPUTE2, https://mathgen.stats.ox.ac.uk/impute/impute_v2.html;
LocusZoom, http://locuszoom.sph.umich.edu/locuszoom/;
MetaXcan, https://github.com/hakyimlab/MetaXcan;
NIAGADS, https://www.niagads.org/;
PLINK2, https://www.cog-genomics.org/plink2;
SHAPEIT, https://mathgen.stats.ox.ac.uk/genetics_software/shapeit/
shapeit.html;
UCSC Genome Browser https //genome ucsc edu/cgi bin/hgGateway PLINK2, https://www.cog-genomics.org/plink2; SHAPEIT, https://mathgen.stats.ox.ac.uk/genetics_software/shapeit/
shapeit.html; SHAPEIT, https://mathgen.stats.ox.ac.uk/genetics_software/shapeit/
shapeit.html; UCSC Genome Browser, https://genome.ucsc.edu/cgi-bin/hgGateway; Acknowledgements We acknowledge the contributions of Alice Zhang, who has helped with
acquiring controls genotyping; Margaret Chu, for administrative support; and
all of the patients and their families, to whom this work is dedicated. Abbreviations AD: Alzheimer’s disease; ALS: Amyotrophic lateral sclerosis; bvFTD: Behavioral
variant frontotemporal dementia; eQTL: Expression quantitative trait loci;
FTD: Frontotemporal dementia; GWAS: Genome-wide association study;
PCA: Principal component analysis; PD: Parkinson’s disease; PSP: Progressive
supranuclear palsy; QC: Quality control; SNP: Single nucleotide polymorphism Conclusion
h Summary measures will be made available in the NIAGADS database (https://
www.niagads.org/). Additional data obtained from the NIAGADS and dbGAP
databases are available with access restrictions from the corresponding
databases. Here, we have increased the number of significant
genetic risk locus for PSP, an important advance for
understanding its pathophysiology. The power of this
study to identify novel loci at genome wide signifi-
cance and a large unexplained heritability suggests
that PSP may be highly amenable to genetic associ-
ation studies in larger sample cohorts using next
generation sequencing. Overall, by establishing the
genetic correlations of PSP with PD and ALS and
identifying novel genome-wide significant and sug-
gestive associations, we shed insight into the mecha-
nisms of neurodegenerative disease. Discussion The NNIPPS genotyping and analysis was
supported under the aegis of JPND – (www.jpnd.eu, United Kingdom), Med-
ical Research Council (MR/L501529/1; MR/R024804/1) and Economic and So-
cial Research Council (ES/L008238/1). A.A.C. receives salary support from the
National Institute for Health Research (NIHR) Dementia Biomedical Research
Centre at South London and Maudsley NHS Foundation Trust and King’s Col-
lege London. The work leading up to this publication was funded by the
European Community’s Health Seventh Framework Programme (FP7/2007–
2013; grant agreement number 259867) and Horizon 2020 Programme
(H2020-PHC-2014-two-stage; grant agreement number 633413). We thank
the UCLA Neuroscience Genomics Core (www.semel.ucla.edu/ungc) for as-
sistance with genotyping data generation. Samples from the National Cell
Repository for Alzheimer’s Disease (NCRAD), which receives government sup-
port under a cooperative agreement grant (U24 AG021886) awarded by the
National Institute on Aging (NIA), were used in this study. We thank contribu-
tors who collected samples used in this study, as well as patients and their
families, whose help and participation made this work possible. Authors’ contributions Additional file 1: Supplementary Tables S1-S5. (XLSX 636 kb)
Additional file 2: Supplementary Figures S1-S5, Supplementary
Methods. (DOCX 8021 kb) Additional file 1: Supplementary Tables S1-S5. (XLSX 636 kb)
Additional file 2: Supplementary Figures S1-S5, Supplementary
Methods. (DOCX 8021 kb) JAC contributed to study design, data collection, experimental design,
statistical analysis, and drafting and revision of the manuscript. ZC
contributed to data collection, statistical analysis, and drafting of the
manuscript. HW contributed to data collection and statistical analysis. AYH
contributed to study design and statistical analysis. JKL, KW, GB, PNL, CP, AS,
ARJ, CML, WL, PD, PA, CT, J-FD, AL, ALB, and JMB contributed to subject
recruitment, data collection, experimental design, and revision of the
manuscript. JSY contributed to data collection and statistical analysis. AA-C
and DHG contributed to supervision of the project, subject recruitment, data
collection, statistical analysis, experimental design, and revision of the manu-
script. GC contributed to supervision of the project, subject recruitment, data
collection, statistical analysis, experimental design, and drafting and revision
of the manuscript. All authors read and approved the final manuscript. Ethics approval and consent to participate Written informed consent was obtained for all patients participating in the
Allon Therapeutics davunetide trial and the NNIPPS trial, and IRB approval
was obtained from the corresponding Institutional Review Boards. For the
NNIPPS study, The protocol and amendments were reviewed and approved
by the Comité de Protection des Personnes of Pitié-Salpêtrière Hospital
(France), the UK Multicentre Research Ethics Committee (MREC) (UK),
Ethikkommission of the University of Ulm (Germany) and by local
Institutional Review Boards (Ethics Committees) where appropriate (UK,
Germany). For the BBBIPPS study, the protocol and amendments were
reviewed and approved by the Comité de Protection des Personnes of Pitié-
Salpêtrière Hospital (France). Additional data was obtained via NIAGADS and
dbGAP in accordance with data access policies. The Institutional Review Discussion While this design allows for matching by
array platforms and avoids stratification due to technical
artifacts, stratification based on the ancestral differences
may be present. The potential for stratification was
reduced by strict filtering based on multidimensional
scaling to limit the sample to subjects of European ances-
try, and linear mixed model methods to further reduce
confounding. Combining multiple cohorts as we have
done may also reduce the degree of population stratifica-
tion in the joint sample. Overall, the genomic inflation fac-
tor (λ = 1.05) suggested an acceptable level of population
stratification. The validity of the results and replication of
the
original
GWAS
are
further
reinforced
by
the
consistency of the identified associations across the mul-
tiple platform-matched sub-cohorts. study was supported by the French Health Ministry, Programme Hospitalier
de Recherche Clinique (AOM04035). The Assistance Publique - Hôpitaux de
Paris (France) was the sponsor of the study. The protocol and amendments
were reviewed and approved by the Comité de Protection des Personnes of
Pitié-Salpêtrière Hospital (France). The NNIPPS genotyping and analysis was
supported under the aegis of JPND – (www.jpnd.eu, United Kingdom), Med-
ical Research Council (MR/L501529/1; MR/R024804/1) and Economic and So-
cial Research Council (ES/L008238/1). A.A.C. receives salary support from the
National Institute for Health Research (NIHR) Dementia Biomedical Research
Centre at South London and Maudsley NHS Foundation Trust and King’s Col-
lege London. The work leading up to this publication was funded by the
European Community’s Health Seventh Framework Programme (FP7/2007–
2013; grant agreement number 259867) and Horizon 2020 Programme
(H2020-PHC-2014-two-stage; grant agreement number 633413). We thank
the UCLA Neuroscience Genomics Core (www.semel.ucla.edu/ungc) for as-
sistance with genotyping data generation. Samples from the National Cell
Repository for Alzheimer’s Disease (NCRAD), which receives government sup-
port under a cooperative agreement grant (U24 AG021886) awarded by the
National Institute on Aging (NIA), were used in this study. We thank contribu-
tors who collected samples used in this study, as well as patients and their
families, whose help and participation made this work possible. study was supported by the French Health Ministry, Programme Hospitalier
de Recherche Clinique (AOM04035). The Assistance Publique - Hôpitaux de
Paris (France) was the sponsor of the study. The protocol and amendments
were reviewed and approved by the Comité de Protection des Personnes of
Pitié-Salpêtrière Hospital (France). Author details
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Received July 10, 2020, accepted July 27, 2020, date of publication July 29, 2020, date of current version August 11, 2020. Received July 10, 2020, accepted July 27, 2020, date of publication July 29, 2020, date of current version August 11, 2020. Digital Object Identifier 10.1109/ACCESS.2020.3012709 GUIDONG ZHANG
1, (Member, IEEE), NA JIN1, LILI QU
2,
AND SAMSON SHENGLONG YU
3, (Member, IEEE)
1School of Automation, Guangdong University of Technology, Guangzhou 510006, China
2School of Automation, Foshan University, Foshan 528225, China
3School of Engineering, Deakin University, Melbourne, VIC 3216, Australia
C
di
h
Lili Q (
lili@f
d
) GUIDONG ZHANG
1, (Member, IEEE), NA JIN1, LILI QU
2,
AND SAMSON SHENGLONG YU
3, (Member, IEEE)
1School of Automation, Guangdong University of Technology, Guangzhou 510006, China
2School of Automation, Foshan University, Foshan 528225, China
3School of Engineering, Deakin University, Melbourne, VIC 3216, Australia
Corresponding author: Lili Qu (qulili@fosu edu cn) This work was supported in part by the National Natural Science Foundation of China under Grant 51907032 and Grant 61733015; in part
by the Natural Science Foundation of Guangdong Province under Grant 2018A030313365; and in part by the Science and Technology
Planning Project of Guangzhou under Grant 201804010310. ABSTRACT With an ever-growing number of batteries being integrated into the electric grid, bidirectional
converters with non-pulsating dc input current are required to replace the existing bidirectional converters
so as to extend the lifetime of such dc power suppliers. Bidirectional DC-DC converters are increasingly
used in a variety of applications including uninterruptable power supplies, electric vehicles and renewable
energy systems. In this study, we propose a new topology of bidirectional dc-dc converter with inherently
non-pulsating input current (NPIC), which is intended to be used for battery energy storage systems. With simple modifications from the conventional converters, the proposed NPIC converter has no inherent
pulsating input current in the step-down (buck) mode under both continuous and boundary conduction
modes (CCM and BCM), whereas in the step-up (boost) mode, the proposed NPIC converter retains the
desired voltage gain. Underpinning theories, operating principles and steady-state performances are analyzed
and presented in detail, which are then corroborated by simulation and experimentation. The proposed
converter topology, with simple design principle and ease for implementation, is likely to have wide-ranging
applicability in interfacing electrochemically functioned dc sources to modern power systems. INDEX TERMS Bidirectional dc-dc converter, non-pulsating input current, step-up mode, step-down mode,
continuous conduction mode, boundary conduction mode. INDEX TERMS Bidirectional dc-dc converter, non-pulsating input current, step-up mode, step-down mode,
continuous conduction mode, boundary conduction mode. The associate editor coordinating the review of this manuscript and
approving it for publication was Zhilei Yao
. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0 I. INTRODUCTION ated by the converter cannot be recovered, causing a low
efficiency. Compared to isolated bidirectional dc-dc con-
verter, non-isolated converters have smaller volume and
higher efficiency due to the absence of transformer [8]–[10]. Conventional buck/boost converters are widely used due
to their simple topologies and required control strategies,
but these converters cannot operate in extensively wide
voltage-conversion range [11], [12]. For zeta/sepic and
cuk/cuk type converters, energy transfer efficiency is low
because of their two-stage structures [13]–[15]. In order to
improve the efficiency and increase the voltage-conversion
range, many new non-isolated bidirectional dc-dc con-
verters have been proposed. Authors in [16] proposed a
non-isolated bidirectional dc-dc converter with high volt-
age gain comprised of two boost converters; authors in
[17] and [18] proposed two non-isolated bidirectional dc–dc
converter topologies, with a simple circuit structure. Their In renewable energy integrated hybrid power systems, hybrid
vehicle energy systems and uninterruptible power sup-
ply systems, multiple dc power sources need to trans-
fer energy to other dc energy storage systems, which
requires bi-directional dc-dc converters [1]–[4]. The topol-
ogy of bidirectional dc-dc converter can be divided into
isolated (transformer-integrated) and non-isolated (without
transformer) types. Flyback converters, forward-flyback con-
verters, half-bridge converters and full-bridge converters
are typical bidirectional isolated dc-dc converters [5]–[7]. By adjusting the turns ratio of the transformer, this con-
verter family can obtain large voltage gains in both step-up
and step-down modes. However, the power leakage gener- The associate editor coordinating the review of this manuscript and
approving it for publication was Zhilei Yao
. is work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ 140293 VOLUME 8, 2020 G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems FIGURE 1. (a) Proposed converter. (b) Equivalent circuit in the step-down
mode. (c) Equivalent circuit in the step-up mode. experimentally proven superior voltage step-up performances
have extended their applicability to wide-ranging voltage
levels than conventional converters. However, the above-mentioned converters only consider
voltage gain and device stress, without taking into account
the impact of dc pulsating input current on the input power
supply. II. OPERATING PRINCIPLE OF THE PROPOSED NPIC
CONVERTER II. OPERATING PRINCIPLE OF THE PROPOSED NPIC
CONVERTER I. INTRODUCTION For example, the buck/boost bidirectional converter
operating in step-down mode is equivalent to a buck con-
verter, and input current of the converter is equal to the
inductor current when the switch is turned on and becomes
zero when the switch is turned off, causing the converter
to have a pulsating input current [19]. The pulsating input
current of these bidirectional converters may degrade the
performance and lifespan of the electrochemically functioned
power sources or energy storage systems, because the pul-
sating current can lead to an accelerated aging rate of the
electrodes [20]–[22], which may also cause problems like
electromagnetic interference, slow transient response, and
reduction of energy efficiency. It is well known that large electrolytic capacitors con-
nected to the input port can cope with the inherent pulsating
input current issue, however, this solution is executed at the
expense of deterioration of the electrolytic capacitor, which
can shorten the lifespan of the overall converter system [22],
[23]. Another feasible option may be replacing the bulky
electrolytic capacitor with an LC filter, which, however, may
degrade energy efficiency and affect the dynamic perfor-
mance of the converter [24], [25]. Interleaved converter could
be another reasonable solution to alleviate the pulsating input
current issue, but this method has been proven to be not
suitable for bidirectional dc–dc converters [26], [27]. Knowing the need of non-pulsating input current convert-
ers and the shortage of a proper design, in this study, we pro-
pose a novel bidirectional dc-dc converter, which, compared
to its conventional counterparts, is able to draw NPIC from
the dc source while operating in the buck mode. At the same
time, the proposed NPIC bidirectional converter is also able
to retain its high voltage gain when working in the boost
mode. Simulation and experimentation are conducted in this
study, which verify the functionality and effectiveness and the
proposed converter structure. The proposed NPIC converter
has a simple topological design, and is easy to implement and
apply to industrial uses. FIGURE 1. (a) Proposed converter. (b) Equivalent circuit in the step-down
mode. (c) Equivalent circuit in the step-up mode. connected in series with the input source, with contributes
to the NPIC feature of the proposed converter. I. INTRODUCTION In order to
simplify the analysis, some reasonable assumptions are made
as: a) all semi-conductor devices and passive components
are considered ideal; b) capacities of the capacitors are large
enough to keep the capacitor voltages nearly constant. The remainder of the paper is organized as follows. The
structure and operating principle in buck and boost modes are
detailed in Section II. Simulations and experiments are con-
ducted with their results shown in Section III and Section IV
respectively. Finally, a conclusion is drawn in Section V. A. STEP-DOWN MODE OF THE PROPOSED NPIC
CONVERTER Fig. 1 shows the configuration of the proposed converter,
which mainly consists of two inductors L1 and L2, one capac-
itor Cb, two switches S1 and S2. Steady-state analysis of
the proposed converter in step-up and step-down modes is
discussed as follows. It is worth-noting that inductor L1 is The proposed converter in step-down mode is shown in
Fig. 1(b). Characteristic waveforms of the proposed converter
in CCM are depicted in Fig. 3, and the current flow path in
one switching period is illustrated in Fig. 2(a). 140294 VOLUME 8, 2020 G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems FIGURE 2. Key waveforms of the proposed converter in the step-down mode: (a) CCM; (b) BCM. FIGURE 2. Key waveforms of the proposed converter in the step-down mode: (a) CCM; (b) BCM. source delivers energy to inductor L1 and the load. Therefore,
inductor L1 starts to store energy, and accordingly the current
flowing through inductor L1 (iL1) increases. At the same time,
energy saved in capacitor Cb is released to inductor L2 and
the load. Correspondingly, the current of inductor L2 (iL2)
starts increasing. In addition, current and voltage stresses of
switches S1 and S2 in this mode can be found in Fig. 2 (a). Accordingly, the following equations among the components
during this time interval can be obtained, FIGURE 3. Equivalent circuits in step-down mode: (a) mode 1;
(b) mode 2.
iL1(t) = IL1V + vH −vL
L1
t,
iL2(t) = IL2V + vCb −vL
L2
t,
iCb(t) = −IL2V −vCb −vL
L2
t,
iCL(t) = IL1V + IL2V + (vH −vL
L1
+ vCb −vL
L2
)t −io,
(1) (1) where vH is the input voltage, vCb is the voltage of the
capacitor Cb, vL is the output voltage and IL1V and IL2V are
the minimum values of the currents flowing through inductors
L1 and L2, respectively. FIGURE 3. Equivalent circuits in step-down mode: (a) mode 1;
(b) mode 2. Mode 2 [t1 < t < t2]: In this mode, S1 is turned off and S2
is turned on at t = t1 as shown in Fig. 3 (b). During this mode,
energy stored in L1 and the input power source is delivered
to the load and Cb. Hence, the current flowing through L1
decreases. Meanwhile, inductor L2 releases energy to the
load and the current flowing through it keeps decreasing as 2) INDUCTOR CURRENTS (19) Applying the ampere-second balance theorem for the capac-
itor current, we can have Similarly, the critical inductance of inductor L2 can be
derived as, Z Ts
0
iC(t)dt = 0,
(6) (6) L2c = VLRL
2VHfs
. (20) (20) where Ts is the switching period. where Ts is the switching period. where Ts is the switching period. Considering (1), (2), (4) and (6), the maximum and mini-
mum values of the inductor currents can be calculated as, When inductances L1 and L2 are equal to critical values, the
converter operates in the boundary-conduction-mode (BCM)
mode. Fig. 2 (b) shows some typical waveforms in BCM
operation. IL1P = DIo + VH −VL
2L1
DTs,
(7)
IL1V = DIo −VH −VL
2L1
DTs,
(8)
IL2P = (1 −D)Io + VH −VL
2L2
DTs,
(9)
IL2V = (1 −D)Io −VH −VL
2L2
DTs,
(10) (7) (7) (8) Then, current ripples of the inductors can be deduced as, Then, current ripples of the inductors can be deduced as, depicted in Fig. 2 (a). Main equations among the components
during this time interval can be deduced as, depicted in Fig. 2 (a). Main equations among the components
during this time interval can be deduced as, 1iL1 = VH −VL
L1
DTs,
(13)
1iL2 = VH −VL
L2
DTs. (14) (13)
iL1(t) = IL1P −vH −vCb −vL
L1
t,
iL2(t) = IL2P −vL
L2
t,
iCb(t) = IL1P −vH −vCb −vL
L1
t,
iCL(t) = IL1P + IL2P −(vH −vCb −vL
L1
+ vL
L2
)t −io,
(2) (14) (2) 4) CRITICAL INDUCTANCE L1c AND L2c (
VCb = VH,
VL = DVH. (4) (4) When IL1V = 0, the critical inductance of the inductor L1 can
be derived as, According to (4), the voltage conversion ratio of the pro-
posed converter can be expressed as, L1c = RL(1 −D)Ts
2D
,
(18) (18) Gstep−down = VL
VH
= D. (5) where RL = VL/Io. Incorporating (4), (18) can be rewritten
as, where RL = VL/Io. Incorporating (4), (18) can be rewritten
as, (5) as, L1c = RL(VH −VL)
2VLfs
. (19) 3) VOLTAGE AND CURRENT STRESSES OF POWER DEVICES According to the above analysis, it can be obtained that
the voltage stresses of switches S1 and S2 in the proposed
converter, namely, VS1 and VS2, are equal to the voltage of
the capacitor Cb, i.e., VCb, as shown in (4). where IL1P and IL2P are the maximum values of the currents
flowing through the inductors L1 and L2, respectively. b
According to Fig. 2(a), peak currents flowing through
switches S1 and S2 can be derived as, Assuming the converter is a lossless system and the voltage
ripples are ignorable in a switching period. Based on the
mode analysis and applying volt-second balance principle to
inductors L1 and L2, steady-state voltage relationships among
input voltage, output voltage, and capacitor voltages can be
obtained as, IS1
peak = IL1P + IL2P,
(15)
IS2
peak = IL1P + IL2P. (16) (15)
(16) (15) (15)
(16) (16) By applying (5), (7), and (9) to (15) and (16), equa-
tions (15) and (16) can be rewritten as follows, (
(VH −VL)D + (VH −VCb −VL)(1 −D) = 0,
(VCb −VL)D −VL(1 −D) = 0,
(3) (3) IS1
peak = IS2
peak = Io + VL(VH −VL)
2LefsVH
,
(17) (17) where D is the duty cycle of switch S1. where D is the duty cycle of switch S1. where Le = L1//L2. where Le = L1//L2. From (3), the voltage of capacitor Cb and voltage of capac-
itor CL, i.e., the output voltage, can be derived as, 4) CRITICAL INDUCTANCE L1c AND L2c 1) OPERATION MODES Mode 1 [t0 < t < t1]: As shown in Fig. 3 (a), switch S1
is turned on at t = t0 and switch S2 is turned off. The input 140295 VOLUME 8, 2020 G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems depicted in Fig. 2 (a). Main equations among the components
during this time interval can be deduced as
Then, current ripples of the inductors can be deduced as, B. STEP-UP MODE OF THE PROPOSED CONVERTER The proposed converter in step-up mode is shown in Fig. 1(c),
whose equivalent circuits in CCM mode are depicted in
Fig. 5, and the characteristic waveforms are illustrated in
Fig. 4(a).
iL1(t) = IL1V + vH −vL −vCb
L1
t,
iL2(t) = IL2V + vL
L2
t,
iCb(t) = −(IL1V + vH −vL
L1
t),
iCH(t) = IL1V + vH −vL
L1
t −io. (27) (27) 5) OUTPUT VOLTAGE RIPPLE ANALYSIS IN CCM According to the characteristics of the capacitor, voltage
ripples of the capacitor can be calculated as, (9) vrip
C = 1
C
Z t02
t01
iC(t)dt,
(21) (10) (21) where Io is the load current. The average current of each
inductor can be derived as, where t01 is the time that the current flowing through it
changes from positive to negative, and t02 is the time that the
current flowing through the capacitor changes from negative
to positive. ¯IL1 = DIo,
(11)
¯IL2 = (1 −D)Io. (12) (11)
(12) (12) VOLUME 8, 2020 VOLUME 8, 2020 140296 140296 G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems FIGURE 4. Key waveforms of the proposed converter in the step-up mode: (a) CCM.; (b) BCM. FIGURE 4. Key waveforms of the proposed converter in the step-up mode: (a) CCM.; (b) BCM. For capacitor CL, the voltage ripple can be calculated as, quantities of the components during this time interval can be
obtained, vCCM
CL−rip = 1
CL
Z t02
t01
iCL(t)dt,
(22) (22)
iL1(t) = IL1P −vH −vL
L1
t,
iL2(t) = IL2P −vCb −vL
L2
t,
iCb(t) = IL2P −vCb −vL
L2
t,
iCL(t) = IL1P −vH −vL
L1
t −io. (26) where t01 and t02 can be calculated as, (26) t01 = Le(Io −IL1V −IL2V )
VH −VL
= DTs
2 ,
(23)
t02 = Le(Io −IL1P −IL2P)
VL
= −(1 −D)Ts
2
. (24) (23) Mode 2 [t1 < t < t2]: As shown in Fig. 5 (b), switch
S1 is turned off and the switch S2 is turned on. During this
mode, VL charges L1, L2 and Cb while releasing energy to
the load. So, iL1, iL2 and iCb start to increase. The main
equations relating the components during this time interval
can be deduced as, From (21)–(24), the voltage ripple can be calculated as, From (21)–(24), the voltage ripple can be calculated as, vCCM
CL−rip = VL(VH −VL)
8LeCLVHf 2s
. (25) (25) 5) OUTPUT VOLTAGE RIPPLE ANALYSIS IN CCM
According to (21), voltage ripples of CH can be calcul 5) OUTPUT VOLTAGE RIPPLE ANALYSIS IN CCM
According to (21), voltage ripples of CH can be calculated as, (
VCb = VH,
VL = DVH. (29) )
According to (21), voltage ripples of CH can be calculated as, According to (21), voltage ripples of CH can be calcu (29) vCCM
CH−rip = 1
CH
Z t02
t01
iCH(t)dt = (VH −VL)3
8L1CHV 2
Hf 2s
,
(42) (42) According to (29), the voltage conversion ratio of the
proposed converter can be expressed as, 2) CURRENTS OF INDUCTORS Combining (26), (27), (29) and (6), the maximum and mini-
mum values of the inductor currents can be calculated as, IL1P = Io + VH −VL
2L1
(1 −D)Ts,
(31)
IL1V = Io −VH −VL
2L1
(1 −D)Ts,
(32)
IL2P = 1 −D
D
Io + VH −VL
2L2
(1 −D)Ts,
(33)
IL2V = 1 −D
D
Io −VH −VL
2L2
(1 −D)Ts. (34) (31) (32) (32) 1) OPERATION MODES Mode 1 [t0 < t < t1]: As shown in Fig. 5 (a), S1 is turned on
and S2 is turned off. Input source VL and L1 and L1 supply
power to the load, so iL1 decreases. At the same time, L2 and
Cb release energy to the load, so iL2 and iCb also decrease. Therefore, the following equation set relating the electric Based on the mode analysis and applying volt-second bal-
ance principle to inductors L1 and L2, voltage relationships
in steady state among the input voltage, output voltage, and 140297 VOLUME 8, 2020 G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems FIGURE 5. Equivalent circuits in step-up mode: (a) mode 1; (b) mode 2. C. COMPARION OF THE PROPOSED NPIC CONVERTER
AND OTHER BI-DIRECTIONAL DC-DC CONVERTERS Gstep−up = VH
VL
= 1
D. (30) (30) Comparisons between the NPIC converter proposed in this
paper and other commonly used and previously proposed
bidirectional converters are shown in TABLE 1. Compared
with these converters, the proposed NPIC converter has the
advantages of continuous input current in the step-down
mode, simple structure with the fewest number of additional
electronic devices. 3) VOLTAGE AND CURRENT STRESSES OF POWER DEVICES 3) VOLTAGE AND CURRENT STRESSES OF POWER DEVICES Based on the previous discussions, voltage stresses of the
switches VS1 and VS2 are equal to the voltage of the capacitor
Cb i.e., VCb, which is shown in (29). According to Fig. 4(a), the peak value of the currents
flowing through switches S1 and S2 can be derived as, IS1
peak = IL1P + IL2P,
(37)
IS2
peak = IL1P + IL2P. (38) (37) (38) By applying (30), (31), (33) to (37) and (38), we can have By applying (30), (31), (33) to (37) and (38), we can have IS1
peak = IS2
peak = 1
DIo + (VH −VL)2
2LefsVH
,
(39) (39) where Le = L1//L2. 4) CALCULATION OF CRITICAL INDUCTANCE L1c AND L2c
When IL1V = 0, critical inductance of inductor L1 can be
derived as, 4) CALCULATION OF CRITICAL INDUCTANCE L1c AND L2c
When IL1V = 0, critical inductance of inductor L1 can be
derived as, 4) CALCULATION OF CRITICAL INDUCTANCE L1c AND L2c
When IL1V = 0, critical inductance of inductor L1 can be
derived as, L1c = (VH −VL)(1 −D)Ts
2Io
= RH(VH −VL)2
2V 2
Hfs
. (40) (40) Similarly, critical inductance of inductor L2 can be derived
as, FIGURE 5. Equivalent circuits in step-up mode: (a) mode 1; (b) mode 2. as, L2c = RHVL(VH −VL)
2V 2
Hfs
. (41) capacitor voltages can be obtained as, (41) (
(VL −VH)D + (VL + VCb −VH)(1 −D) = 0,
(VCb −VL)D −VL(1 −D) = 0. (28) When L1 and L2 are equal to their critical values, the
converter operates in the BCM mode. Fig. 4 (b) shows some
typical waveforms in BCM operation. (28) From (28), the voltage of capacitor Cb and the voltage of
capacitor CL, i.e., the output voltage, can be derived as, III. SIMULATION VERIFICATION In order to verify the effectiveness of the proposed converter,
the converter topology is simulated in PSIM software envi-
ronment with useful parameters shown in TABLE 2. (33) Step-down
simulation
results
are
demonstrated
in
Fig. 6 (a) and (b), which comprise the driving signals (vgs1
and vgs2), input current (iin), output voltage (vo), inductor and
switch currents (iL1, iL2, iS1 and iS2), switch voltage stresses
(vds1 and vds2), respectively. Since inductor L1 is in series with
the power source, current of L1 is equal to the input current, Then, average currents of L1 and L2 can be derived as Then, average currents of L1 and L2 can be derived as ¯IL1 = Io,
(35)
¯IL2 = 1 −D
D
Io. (36) (35) 140298 VOLUME 8, 2020 VOLUME 8, 2020 G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems
TABLE 1. Performance comparison of various bi-directional dc-dc converters. TABLE 2. Simulation parameters. i.e., iL1 = iin. Fig. 7 shows the input current waveform of the
proposed NPIC converter and the conventional buck/boost
converter with the same simulation parameters. Apparently
the proposed NPIC converter is able to regulate and draw
non-pulsating input current into the converter system, demon-
strating its superiority its conventional counterparts. The step-up simulation results are demonstrated in
Fig. 6 (c) and (d), which comprise the driving signals (vgs1
and vgs2), input current (iin), output voltage (vo), inductor and
switch currents (iL1, iL2, iS1 and iS2), switch voltage stresses
(vds1 and vds2), respectively. According to Fig. 6 (c) and (d),
this NPIC converter can obtain the same voltage gain as
buck/boost bidirectional converter when working in the
step-up mode. TABLE 3. Experimental platform. TABLE 4. Experimental parameters. with the physical prototype and experimental en
shown in Fig. 8 and TABLE 3, and the paramete
the experiment are shown in TABLE 4. A STEP DOWN MODE TABLE 1. Performance comparison of various bi-directional dc-dc converters. TABLE 1. Performance comparison of various bi-directional dc-dc converters. 1. Performance comparison of various bi-directional dc-dc converters. TABLE 3. Experimental platform. TABLE 4. Experimental parameters. TABLE 3. Experimental platform. TABLE 2. Simulation parameters. TABLE 2. Simulation parameters. TABLE 4. Experimental parameters. i.e., iL1 = iin. Fig. 7 shows the input current waveform of the
proposed NPIC converter and the conventional buck/boost
converter with the same simulation parameters. III. SIMULATION VERIFICATION Apparently
the proposed NPIC converter is able to regulate and draw
non-pulsating input current into the converter system, demon-
strating its superiority its conventional counterparts. The step-up simulation results are demonstrated in
Fig. 6 (c) and (d), which comprise the driving signals (vgs1
and vgs2), input current (iin), output voltage (vo), inductor and
switch currents (iL1, iL2, iS1 and iS2), switch voltage stresses
(vds1 and vds2), respectively. According to Fig. 6 (c) and (d),
this NPIC converter can obtain the same voltage gain as
buck/boost bidirectional converter when working in the
step-up mode. with the physical prototype and experimental environment
shown in Fig. 8 and TABLE 3, and the parameters used in
the experiment are shown in TABLE 4. IV. EXPERIMENTAL VERIFICATION Experimental results are demonstrated in Figs. 9∼11, which
consist of the driving signals (vgs1), output voltages (vL),
inductor currents (iL1 and iL2), switch currents (iS1 and iS2) To further validate the effectiveness of the proposed con-
verter, a prototype of the converter is built in this study, VOLUME 8, 2020 140299 VOLUME 8, 2020 G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems FIGURE 6. Simulation results for the proposed NIPC converter: (a) CCM in
step-down mode; (b) BCM in step-down mode; (c) CCM in step-up mode;
(d) BCM in step-up mode. f h
d
d
FIGURE 8. Prototype with the experimental environment. FIGURE 9. Experimental waveforms of the driving signal, output voltage,
and the currents of inductors L1, L2 in step-down mode: (a) CCM;
(b) BCM. FIGURE 10. Experimental waveforms of the currents of switches S1 and
S2 in step-down mode: (a) CCM; (b) BCM. FIGURE 8. Prototype with the experimental environment. FIGURE 9. Experimental waveforms of the driving signal, output voltage,
and the currents of inductors L1, L2 in step-down mode: (a) CCM;
(b) BCM. FIGURE 8. Prototype with the experimental environment. FIGURE 8. Prototype with the experimental environment. FIGURE 9. Experimental waveforms of the driving signal, output voltage,
and the currents of inductors L1, L2 in step-down mode: (a) CCM;
(b) BCM. FIGURE 9. Experimental waveforms of the driving signal, output voltage,
and the currents of inductors L1, L2 in step-down mode: (a) CCM;
(b) BCM. FIGURE 10. Experimental waveforms of the currents of switches S1 and
S2 in step-down mode: (a) CCM; (b) BCM. FIGURE 6. Simulation results for the proposed NIPC converter: (a) CCM in
step-down mode; (b) BCM in step-down mode; (c) CCM in step-up mode;
(d) BCM in step-up mode. FIGURE 7. Input current of the proposed NPIC converter and
conventional buck/boost converter in step-down mode. FIGURE 10. Experimental waveforms of the currents of switches S1 and
S2 in step-down mode: (a) CCM; (b) BCM. FIGURE 11. Experimental waveforms of the voltage stresses of the switch
S1 and S2 in step-down mode: (a) CCM; (b) BCM. FIGURE 7. Input current of the proposed NPIC converter and
conventional buck/boost converter in step-down mode. FIGURE 11. Experimental waveforms of the voltage stresses of the switch
S1 and S2 in step-down mode: (a) CCM; (b) BCM. and switch voltage stresses (vds1 and vds2), respectively. As can be seen from Fig. V. CONCLUSION In this study, a novel bidirectional dc-dc converter converter
with no inherent pulsating input current in step-down mode
has been proposed. The proposed converter has lesser number
of active and passive components. And in the step-down
mode, the input current of the proposed converter in both
CCM mode and BCM mode is greater than 0. In the step-up
mode can retain the desired voltage gain. With the increase
of the output power, the efficiency of the proposed converter
promotes gradually. As it can be seen that the analysed effi-
ciency is about 90-91.5% in the step-up mode and 91-94%
in the step-down mode. This converter is a good candidate
for applications like battery storage systems, which require
continuous input current. Compared to traditional boost/buck
bidirectional converters whose input current is inherently
intermittent or pulsating, the proposed NPIC converter is able
to produce a continuous non-pulsating input current in the
step-down mode, overcomes the limitations of conventional
converters and can better reserve the lifetime of electrochem-
ically functioned dc sources such as batteries and fuel cells. The operating principle and steady-state performance of the
proposed NPIC current have been first demonstrated in detail,
which have then been corroborated through both simulation
and experimentation, verifying the functionality and unique
features of the proposed NPIC converter topology. It has also
been proven in this study that the proposed converter has
low output voltage ripples, high efficiency and the ability to
remain the desired voltage gain in the step-up mode. FIGURE 13. Experimental waveforms of the currents of switches S1 and
S2 in step-up mode: (a) CCM; (b) BCM. FIGURE 14. Experimental waveforms of the voltage stresses of the switch
S1 and S2 in step-up mode: (a) CCM; (b) BCM. FIGURE 14. Experimental waveforms of the voltage stresses of the switch
S1 and S2 in step-up mode: (a) CCM; (b) BCM. FIGURE 15. Efficiency of proposed converter with different operation
power. REFERENCES [1] G. Zhang, Z. Li, B. Zhang, and W. A. Halang, ‘‘Power electronics con-
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agree well with the theoretical analyses and stimulation VOLUME 8, 2020 G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems FIGURE 12. Experimental waveforms of the driving signal, output voltage,
and currents of the inductors L1, L2 in step-up mode: (a) CCM; (b) BCM. FIGURE 13. Experimental waveforms of the currents of switches S1 and
S2 in step-up mode: (a) CCM; (b) BCM. FIGURE 12. Experimental waveforms of the driving signal, output voltage,
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the desired voltage gain in the step-up mode while producing
non-pulsating input current in the step-down mode. Fig. 15 shows efficiency curves of the proposed converter
under different operation modes and power output, which
demonstrates that the proposed converter has high efficiency. when the output power varies from 25 W to 125 W, the
efficiency of the proposed converters is over 90%. FIGURE 12. Experimental waveforms of the driving signal, output voltage,
and currents of the inductors L1, L2 in step-up mode: (a) CCM; (b) BCM. FIGURE 12. Experimental waveforms of the driving signal, output voltage,
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Figs. 12∼14, including driving signals (vgs1), output voltages
(vH), inductor currents (iL1 and iL2), switch currents (iS1 and
iS2) and switch voltage stresses (vds1 and vds2), respectively. It
can be seen from Figs. 12∼14 that the converter can achieve
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May 2009. 140301 VOLUME 8, 2020 G. Zhang et al.: Inherently NPIC DC–DC Converter for Battery Storage Systems GUIDONG ZHANG (Member, IEEE) was born in
Guangdong, China, in 1986. He received the B.Sc. degree from the Xi’an University of Technology,
in 2008, and the two Ph.D. degrees from the South
China University of Technology and FernUniver-
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of Technology, Guangzhou. His research inter-
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of novel snubberless bidirectional naturally clamped ZCS/ZVS current-fed
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Guangdong, China, in 1986. He received the B.Sc. degree from the Xi’an University of Technology,
in 2008, and the two Ph.D. degrees from the South
China University of Technology and FernUniver-
sität, Hagen, in 2014 and 2015, respectively. [8] G. Chen, Y. Deng, L. Chen, Y. Hu, L. Jiang, X. He, and Y. Wang,
‘‘A family of zero-voltage-switching magnetic coupling nonisolated bidi-
rectional DC–DC converters,’’ IEEE Trans. Ind. Electron., vol. 64, no. 8,
pp. 6223–6233, Aug. 2017. He is currently an Associate Professor with
the School of Automation, Guangdong University
of Technology, Guangzhou. His research inter-
est includes power electronics topology and their
applications. He is currently an Associate Professor with
the School of Automation, Guangdong University
of Technology, Guangzhou. His research inter-
est includes power electronics topology and their
applications. [9] L. Zhao, J. Chen, T. Chen, Y. Shi, Z. B. STEP-UP MODE degree in high-voltage
technology and equipment from the Huazhong
University of Science and Technology, Wuhan,
Hubei, in 1989, the M.S. degree in control sci-
ence and engineering from Xi’an Jiaotong Univer-
sity, Xi’an, Shanxi, China, in 2002, and the Ph.D. degree in power electronics from the South China
University of Technology, Guangzhou, Guang-
dong, China, in 2009. She is currently a Professor at Foshan University, China. Her research
interests include power electronic systems and devices, switching power
supply, and circuit analysis technology and its applications. LILI QU was born in Hubei, China, in 1968. She received the B.S. degree in high-voltage
technology and equipment from the Huazhong
University of Science and Technology, Wuhan,
Hubei, in 1989, the M.S. degree in control sci-
ence and engineering from Xi’an Jiaotong Univer-
sity, Xi’an, Shanxi, China, in 2002, and the Ph.D. degree in power electronics from the South China
University of Technology, Guangzhou, Guang-
dong, China, in 2009. LILI QU was born in Hubei, China, in 1968. She received the B.S. degree in high-voltage
technology and equipment from the Huazhong
University of Science and Technology, Wuhan,
Hubei, in 1989, the M.S. degree in control sci-
ence and engineering from Xi’an Jiaotong Univer-
sity, Xi’an, Shanxi, China, in 2002, and the Ph.D. degree in power electronics from the South China
University of Technology, Guangzhou, Guang-
dong, China, in 2009. She is currently a Professor at Foshan University, China. Her research
interests include power electronic systems and devices, switching power
supply, and circuit analysis technology and its applications. [17] C.-C. Lin, L.-S. Yang, and G. Wu, ‘‘Study of a non-isolated bidirectional
DC–DC converter,’’ IET Power Electron., vol. 6, no. 1, pp. 30–37, 2013. [18] H. Ardi, R. R. Ahrabi, and S. N. Ravadanegh, ‘‘Non-isolated bidirectional
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verter with extra operation zone,’’ IET Power Electron., vol. 13, no. 10,
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She is currently a Professor at Foshan University, China. Her research
interests include power electronic systems and devices, switching power
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ripple-free flyback-type converter with passive pulsating ripple canceling
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fuel cell converter with direct double-frequency ripple current control,’’
IEEE Trans. Ind. Appl., vol. 51, no. 1, pp. 297–308, Jan. 2015. SAMSON SHENGLONG YU (Member, IEEE)
received the master’s degree (Hons.) in electrical
and electronic engineering and the Ph.D. degree
in electrical power engineering from The Uni-
versity of Western Australia (UWA), Perth, WA,
Australia, in 2014 and 2017, respectively. SAMSON SHENGLONG YU (Member, IEEE)
received the master’s degree (Hons.) in electrical
and electronic engineering and the Ph.D. degree
in electrical power engineering from The Uni-
versity of Western Australia (UWA), Perth, WA,
Australia, in 2014 and 2017, respectively. [23] Y. Ohnuma, K. Orikawa, and J.-I. Itoh, ‘‘A single-phase current-source PV
inverter with power decoupling capability using an active buffer,’’ IEEE
Trans. Ind. Appl., vol. 51, no. 1, pp. 531–538, Jan. 2015. [24] W.-T. Fan, K. K.-F. Yuen, and H. S.-H. Chung, ‘‘Power semiconduc-
tor filter: Use of series-pass device in switching converters for filtering
input current harmonics,’’ IEEE Trans. Power Electron., vol. 31, no. 3,
pp. 2053–2068, Mar. 2016. From 2017 to 2019, he served as a Postdoc-
toral Research Fellow at UWA. He is currently
an Assistant Professor at Deakin University, Mel-
bourne, VIC, Australia. His research interests
include power system analysis, renewable energy integration and forecasting,
and power electronics and its applications and control. He received the first
and second Best Paper Awards in the IEEE Australia Paper Competition,
in 2016 and 2017, the Best Reviewer Award for the IEEE TRANSACTIONS ON
SMART GRID, in 2018, and the Featured Article in Chaos: An Interdisciplinary
Journal for Nonlinear Science, in 2019. [25] V. Beldjajev, T. Lehtla, and J. Zakis, ‘‘Impact of component losses on the
efficiency of the LC-filter based dual active bridge for the isolation stage of
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bidirectional converter made with many interleaved buck stages,’’ IEEE
Trans. Power Electron., vol. 21, no. 3, pp. 578–586, May 2006. [27] M. Ilic and D. B. STEP-UP MODE Fan, and Z. Zhuang, ‘‘Zero-voltage
and zero-current-switching dual-transformer-based full-bridge converter
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May 25, 2020, doi: 10.1109/TPEL.2020.2997017. [10] J. Huang, Y. Wang, Z. Li, and W. Lei, ‘‘Unified triple-phase-shift control
to minimize current stress and achieve full soft-switching of isolated
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GaN-based interleaved CRM bidirectional buck/boost converter with
inverse coupled inductor,’’ IEEE Trans. Power Electron., vol. 31, no. 6,
pp. 4343–4352, Jun. 2016. NA JIN was born in Zhanjiang, Guangdong,
China, in 1996. She received the B.Sc. degree from
the Guangdong Normal University of Technology,
Guangzhou, China, in 2019. She is currently pur-
suing the M.Sc. degree in electrical engineering
with the School of Automation, Guangdong Uni-
versity of Technology, Guangzhou. NA JIN was born in Zhanjiang, Guangdong,
China, in 1996. She received the B.Sc. degree from
the Guangdong Normal University of Technology,
Guangzhou, China, in 2019. She is currently pur-
suing the M.Sc. degree in electrical engineering
with the School of Automation, Guangdong Uni-
versity of Technology, Guangzhou. [12] D. Baolei, L. Tao, H. Jun, J. Yang, and W. Xiao, ‘‘High-efficiency
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rectional ZVS PWM sepic/zeta DC-DC converter,’’ in Proc. IEEE ISIE,
Jun. 2007, pp. 555–560. Her current research interest includes power
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converters to improve voltage conversion ratio,’’ IEEE Trans. Power Elec-
tron., vol. 35, no. 4, pp. 3679–3687, Apr. 2020, doi: 10.1109/TPEL.2019. 2934726. [15] E. Babaei and M. E. Seyed Mahmoodieh, ‘‘Analysis and investigation
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converter,’’ IET Power Electron., vol. 7, no. 4, pp. 819–828, Apr. 2014. [16] H. Ardi, A. Ajami, F. Kardan, and S. N. Avilagh, ‘‘Analysis and imple-
mentation of a nonisolated bidirectional DC–DC converter with high
voltage gain,’’ IEEE Trans. Ind. Electron., vol. 63, no. 8, pp. 4878–4888,
Aug. 2016. LILI QU was born in Hubei, China, in 1968. She received the B.S. B. STEP-UP MODE Maksimovic, ‘‘Interleaved zero-current-transition buck
converter,’’ IEEE Trans. Ind. Appl., vol. 43, no. 6, pp. 1619–1627,
Nov./Dec. 2007. 140302 140302 VOLUME 8, 2020
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In-Depth Transcriptome Analysis Reveals Novel TARs and Prevalent Antisense Transcription in Human Cell Lines
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PloS one
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Experimental outline In this study we investigate the transcriptome of three cell lines,
A431, U-2 OS and U251, by applying the massive SOLiD DNA
sequencing technology facilitating sense/antisense identification of
reads. The cell lines were chosen to represent three different
lineages; epithelial, mesenchymal and glia cells. A total of 10 to 15
million high quality 50-basepair reads were obtained for each cell
line. The reads were mapped onto the human reference genome
(hg18), after which reads were aggregated for each gene. An
expression value was calculated based on the number of reads per
kilobase gene and million reads in each sample (RPKM) [12]. Analysis of the gene expression pattern demonstrated that 66% to
69% of all genes are expressed in each cell line of which 85% to
88% were shared for all three cell lines (figure S1). Several studies [3] have shown that antisense transcription is
prevalent and likely to have a regulatory function. Studies indicate
that 20% to 90% of all human protein-coding genes can generate
transcripts with potential to form sense-antisense pairs [4–6] and
that these generally are arranged in a tail-to-tail pattern. Recently,
short fragments of RNA have been detected in the antisense
direction in regions just upstream protein-coding genes [7–9]. In parallel to experimental discovery of regulatory RNAs, compu-
tational methods are being developed to identify conserved structural
RNA elements likely to be involved in transcriptional and trans-
lational control [10]. These approaches aim to make in silico predic-
tions of regulatory sites in the human genome that can be validated
by the on-going massive transcriptome sequencing (RNA-Seq) efforts
on cells, tissues and organs [11], however, more development is
needed to make these algorithms more accurate and efficient. Abstract Competing Interests: The authors have declared that no competing interests exist. ¤a Current address: Department of Medical Epidemiology and Biostatistics, Karolinska Institute, Stockholm, Sweden
¤b Current address: Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospita lines. We show that approximately 20% of all protein-coding genes
have antisense transcription coupled to them and that antisense
transcription is prevalent in introns. Introduction Less than 2% of the human genome encodes for proteins, yet a
large fraction, recently estimated to 60% to 90% of the genome
can be transcribed [1]. The functions of the majority of these novel
uncharacterized
transcriptionally
active
regions
(TARs)
are
currently unknown, but they are believed to be of regulatory
importance. For example, Ebisuya and colleagues showed that
‘‘transcriptional ripples’’ can propagate along the genome and
mediate regulation of genes several tens of kilobases away [2]. Daniel Klevebring¤a, Magnus Bjursell¤b, Olof Emanuelsson, Joakim Lundeberg*
Di i i
f G
T
h
l
S h
l
f Bi
h
l
Alb N
U i
i
C
R
l I
i
f T
h
l
S
kh l
S
d Daniel Klevebring¤a, Magnus Bjursell¤b, Olof Emanuelsson, Joakim Lundeberg*
Division of Gene Technology, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology, Stockholm, Sweden on of Gene Technology, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology, Stockholm, Sweden PLoS ONE | www.plosone.org Abstract Several recent studies have indicated that transcription is pervasive in regions outside of protein coding genes and that
short antisense transcripts can originate from the promoter and terminator regions of genes. Here we investigate
transcription of fragments longer than 200 nucleotides, focusing on antisense transcription for known protein coding genes
and intergenic transcription. We find that roughly 12% to 16% of all reads that originate from promoter and terminator
regions, respectively, map antisense to the gene in question. Furthermore, we detect a high number of novel
transcriptionally active regions (TARs) that are generally expressed at a lower level than protein coding genes. We find that
the correlation between RNA-seq data and microarray data is dependent on the gene length, with longer genes showing a
better correlation. We detect high antisense transcriptional activity from promoter, terminator and intron regions of protein-
coding genes and identify a vast number of previously unidentified TARs, including putative novel EGFR transcripts. This
shows that in-depth analysis of the transcriptome using RNA-seq is a valuable tool for understanding complex
transcriptional events. Furthermore, the development of new algorithms for estimation of gene expression from RNA-seq
data is necessary to minimize length bias. Citation: Klevebring D, Bjursell M, Emanuelsson O, Lundeberg J (2010) In-Depth Transcriptome Analysis Reveals Novel TARs and Prevalent Antisense
Transcription in Human Cell Lines. PLoS ONE 5(3): e9762. doi:10.1371/journal.pone.0009762 Editor: Fatah Kashanchi, George Washington University, United States of America Received December 3, 2009; Accepted February 22, 2010; Published March 25, 2010 Received December 3, 2009; Accepted February 22, 2010; Published March 25, 2010 Copyright: 2010 Klevebring 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 Swedish Scientific Council, and the Knut and Alice Wallenberg Foundation. 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. Funding: This work was supported by the Swedish Scientific Council, and the Knut and Alice Wallenberg Foundation. 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. Investigation of antisense expression The vast majority of all reads originate from the sense strand of
protein-coding genes (figure 2A and figure S3). A large fraction of
the reads also originate from the introns of protein-coding genes,
but when normalized to the length of the introns, the relative
expression levels of introns are very low (figure 2B and figure S3,
S4, S5). We also note that many reads map to regions distant from
protein-coding genes (here denoted as ‘‘Other’’), which to some
extent can be expected since this includes many long non-protein-
coding genes. Previous studies have described a class of short
transcripts (20–90 nucleotides) that originate from the antisense
strand in the promoter regions of genes [7–9,15]. We investigated
tag densities in promoter and terminator regions (defined as 1000
base pairs upstream and downstream of genes, respectively) and
are unable to detect an increased density upstream of genes. This
is expected since our extraction method does not capture
fragments shorter than roughly 200 nucleotides. In the terminator
regions, however, the relative antisense tag density is higher than
that in exons and promoter regions (figure 2B and figure S3). This
indicates that transcription of long RNAs in terminator regions
could represent a regulatory mechanism for termination of
transcription. We investigated the sense-to-antisense ratio for Even though most novel TARs seem to be lowly expressed, we
find a few interesting instances among these. Several clusters are
detected downstream of a tRNA pseudogene on chromosome 3. We believe that this is the result of transcription which has been
initiated upstream of the pseudogene and continues downstream
(Figure 4A). The pseudogene itself has 100% sequence identity to
another region in the genome (chr5:79,982,623–79,982,691), and
since only reads that map uniquely to the genome were used in this
analysis, this gene appears not to be expressed. Very high
expression of EGFR is one of the hallmarks for the A431 cell line
[16]. In figure 4B, a prolonged exon of the epidermal growth
factor receptor (EGFR) is shown, along with two small clusters
several tens of kilobases away. Whether or not the two small Figure 1. Scatter plot of RNA-seq (x-axis) versus microarray log2(fold change) (y-axis) for all protein coding genes (A), genes
shorter than 2000 nts (B) and genes longer than 10 000 nts (C). Colors dots indicate the most similar microarray probe (black), the mean
probe ratio (red) and median ratio (green). Identification of novel TARs To identify novel TARs, we merged reads from all three cell
lines and created clusters from overlapping and nearby reads. After subtraction of known genes and non-coding RNA genes, we
identify approximately 40,000 novel TARs, of which most are
short (figure 3A). In fact, only 1360 TARs are longer than 500
base pairs and only 508 are longer than 1000 base pairs. Expression values for all TARs were calculated using the same
approach as for protein-coding genes. This showed that most
TARs are lowly expressed and covered by few reads. In A431,
only approximately 10% (4144 TARs) are detected by 10 reads or
more (figure 3B). The corresponding number in protein-coding
genes is 33%, but this number is likely biased by the fact that
protein-coding genes are generally longer than the putative novel
TARs (data not shown). The method for RNA extraction used in
this study excludes fragments shorter than approximately 200
nucleotides. This suggests that the majority of TARs identified in
this study are in fact 200 nucleotides or longer and that deeper
sequencing is needed to cover the entire TARs in order to define
their boundaries. Comparison of RNA-seq and microarray gene expression
data To validate the results obtained from RNA-seq, we compared
the data to gene expression data from the A431 and U251 cell
lines obtained using microarrays (no data was available for U-2 In this study, we use massive DNA sequencing to investigate
RNA longer than 200 nucleotides from three human cancer cell March 2010 | Volume 5 | Issue 3 | e9762 1 Human Antisense Transcription OS). Since the microarray platform only generates relative
expression values, the correlation between the RNA-seq data
and the microarray data was calculated using the log2 value of the
ratio between A431 and U251, which in the RNA-seq case yields
one value per Ensembl-gene. Since one gene can be represented
by several microarray probes, we used three different methods to
convert these to a single value that could be compared to the
RNA-seq data (mean, median and best probe, see Materials and
Methods for details). The Spearman correlation was determined to
0.55, 0.55 and 0.64 for the three methods respectively, values in
the same range as those described earlier [13]. Oshlack and
Wakefield recently showed that the variance estimation of the
RPKM measure is dependent on the gene length [14]. Thus, we
hypothesized that the correlation between microarray data and
RNA-seq data would share this dependence, since the log2-fold
change in RNA-seq will have lower variance for longer genes that
for shorter genes. This assumption turned out to be correct; for
genes shorter than 2000 bases, the correlation was 0.48 to 0.52
depending on method, while for genes longer than 10 kb, this
range was 0.59 to 0.71 (figure 1B–C and figure S2). different regions of the genome. In protein-coding exons, 98% to
99.5% of the reads originate from the sense strand, indicating that
antisense transcripts are present at very low levels (figure 2C and
figure S3). Interestingly, the sense-to-antisense ratio is markedly
increased for promoter and terminator regions. In promoter
regions, about 12% of the reads originate from the antisense
strand, and in terminator regions, the fraction is 16%. In introns,
the corresponding number increases to approximately 50%
(figure 2C and figure S3). Investigation of antisense expression Longer genes correlate better with microarray data than short genes. doi:10.1371/journal.pone.0009762.g001 Figure 1. Scatter plot of RNA-seq (x-axis) versus microarray log2(fold change) (y-axis) for all protein coding genes (A), genes
shorter than 2000 nts (B) and genes longer than 10 000 nts (C). Colors dots indicate the most similar microarray probe (black), the mean
probe ratio (red) and median ratio (green). Longer genes correlate better with microarray data than short genes. doi:10.1371/journal.pone.0009762.g001 March 2010 | Volume 5 | Issue 3 | e9762 PLoS ONE | www.plosone.org 2 Human Antisense Transcription Human Antisense Transcription Figure 2. (A) Pie chart describing read mappings to different genomic regions. Almost half of all reads map to known genes (5 0 UTRs, CDS
and 3 0 UTRs). A large fraction of reads map to regions outside promoters, known genes or terminator regions (red). A very low fraction of all reads
map to antisense to protein coding genes. (B) Relative expression densities in different genomics regions. For the antisense strand, a small increase
can be seen in promoter and terminator regions. Error bars are one standard deviation calculated across all three samples. (C) Fraction of reads that
map to different regions in the genome. In introns, roughly half of all reads map to the antisense strand. Sense in light gray, antisense in dark gray. doi:10.1371/journal.pone.0009762.g002 Figure 2. (A) Pie chart describing read mappings to different genomic regions. Almost half of all reads map to known genes (5 0 UTRs, CDS
and 3 0 UTRs). A large fraction of reads map to regions outside promoters, known genes or terminator regions (red). A very low fraction of all reads
map to antisense to protein coding genes. (B) Relative expression densities in different genomics regions. For the antisense strand, a small increase
can be seen in promoter and terminator regions. Error bars are one standard deviation calculated across all three samples. (C) Fraction of reads that
map to different regions in the genome. In introns, roughly half of all reads map to the antisense strand. Sense in light gray, antisense in dark gray. doi:10.1371/journal.pone.0009762.g002 clusters are in fact novels exons remains to be investigated. In
figure 4C, transcription is detected from both strands of a 1.3 Mb-
region surrounding Peroxisome Proliferator-activated Receptor c
Coactivator-1 a (PPARGC1A) on chromosome 4. Investigation of antisense expression Expression from
this regions is detected at high levels in A-431 cells, but is almost
completely shut off in U-2 OS and U-251 cells. This provides an
intriguing example of complex transcription, and could indicate
gene regulation through antisense transcript expression. This gene
has been implicated in diabetes where lower expression has been
linked to insulin resistance and DNA damage [17]. What
functional role the antisense transcript plays remains to be
elucidated. increased precision in expression level measurements for longer
genes due to the fact that a higher number of reads will map to
longer genes than short, as described earlier [14]. In this study, this
means that the correlation between DNA microarrays and RNA-
seq ranges from approximately 0.48 to 0.71 depending on which
gene length and microarray probe selection method is used. However, this points to a larger statistical issue when using RNA-
seq data to assess differential expression, since long genes will bias
for example lists of differentially expressed genes between samples,
and thus influence the power of gene set enrichment analysis
negatively. Future research in this area will certainly have to
address this issue, for example by improved statistical methods or
by limiting the analysis to reads mapping to the 3 0 part of the gene
with length equal to the shortest gene included in the analysis. Discussion We show that for approximately 20% of all human protein-
coding genes, there is at least weak antisense transcription to
exonic sequences. We also show that many of the antisense
signatures overlap between the investigated cell lines (figure S3b). During recent years, several studies have indicated that 20–90% of In the current study we have investigated the transcriptional
levels of three human cancer cell lines using RNA-seq. We show
that the correlation between DNA microarray data and RNA-seq
data depends on gene length, and that the reason for this is the Figure 3. (A) Histogram of detected TAR lengths. Many regions appear as shorter than 200 base pairs which is likely caused by the fact that
they are very lowly expressed. The RNA extraction method captures fragments longer than 200 nucleotides, indicating that the majority of the
detected TARs are in fact longer than we detect. (B) Bar plot describing number of reads in the detected TARs. Roughly 5 000 of the 40 000 TARs are
detected by more than 10 reads. Error bars are one standard deviation calculated across all three samples. doi:10.1371/journal.pone.0009762.g003 Figure 3. (A) Histogram of detected TAR lengths. Many regions appear as shorter than 200 base pairs which is likely caused by the fact that
they are very lowly expressed. The RNA extraction method captures fragments longer than 200 nucleotides, indicating that the majority of the
detected TARs are in fact longer than we detect. (B) Bar plot describing number of reads in the detected TARs. Roughly 5 000 of the 40 000 TARs are
detected by more than 10 reads. Error bars are one standard deviation calculated across all three samples. doi:10.1371/journal.pone.0009762.g003 PLoS ONE | www.plosone.org March 2010 | Volume 5 | Issue 3 | e9762 3 Human Human Antisense Transcription PLoS ONE | www.plosone.org
4
March 2010 | Volu March 2010 | Volume 5 | Issue 3 | e9762 PLoS ONE | www.plosone.org Human Antisense Transcription Human Antisense Transcription Figure 4. (A) Transcription downstream of a known tRNA pseudogene. Several TARs are detected in all three cell lines. The pseudogene
itself has perfect identity to another region in the genome, so reads derived from is will not map unambiguously are are discarded. (B) Several TARs
are detected downstream the gene EGFR. A group of them likely describe a prolonged 3 0 UTR. Two TARs further downstream could represent new
exons. Materials and Methods A431, U-2 OS and U251 cells were grown as described earlier
[21]. Cells were harvested and RNA was extracted using the
RNeasy mini kit (Qiagen, Valencia, CA) following the manufac-
turer’s instructions, and 15 m g of total RNA was used as input
material for the SOLiD Whole Transcriptome kit (Applied
Biosystems Inc., Foster City, CA) and 14 372 246, 10 547 681
and 11 449 673 reads (each 50 nucleotides) passed quality filters
including filtering against adaptors. The reads were mapped to
chromosomes 1–22, X and Y of the human genome (hg18) using
Corona lite with default parameters (Life Technologies/Applied
Biosystems). Identification of novel transcriptionally active regions To identify putative novel transcriptionally active regions, we
clustered reads (using the online-version of Galaxy, [24] allowing
for reads to be 15 bases apart and require at least three reads to be
present to form a cluster. These first clusters were then merged
across cell lines. We then subtracted clusters that overlap with
known genes (as defined by Ensembl genes) as well as non-coding
RNA genes (RNA genes, UCSC Genome Browser). Supporting Information Figure S1
Overlap of sense and antisense expression between
the cell lines. Found
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doi:10.1371/journal.pone.0009762.s001
(0.23
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at:
doi:10.1371/journal.pone.0009762.s001
(0.23
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Correlation to microarray data, binned per gene
length in intervals of 2000 bps. See main text for discussion. Found at: doi:10.1371/journal.pone.0009762.s002 (11.70 MB
TIF) Figure S2
Correlation to microarray data, binned per gene
length in intervals of 2000 bps. See main text for discussion. Found at: doi:10.1371/journal.pone.0009762.s002 (11.70 MB
TIF) Figure S3
Information on read mappings for additional cell
lines. (D, G) Fraction of reads mapping to different regions. (E, H)
Relative tag density in different regions. (F, I) Fraction reads
mapping to the sense and antisense strand for different regions. See main text for discussion. Found
at:
doi:10.1371/journal.pone.0009762.s003
(0.20
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PDF) Figure S3
Information on read mappings for additional cell
lines. (D, G) Fraction of reads mapping to different regions. (E, H)
Relative tag density in different regions. (F, I) Fraction reads
mapping to the sense and antisense strand for different regions. See main text for discussion. Sense versus antisense expression regions To investigate the sense and antisense expression in different
genomic regions, we calculated the number of reads that map to
each region of interest. Some regions (coding regions, introns, 5 0
UTRs and 3 0 UTRs of protein-coding genes) were downloaded as
BED-files
from
the
UCSC
table
browser. Promoter
and
terminator regions were defined as 1 kb upstream or downstream
of a protein-coding gene, respectively, similar to what has been
used earlier [7]. If a neighbouring gene resides within the
promoter or terminator region, the overlap with this gene was
removed from the promoter or terminator region. For each region
type, we calculated the expression density by counting number of
reads that map entirely within the region type and normalized to
the total length of the regions and the total amount of sense or
antisense reads. This procedure yields one relative tag density
value for each region type, sense and sample. We also calculated
the sense-to-antisense ratio for each regions type and sample. The ENCODE project showed that transcription was frequent
even outside of protein-coding genes [1], and with the recent
emerge of new sequencing technologies, vast numbers of new
transcriptionally active units have been detected. These TARs are
situated in a non-random pattern along the chromosomes,
indicating that they are not general background transcription. Some also show patterns of differential expression (figure 4). As
more in-depth transcriptome studies deposit their data into
publically
available
warehouses,
such
as
Gene
Expression
Omnibus (http:www.ncbi.nlm.nih.gov/geo), more regions like
these will likely be detected and characterized. It will be of great
importance to functionally characterize these novel non-protein-
coding transcripts and their potential role in gene regulation. Discussion The gene and TARs are detected at higher levels in A431 than the other samples, which agrees with previous knowledge [16]. (C) Massive
transcription from a region around PPARGC1A. Transcription is detected from the sense strand several hundred kilobases downstream of the gene, as
well as from the antisense strand several hundred kilobases upstream in A431. Transcription from this region is almost completely shut off in U251
and U-2 OS. d i 10 1371/j
l
0009762 004 doi:10.1371/journal.pone.0009762.g004 doi:10.1371/journal.pone.0009762.g004 for the ratio A431 versus U251. Since one gene can be
interrogated with several microarray probes, three different
methods were used transform the microarray expression data to
one value per gene; the mean of all probes, the median of all
probes or the probe with the value closest to the RNA-seq data. We used Spearman’s rho to quantify the correlation between the
two platforms. all human genes can generate antisense transcripts that can
mediate regulation of the sense transcript [4–6]. Our study falls
into the lower end of that interval, possibly indicating that deeper
sequencing is required to investigate this phenomenon further. We
also investigate antisense transcription in different regions of the
genome. He and colleagues demonstrated that antisense tran-
scription was prevalent upstream of transcription start sites, and
Preker and colleagues showed that these transcripts are polyad-
enylated and short (20–90 nucleotides) [7,18]. We do not identify
such a pattern, which is likely explained by the fact that our study
targets transcripts longer than 200 nucleotides. After clustering
reads, we identify many novel TARs, most of which are shorter
than 200 base pairs. This is likely due to the fact that they are
generally lowly expressed, and a deeper sequencing of these
samples would likely reveal the remaining parts of these TARs. Interestingly, we see approximately equal levels of transcription
from both strands of introns of protein-coding genes. Non-protein
coding intronic transcripts have been shown to be enriched in
genes related to transcription regulation and interact with
promoters to mediate regulation [19,20]. References 14. Oshlack A, Wakefield MJ (2009) Transcript length bias in rna-seq data
confounds systems biology. Biol Direct 4: 14. 1. Birney E, Consortium EP, Stamatoyannopoulos JA, Dutta A, Guigo´ R, et al. (2007) Identification and analysis of functional elements in 1% of the human
genome by the encode pilot project. Nature 447: 799–816. 15. Core LJ, Waterfall JJ, Lis JT (2008) Nascent rna sequencing reveals widespread
pausing and divergent initiation at human promoters. Science 322: 1845–1848. 2. Ebisuya M, Yamamoto T, Nakajima M, Nishida E (2008) Ripples from
neighbouring transcription. Nat Cell Biol. 16. Lin CR, Chen WS, Kruiger W, Stolarsky LS, Weber W, et al. (1984) Expression
cloning of human egf receptor complementary dna: gene amplification and three
related messenger rna products in a431 cells. Science 224: 843–8. 3. Lapidot M, Pilpel Y (2006) Genome-wide natural antisense transcription:
coupling its regulation to its different regulatory mechanisms. EMBO Rep 7:
1216–22. 17. Lai CQ, Tucker KL, Parnell LD, Adiconis X, Garcı´a-Bailo B, et al. (2008)
Ppargc1a variation associated with dna damage, diabetes, and cardiovascular
diseases: the boston puerto rican health study. Diabetes 57: 809–16. 4. Chen J, Sun M, Kent WJ, Huang X, Xie H, et al. (2004) Over 20% of human
transcripts might form sense-antisense pairs. Nucleic Acids Research 32:
4812–20. p
y
18. Preker P, Nielsen J, Kammler S, Lykke-Andersen S, Christensen MS, et al. (2008) Rna exosome depletion reveals transcription upstream of active human
promoters. Science 322: 1851–4. 5. Yelin R, Dahary D, Sorek R, Levanon EY, Goldstein O, et al. (2003)
Widespread occurrence of antisense transcription in the human genome. Nat
Biotechnol 21: 379–86. 19. Nakaya HI, Amaral PP, Louro R, Lopes A, Fachel AA, et al. (2007) Genome
mapping and expression analyses of human intronic noncoding rnas reveal
tissue-specific patterns and enrichment in genes related to regulation of
transcription. Genome Biol 8: R43. 6. Katayama S, Tomaru Y, Kasukawa T, Waki K, Nakanishi M, et al. (2005)
Antisense transcription in the mammalian transcriptome. Science 309: 1564–6. 7. He Y, Vogelstein B, Velculescu VE, Papadopoulos N, Kinzler KW (2008) The
antisense transcriptomes of human cells. Science 322: 1855–7. 20. Louro R, Smirnova AS, Verjovski-Almeida S (2009) Long intronic noncoding
rna transcription: Expression noise or expression choice? Genomics 93:
291–298. 8. Seila AC, Calabrese JM, Levine SS, Yeo GW, Rahl PB, et al. (2008) Divergent
transcription from active promoters. Science 322: 1849–1851. 21. Acknowledgments We would like to thank Dr. Emma Lundberg for providing cells and
Uppsala Genome Center for technical assistance and helpful discussions. Also, Dr. Rickard Sandberg and Daniel Ramskld are acknowledged for
valuable discussion on data analysis and provision of analysis scripts. p
Found
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doi:10.1371/journal.pone.0009762.s004
(1.41
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at:
doi:10.1371/journal.pone.0009762.s004
(1.41
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PDF) Figure S5
Smooth scatterplots of log10(antisense-rpkm) between
samples. Spearman’s rho correlation coefficient is here slightly
lower than that in the sense-case (supplementary figure S4). A
reason for this could be that the majority of antisense transcripts
are lowly expressed. It is also possible that these antisense
transcripts have regulatory function and differ more than the bulk
of mRNAs expressed in a cell. Comparison with microarray data Two-color DNA microarray data for the cell lines A431 and
U251 was provided by Gry et al., and was pre-processed as
described elsewhere [22]. The quality of the arrays has previously
been addressed by comparison with MAQC data [23]. To allow
for comparison to with RNA-seq data, RPKM expression levels
were calculated for every Ensembl gene (http:www.ensembl.org) as
described elsewhere [12], and a log2-fold change was calculated Found
at:
doi:10.1371/journal.pone.0009762.s003
(0.20
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at:
doi:10.1371/journal.pone.0009762.s003
(0.20
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PDF) Figure S4
Smooth scatterplots of log10(rpkm) between samples
along with Spearman’s rho correlation. The correlation is .87 to PLoS ONE | www.plosone.org March 2010 | Volume 5 | Issue 3 | e9762 March 2010 | Volume 5 | Issue 3 | e9762 5 Human Antisense Transcription .88 between all samples. This indicates that most genes have
similar levels across all samples. .88 between all samples. This indicates that most genes have
similar levels across all samples. Author Contributions Conceived and designed the experiments: JL. Performed the experiments:
DK. Analyzed the data: DK MKB OE. Wrote the paper: DK MKB OE
JL. p
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doi:10.1371/journal.pone.0009762.s005
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PDF) References Barbe L, Lundberg E, Oksvold P, Stenius A, Lewin E, et al. (2007) Toward a
confocal subcellular atlas of the human proteome. Molecular & Cellular
Proteomics 7: 499–508. 9. Taft RJ, Glazov EA, Cloonan N, Simons C, Stephen S, et al. (2009) Tiny rnas
associated with transcription start sites in animals. Nat Genet 41: 572–8. 10. Havgaard JH, Torarinsson E, Gorodkin J (2007) Fast pairwise structural rna
alignments by pruning of the dynamical programming matrix. PLoS Comput
Biol 3: 1896–908. 22. Gry M, Rimini R, Stro¨mberg S, Asplund A, Ponte´n F, et al. (2009) Correlations
between rna and protein expression profiles in 23 human cell lines. BMC
Genomics 10: 365. 11. Lindberg J, Lundeberg J (2009) The plasticity of the mammalian transcriptome. Genomics. 23. Klevebring D, Gry M, Lindberg J, Eidefors A, Lundeberg J (2009) Automation
of cdna synthesis and labelling improves reproducibility. Journal of Biomedicine
and Biotechnology 2009: 1–8. 12. Mortazavi A, Williams BA, Mccue K, Schaeffer L, Wold B (2008) Mapping and
quantifying mammalian transcriptomes by rna-seq. Nat Meth. 8 p. 24. Giardine B, Riemer C, Hardison RC, Burhans R, Elnitski L, et al. (2005)
Galaxy: a platform for interactive large-scale genome analysis. Genome Res 15:
1451–5. quantifying mammalian transcriptomes by rna-seq. Nat Meth. 8 p. 13. Fu X, Fu N, Guo S, Yan Z, Xu Y, et al. (2009) Estimating accuracy of rna-seq
and microarrays with proteomics. BMC Genomics 10: 161. PLoS ONE | www.plosone.org March 2010 | Volume 5 | Issue 3 | e9762 6
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Rethinking University Presidencies in Canadian Higher Education
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https://johepal.com
Journal of
Higher Education Policy
And
Leadership Studies
JHEPALS (E-ISSN: 2717-1426)
Rethinking University
Presidencies in Canadian
Higher Education
Lawrence A. White
Faculty of Education,
Unicaf University, CANADA
Email: larry.ookpik@gmail.com
https://orcid.org/0000-0003-4459-4499 https://johepal.com
Journal of
Higher Education Policy
And
Leadership Studies
JHEPALS (E-ISSN: 2717-1426)
Rethinking University
Presidencies in Canadian
Higher Education
Lawrence A. White
Faculty of Education,
Unicaf University, CANADA
Email: larry.ookpik@gmail.com
https://orcid.org/0000-0003-4459-4499 https://johepal.com
Cite article as:
White, L. A. (2023). Rethinking university presidencies in Canadian higher
education. Journal of Higher Education Policy and Leadership Studies, 4(3), 157-
166. https://dx.doi.org/10.61186/johepal.4.3.157
Leadership Studies
JHEPALS (E-ISSN: 2717-1426)
Rethinking University
Presidencies in Canadian
Higher Education
Lawrence A. White
Faculty of Education,
Unicaf University, CANADA
Email: larry.ookpik@gmail.com
https://orcid.org/0000-0003-4459-4499
Article Received
Article Accepted
Published Online
2023/05/27
2023/09/07
2023/09/30 Rethinking University
Presidencies in Canadian
Higher Education Published Online
2023/09/30 Article Accepted
2023/09/07 Published Online
2023/09/30 Article Accepted
2023/09/07 Cite article as: White, L. A. (2023). Rethinking university presidencies in Canadian higher
education. Journal of Higher Education Policy and Leadership Studies, 4(3), 157-
166. https://dx.doi.org/10.61186/johepal.4.3.157 White, L. A. Journal of Higher Education
Policy And Leadership
Studies (JHEPALS)
E-ISSN: 2717-1426
Volume: 4 Issue: 3
pp. 157-166
DOI:
10.61186/johepal.4.3.157 Rethinking University Presidencies in
Canadian Higher Education E-ISSN: 2717-1426
Volume: 4 Issue: 3
pp. 157-166
DOI:
10.61186/johepal.4.3.157 Keywords: Higher Education Leadership; University Presidents; University Culture;
Career Complexity; Leadership Roles Abstract This article discusses the evolving role of university presidents
in Canada. The university president is accountable for the
successful operation of a university and achieving its strategic
goals. The role has become complex due to factors such as the
absence
of
a
standard
definition
of
a
university,
decentralization of funding, and increased competition for
student enrollment and research funding. Universities have
grown considerably, leading to more diverse and activist
populations, larger budgets, and the need for more people to
manage internal and external relationships. University
presidents must now balance the interests of various
stakeholders such as faculty, boards, alumni, governments,
and learners. They must also plan for and address the
challenges created by system modernization and enterprise
technologies,
internationalization,
campus
health
and
security, changing governments, shifting policy directions,
climate change, social justice, and neoliberalism. This article
argues that university presidents must possess strong
communication skills, impeccable management acumen, and
the ability to relate to the evolving realities of learners. This
article concludes that the role of university president has
evolved from being presiding figures to being accountable
leaders who must navigate the complex demands of the
modern university system. Lawrence A. White * Lawrence A. White * Keywords: Higher Education Leadership; University Presidents; University Culture;
Career Complexity; Leadership Roles *Corresponding author’s email: larry.ookpik@gmail.com *Corresponding author’s email: larry.ookpik@gmail.com E-ISSN: 2717-1426 Volume: 4 Issue: 3 DOI: 10.61186/johepal.4.3.157
157 E-ISSN: 2717-1426 Volume: 4 Issue: 3 DOI: 10.61186/johepal.4.3.157 Rethinking University Presidencies “Being president of a university is no way for an adult to make a living. Which is
why so few adults actually attempt to do it. It is to hold a mid-nineteenth-century
ecclesiastical position on top of a late-twentieth-century corporation”. A. Bartlett Giametti, 19th President of Yale University, 1978-1986 The World Changed Unexpectedly, the COVID-19 pandemic occasioned the single largest disruption of education
ever experienced resulting in the closure of learning spaces in some 190 countries and
affecting 1.6 billion learners (United Nations, 2020), as well as educators and families. It
tested and continues to test the resilience, fortitude, and ingenuity of educational leaders
worldwide. As the world becomes more globalized and interconnected, as recognition grows
regarding planetary limits to our civicidal (Hancock, 2019) commodification fetish (Marx,
1887), as violence and environmental disaster lead to mass migration and refugeeism
(Talbot, 2013), as populism threatens acceptance of science as reality (Wall et al., 2017) and
distorts reality through the “weaponisation of information” (Ireton & Posetti, 2018, p. 15),
as the world reckons with institutions and processes grounded in putative privilege for some
(Carr, 2016; McIntosh, 1988), and as the gap between marginalized populations and the rest
of the world grows (Corbett, 2008; Leal Filho et al., 2020), the world turns to education, and
its leaders, for solutions (Kwauk, 2020; Wals & Benavot, 2017). In fact, the OECD (2018)
suggests that education may be the difference between people embracing or being
defeated by future challenges (Common Worlds Research Collective, 2020). Educational
leadership continues to grow increasingly complex as a profession as does the pressure on
an educational leader to perform and succeed (Cafley, 2015). This paper explores the evolving and increasingly complex role of university presidents
focusing predominantly on the characteristics and attributes fostering efficacy. Specifically,
this paper considers post-secondary education leadership in a Canadian context through
both historical and future visionary lenses. Evolution of a Complex Role Establishing the Dominion of Canada in 1867 under the British North America Act created,
perhaps, the earliest and longest lasting of challenges facing university presidents. To assure
a relatively even distribution of power and authority between federal and provincial
governments, Section 93 of The Act gave the provinces authority over education. To
confound matters further, responsibility for academic research remained with the federal
government. Consequently, presidential advocacy efforts for funding are decentralized. Likewise, there is neither a standard definition of ‘university’ across Canada (Usher, 2018)
nor a standard system of accreditation creating a network of unique educational systems
between which student and credit transferability is not streamlined (Paul, 2011). That said,
decentralization also created stability for university presidents by preventing national
interference in and requiring the collaboration of 13 jurisdictions to make changes to the
mandate for universities (Paul, 2011). Canada remains the only federated country without a
national Ministry of Education. Influenced greatly by French and British colonialism, the majority of Canadian
universities prior to 1960 were faith based (Paul, 2011; Usher, 2018). Since c.1960, however,
most universities are secular, many revising their purpose to take advantage of changes to
ongoing provincial funding requiring a public, secular governance model. In Canada, the
separation of state and church in education is now well established. In the latter half of the twentieth century, the university sector in Canada experienced
tremendous growth both in the number of institutions and the proportion of the population
engaged in university study (Paul, 2011). Growth in demand and numbers of competitors
increases the pressure, challenging educational leaders to increase both depth and breadth
of academic programming and to find ways to distinguish their university from others. Absent a complementary increase in funding, growth also created challenges such as larger
class sizes, greater student/teacher ratios, the growing use of sessional and other part-time
instructors (Paul, 2011), and the seemingly ubiquitous task of ephemeralization. In the early 2000s, more than a few colleges and other institutes made the strategic
transformation to become universities (Usher, 2018). These transitions in status create
greater direct competition between universities for student enrollment, both domestic and
international, and for faculty expertise. At the same time, more universities now vie for
limited funding available for research. University Presidents The university president is the chief executive officer of the organization and is formally
accountable to a Board of Governors or Senate for the fiscal and reputational operation of
a university as well as for the successful achievement of strategic plans, goals, and
objectives. Michael et al. (2001, p. 332) describe the university president as “the most visible
embodiment of institutional mission, vision and culture”. Paul (2011) concurs and views the
role as the leader and manager of a university’s big picture while Woodsworth likens the
role to a translator: "communicating between cultures and across borders, conveying the
sense of the academic enterprise to external stakeholders and board members, and
interpreting the business-related concerns and preoccupations of a responsible board to the
internal community" (2013, p. 134). Journal of Higher Education Policy And Leadership Studies (JHEPALS) 158 Evolution of a Complex Role In their previous iterations as colleges rooted in more
technical and applied learning, the new universities also create dilemmas of culture and self-
identity for others that viewed their central roles as research institutions by blurring the
lines between academic research and career preparedness. In Canada, this often
materializes as conflicts of perspective and motivation among faculty who view research as
their mandate and career preparedness as specifically not their responsibility (Spencer,
2014). Unions for post-secondary faculty and staff, akin to those throughout education in
Canada, are particularly effective in representing these and other interests on behalf of their
members, increasing the complexity and criticality of communication and interpersonal
relations for university presidents. In the past, university communities comprised faculty with longevity and a more
homogeneous full-time student body that resided close to campus. Times were simpler;
organizations less complex. University presidents, usually men with previous Canadian post-
secondary executive experience, presided over their institutions more than they managed E-ISSN: 2717-1426 Volume: 4 Issue: 3 DOI: 10.61186/johepal.4.3.157
159 Rethinking University Presidencies or led (Paul, 2011). The president of a small university might know all students, staff, and
faculty; teach one or two courses; represent the university in the community and with
government; and attend or host community events. The role was somewhat low-key
because “universities were not generally in the public eye” (Paul, 2011, p. 12). Contemporary universities have much larger, more diverse, and activist student, staff,
and faculty populations. Some have operating budgets in excess of $500 million and all have
multiple departments staffed with people to manage internal and external relationships. As
Paul (2011) delineates, university presidents are now accountable to: (a) faculty with
interests in academic freedom, profile, prestige, and reputation; (b) a board of governors or
senate that values integrity, financial stability, impeccable management acumen, and stellar
communication skills; (c) alumni seeking strong and stable, but growing, community
relationships; (d) governments needing change agents to weave academic culture with fiscal
performance and the successful achievement of strategic goals; and (e) learners who,
increasingly, have growing obligations (with university studies being just one of many) and
needing a leader who can relate to their evolving realities. Adding in system modernization
and enterprise technologies; internationalization and globalization; campus health, well-
being, safety, and security; politics, changing governments, and shifting policy directions;
changing and increasingly less reliable funding mechanisms; evolving best practices in
learning and teaching; and competing and conflicting demands from almost every possible
direction, it is clear that presidential workloads are expanding and that expectations for
success are more numerous, diverse, and acute than ever (Khalideen, 2014; Fullan &
Leithwood, 2012; Paul, 2011). The continuously mounting and intensifying responsibility, and concomitant
complexity, required to lead an increasingly public institution led to the development of a
satirical job posting: Wanted: A miracle worker who can do more with less, pacify rival groups,
endure chronic second-guessing, tolerate low levels of support, process large
volumes of paper, and work double shifts (75 nights a year out). He or she will
have carte blanche to innovate, but cannot spend much money, replace any
personnel, or upset any constituency. (Evans, 1995, p. 23) Wanted: A miracle worker who can do more with less, pacify rival groups,
endure chronic second-guessing, tolerate low levels of support, process large
volumes of paper, and work double shifts (75 nights a year out). Rethinking University Presidencies He or she will
have carte blanche to innovate, but cannot spend much money, replace any
personnel, or upset any constituency. (Evans, 1995, p. 23) Challenges of Culture Perhaps the single greatest challenge facing a university president, especially one who may
have recently been appointed from outside the organization, is developing an understanding
about and participating effectively and respectfully within the culture of the university. It
can be a delicate balance between being culturally aware and respectful, and finding ways
to move a new agenda for change positively forward, including finding opportunities to voice
a few key strategic initiatives as frequently as possible (Paul, 2011). Ultimately, the fit of a president within the culture of a university often determines
success going forward (Cafley, 2015; Paul, 2011). Every stakeholder, internal and external,
has a personal perspective on fit and the efficacy of the person in the role. To help navigate
the challenge of innumerable perspectives that, I would argue, is not dissimilar to
simultaneous, diverse, continuous, on-the-job performance evaluations, Bennis (2009)
identifies six core competencies required of a university president: (a) have a strong sense Journal of Higher Education Policy And Leadership Studies (JHEPALS)
160 Journal of Higher Education Policy And Leadership Studies (JHEPALS) Presidencies Looking Forward Presidential roles are influenced significantly by current events, especially if those events
arise from systemic social oppression or systemic consumerism. Freire (2000) advocates
empowering individual consciousness to overcome oppression in education. In Canada, this
is best exemplified by three significant social uprisings: (a) the calls to action arising from
the Truth and Reconciliation Commission’s final report (see Truth and Reconciliation
Commission of Canada, 2015); (b) the rise of Black Lives Matter and its founding principles
of diversity, intersectionality, empathy, and engagement (see Potvin, 2020); and (c) the
#MeToo movement that challenged post-secondary leaders to acknowledge that an
absence of policy and action may have increased vulnerabilities, predominantly those of
female students and staff, on campuses across the country (see MacArthur, 2019). Similarly, Orr (1992, 2020) and others (Common Worlds Research Collective, 2020;
Leal Filho et al., 2020; Reid, 2019; Suzuki, 1998) advocate taking action to mitigate the “long
emergency” (Orr, 2020, p. 13) created by our tendency to overlook anthropocentric causes
of the climate crisis facing the planet. Both the global #FridaysForFuture movement
demanding adults believe climate science and take action against climate change (see
Bowman, 2019; Pickard, 2019), and the fossil fuel divestment movement in Canadian
academia (see Maina et al., 2020) are affecting policy directions and, therefore, affecting
the qualities and characteristics of an effective university president going forward. I argue that, in order to be both relevant and successful in leading universities,
presidents of the 21st century must be responsive to these and other growing public
demands for changes in policy and action, particularly regarding systemic issues. Future
presidents will be those who can capitalize on university culture, build on past
accomplishments of the institution, implement an agenda focused on teaching learners how
to learn, foster agency in learners, and ensure they have a solid foundation of skills to
function and adapt to the 21st century workplace and climate (Bates, 2019; OECD, 2018). In
order to produce graduates who are active citizens that know how to “navigate through
uncertainty” (OECD, 2018, p. 5), presidents must be able to anticipate ambiguous future
global and community needs and to champion this direction within the university
community and among external partners. Furthermore, this should promote the strengths and opportunities that come with
diversity, inclusivity, collaboration, and respect for the valuable contributions each person
brings. Journal of Higher Education Policy And Leadership Studies (JHEPALS) 160 White, L. A. of mission, (b) foster support for the mission among others, (c) establish relationships that
are flexible and adaptable, (d) engender optimism and trust, (e) develop leadership qualities
in others, and (f) achieve results. Cafley (2015) combines the first two of Bennis’
competencies, suggesting that these and success of the organization might be better served
by working toward these collaboratively with the university community. Campbell
(2003/2016) describes fit within organizational culture as the basis for a solid, positive,
successful working relationship with its president. E-ISSN: 2717-1426 Volume: 4 Issue: 3 DOI: 10.61186/johepal.4.3.157 Presidencies Looking Forward Presidents must be prepared to change mindsets; specifically, to promote and instill
a cultural proficiency mindset across the university community (Lindsey et al., 2019). That
is, they must come with some form of vision for changing the general perception of cultural
differences as problematic to one of intercultural interaction that celebrates diversity and, E-ISSN: 2717-1426 Volume: 4 Issue: 3 DOI: 10.61186/johepal.4.3.157
161 E-ISSN: 2717-1426 Volume: 4 Issue: 3 DOI: 10.61186/johepal.4.3.157 Rethinking University Presidencies academically, to one that recognizes the value of cultural uniqueness as a foundation from
which to create learning experiences. Such presidents would be confident enough in
themselves, their beliefs, values, and worldviews to be robustly self-reflective and
sufficiently self-disruptive and open to reconsidering, and changing, deep-rooted meanings. They must model critical thinking, creative problem solving, and communication
transparently, respectfully, and collaboratively. They may wish to surround themselves with a senior executive team that shares and
believes in the mission and strategic vision but that may have perspectives on pathways to
success that differ and which, together, facilitate, foster, and nurture the development of
emergent citizens who are prepared for the challenges of any eventuality (Islam et al., 2006). Learning from the COVID-19 experience, university presidents must come with a plan to
work with the university community and with the executive team, specifically, to strengthen
the capacities for risk and resilience of the institution, to reimagine education and the right
to equitable learning, to remove barriers to learning, and to support the professional
development of all faculty and staff (United Nations, 2020). E-ISSN: 2717-1426 Volume: 4 Issue: 3 DOI: 10.61186/johepal.4.3.157 161 Human Participants p
Human participants were not consulted or otherwise involved in this research. Funding Funding
This research received no specific grant from any funding agency in the public, commercial,
or not-for-profit sectors. Conclusion From climate to technology, politics to academics, and from global health to individual
people, the education landscape today is shifting and evolving at an ever-increasing pace. Canadian Prime Minister Justin Trudeau commented at the 2018 World Economic Forum in
Davos, Switzerland, that “the pace of change has never been this fast, yet it will never be
this slow again” (World Economic Forum, 2018, 5:34). The roles and spheres of influence of universities have expanded greatly in recent
decades. No longer can the role of a university president appear as a concise list of
management and leadership duties on a single page job posting. “Leadership within a system
of education that has been comfortable, predictable and ordered, to one that is new,
uncertain and chaotic is no small task” (Khalideen, 2014, p. 44). Instead, in the coming decades, university presidents will be required to work and
interact with stakeholders simultaneously at the micro, meso, and macro levels and to
develop autonomy and agency across a team, indeed across an entire community, in order
to support them in this work (Fullan & Leithwood, 2012). They must demonstrate an overtly
high degree of social respect, emotional intelligence, spiritual wisdom, integrity, and
maturity (Astin & Astin, 2000) as well as sufficient self-awareness, self-efficacy, and self-
disruptive openness to consider changing their own deep-rooted belief systems and
worldviews. The future requires a university leader who genuinely understands, in a deeply
personal way, the value of respectful and equitable collaboration in the co-creation of
knowledge (Harris et al., 2019) and “learning to become with the world” (Common Worlds
Research Collective, 2020). I conclude with a word of caution. In moving in these directions and in having these
expectations of university leaders, we run the risk of expecting them to be “all things to all
people” (Evans, 1995, para. 8). With the risks of professional burnout or making university
presidencies unattainable, we need to temper our enthusiasm and find ways to support and
help leaders to cope in the challenging times ahead. After all, if education is the solution to
global challenges and problems, their success is our success. Journal of Higher Education Policy And Leadership Studies (JHEPALS)
162 Journal of Higher Education Policy And Leadership Studies (JHEPALS) 162 g
The author declares that there is no conflict of interest. g
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content/uploads/2018/08/HESA SPEC 2018 final pdf Rethinking University Presidencies Rethinking University Presidencies Mr. Lawrence A. White is an advanced graduate student pursuing a Doctor of Education (EdD) at Unicaf
University. His current research interests lie at the intersection of curriculum and pedagogical design and
development with supporting learner mental health. In a grounded theory study rooted in lived experience
and pedagogical journey mapping, he hopes to identify new pedagogical approaches that mindfully reduce
or remove academic stressors that students invariably experience on their academic journeys in the hope
of reducing challenges to mental well-being and improving academic success. White also holds a Master of
Distance Education (MDE) and a Bachelor of Environmental Studies (BES). He worked for 20+ years in various
increasingly senior leadership roles in Canadian colleges and universities throughout his career, is active
with several global research projects related to mental health, and volunteers with the Global Mental Health
Peer Network. He now lives in Yellowknife, Northwest Territories, Canada. Mr. Lawrence A. White is an advanced graduate student pursuing a Doctor of Education (EdD) at Unicaf
University. His current research interests lie at the intersection of curriculum and pedagogical design and
development with supporting learner mental health. In a grounded theory study rooted in lived experience
and pedagogical journey mapping, he hopes to identify new pedagogical approaches that mindfully reduce
or remove academic stressors that students invariably experience on their academic journeys in the hope
of reducing challenges to mental well-being and improving academic success. White also holds a Master of
Distance Education (MDE) and a Bachelor of Environmental Studies (BES). He worked for 20+ years in various
increasingly senior leadership roles in Canadian colleges and universities throughout his career, is active
with several global research projects related to mental health, and volunteers with the Global Mental Health
Peer Network. He now lives in Yellowknife, Northwest Territories, Canada. This is an open access article distributed under the terms of the Creative Commons
Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) which allows reusers to distribute, remix, adapt,
and build upon the material in any medium or format for non-commercial purposes only, and only so long as
attribution is given to the creator. Journal of Higher Education Policy And Leadership Studies (JHEPALS)
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Canada [Video]. YouTube. https://www.youtube.com/watch?v=WQdLDMLrYIA E-ISSN: 2717-1426 Volume: 4 Issue: 3 DOI: 10.61186/johepal.4.3.157 165 Rethinking University Presidencies
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Age aspects of the effects of the new coronavirus infection (COVID 19) on certain biochemical blood parameters
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М.М. Алимурзаева1,2, С.М. Изудинова2, А.М. джафарова1,2, Р.А. Халилов1 1Дагестанский государственный университет, Махачкала, Дагестан
2Медико-санитарная часть МВД по Республике Дагестан, Махачкала, Дагестан
Автор, ответственный за переписку: Альбина Мехьядиновна Джафарова, albina19764@mail.ru Аннотация. Цель: изучение влияния новой коронавирусной инфекции на ряд стандартных биохимических пока
зателей крови у больных разного возраста. Материалы и методы: исследовано 514 пациентов трёх возрастных групп
(I группа — 25-35лет, II группа — 36-50 лет, III группа — 51-75 лет) с диагнозом «Внебольничная пневмония, вызван
ная инфекцией COVID-19». Определение всех биохимических параметров крови больных производили на биохимиче
ском анализаторе Indiko. Результаты: исследование показателей азотистого метаболизма показало, что у больных всех
возрастных групп содержание общего белка и мочевины не претерпевает существенных изменений, однако при этом
происходит повышение концентрации креатинина и мочевой кислоты, наиболее выраженное у пациентов III группы. Исследование активностей АСТ, АЛТ в крови больных показало существенное их повышение у всех возрастных кате
горий, более значимое — у лиц II и III возрастных групп. При этом с возрастном увеличиваются активность а-амилазы,
концентрации билирубина и снижаются уровни железа, достигая патологических значений. Содержание маркера вос
паления (СРБ) демонстрирует наиболее выраженную зависимость от возраста больных: у больных I группы оно уве
личивается в 6,9 раз, у II — в 12,3 раза, у лиц старше 51 года — в 17,25 раз. Вывод: с увеличением возраста больных
COVID-19 отклонения многих биохимических показателей от соответствующих каждой группе контрольных уровней
становятся более выраженными и достигают значений, существенно отклоняющихся от референсных. К
COVID 19
б
б Ключевые слова: COVID-19, возраст больных, кровь, биохимические параметры. Финансирование. Исследование не имело спонсорской поддержки. ,
р
, р
,
р
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Финансирование. Исследование не имело спонсорской поддержки. Для цитирования: Алимурзаева М.М., Изудинова С.М., Джафарова А.М., Халилов Р.А. Возрастные особенности из
менений биохимических параметров крови при COVID-19. Медицинский вестник Юга России. 2023;14(2):90-96. DOI
10.21886/2219-8075-2023-14-2-90-96 Оригинальная статья
УДК 616.9
https://doi.org/10.21886/2219-8075-2023-14-2-90-96 Оригинальная статья
УДК 616.9
https://doi.org/10.21886/2219-8075-2023-14-2-90-96 УДК 616.9
https://doi.org/10.21886/2219-8075-2023-14-2-90-96 Медицинский вестник Юга России
2023; 14(2):90-96 © М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов, 2023 М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов
ВОЗРАСТНЫЕ ОСОБЕННОСТИ ИЗМ ЕНЕНИЙ БИОХИМИЧЕСКИХ
ПАРАМЕТРОВ КРОВИ ПРИ COVID-19 М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов
ВОЗРАСТНЫЕ ОСОБЕННОСТИ ИЗМ ЕНЕНИЙ БИОХИМИЧЕСКИХ
ПАРАМЕТРОВ КРОВИ ПРИ COVID-19 ИНФЕКЦИОННЫЕ БОЛЕЗНИ
3.1.22 М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов
ВОЗРАСТНЫЕ ОСОБЕННОСТИ ИЗМ ЕНЕНИЙ БИОХИМИЧЕСКИХ
ПАРАМЕТРОВ КРОВИ ПРИ COVID-19 М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов
ВОЗРАСТНЫЕ ОСОБЕННОСТИ ИЗМ ЕНЕНИЙ БИОХИМИЧЕСКИХ
ПАРАМЕТРОВ КРОВИ ПРИ COVID-19 ИНФЕКЦИОННЫЕ БОЛЕЗНИ
3.1.22 Материалы и методы Ретроспективное исследование проводили на базе
Медико-санитарной части МВД по РД (г. Махачкала). В период начала пандемии коронавирусной инфекции
(апрель-май 2020 г.) в госпитале было организовано отде
ление для пациентов с диагнозом «внебольничная пнев
мония, вызванная инфекцией COVID-19» (подтверж
дённая обнаружением РНК SARS-CoV-2 или, в редких
случаях — только данными КТ). Исследованы биохими
ческие показатели крови больных на момент госпитали
зации в стационар на 6,5±2,5 день болезни. Критериями
включения в группы для проведения сравнительного ана
лиза были 1-й день госпитализации больных, мужской
пол, средняя степень тяжести процесса, поражение лёг
ких, по данным КТ, от 20% до 50%. Критериями исключе
ния из исследования служили наличие в анамнезе пере
несённого инфаркта миокарда и инсульта, нестабильной
стенокардии, сердечной недостаточности II-IV степени,
эндокринных заболеваний. Вирус может проникать в клетки эпителия желудка,
тонкой и толстой кишки. Имеются сведения о специфи
ческом поражении сосудов (эндотелия), миокарда, по
чек и других органов. Установлено, что диссеминация
SARS-CoV-2 из системного кровотока или через пла
стинку решетчатой кости может привести к поражению
головного мозга. Обнаружена возможность специфиче
ского поражения лимфоцитов и гиперактивности ма
крофагов [3]. Исследовано три группы разных возрастных кате
горий: I группа — 109 пациентов возрастом 25-35 лет,
II группа — 310 пациентов возрастом 36-50 лет, III груп
па — 95 пациентов возрастом 51-75 лет. В качестве кон
троля исследованы биохимические параметры мужчин
тех же возрастных групп, не болеющих COVID-19 и не
имеющих выраженных патологий со стороны сердечно
сосудистой системы, органов пищеварения, эндокринной
системы, выделительной системы. Забор венозной крови
производился утром натощак в первые сутки поступле
ния больных в специальные вакуумные пробирки с акти
ватором свертывания. Определение всех биохимических
параметров в сыворотке крови производили на биохими
ческом анализаторе Indiko (Thermal Fisher, США). При критическом течении COVID-19 вследствие дис-
регуляции синтеза провоспалительных и антивоспали
тельных цитокинов развивается патологическая реакция,
называемая «цитокиновым штормом». Гиперактивация
иммунного ответа при COVID-19 часто ограничивается
лёгочной паренхимой, прилегающей альвеолярной лим
фоидной тканью и ассоциируется с развитием острого
респираторного дисстресс-синдрома (ОРДС). У пациен
тов с критическим течением развивается васкулярная эн
дотелиальная дисфункция, коагулопатия, тромбозы. Ци-
токиновый шторм при COVID-19, как правило, приводит
к полиорганной недостаточности и может стать причи
ной летального исхода [4]. Статистическую
обработку данных производили
с помощью однофакторного дисперсионного анали
за (ANOVA) с использованием пакета прикладных про
грамм Statistica 8.0 (StatSoft, Inc., США). Нормальность
распределения оценивали критерием Шапиро-Уилка. До
стоверность различий между нормально распределенны
ми данными определяли с помощью критерия Фишера на
уровне значимости p < 0.05. Medical Herald of the South of Russia
2023; 14(2):90-96 Введение Цель исследования — изучение влияния новой коро
навирусной инфекции на ряд стандартных биохимиче
ских показателей крови у больных разного возраста В 2019 г. произошла вспышка острой респираторной
инфекции, вызванной вирусом Sars-Cov-2 семейства ко
роновирусов, которая быстро распространилась по все
му миру и привела к глобальной пандемии Covid 19. На
чальным этапом заражения является проникновение
SARS-CoV-2 в клетки-мишени, имеющие рецепторы ан-
гиотензинпревращающего фермента II типа (АСЕ-2) [1]. Рецепторы АСЕ-2 экспрессированы на поверхности раз
личных клеток органов дыхания, пищевода, кишечни
ка, сердца, надпочечников, мочевого пузыря, головно
го мозга (гипоталамуса) и гипофиза, а также эндотелия
и макрофагов. Однако основной и быстро достижимой
мишенью SARS-CoV-2 являются альвеолярные клетки
II типа лёгких, что определяет развитие диффузного аль
веолярного повреждения [2]. INFECTIOUS DISEASES
3.1.22 M.M. Alimurzaeva, S.M. Izudinova, A.M. Dzhafarova, R.A. Khalilov
AGE ASPECTS OF THE EFFECTS OF THE N E w CORONAVIRUS INFECTION (COVID 19)
ON CERTAIN BIOCHEMICAL BLOOD PARAMETERS For citation: Alimurzaeva M.M., Izudinova S.M., Dzhafarova A.M., Khalilov R.A. Age aspects of the effects of the new corona-
virus infection (COVID 19) on certain biochemical blood parameters. Medical Herald of the South of Russia. 2023;14(2):90-96. DOI 10.21886/2219-8075-2023-14-2-90-96 Материалы и методы Данные в таблице приведены
в следующем виде: среднее (M) ± ошибка среднего (m). Высокая
летальность
и
громадные
социально
экономические последствия эпидемии COVID-19 требу
ют анализа имеющихся наблюдений и разработки мето
дов эффективной терапии и профилактики осложнений. Известно, что возраст и сопутствующая патология могут
быть факторами, предопределяющими тяжёлое течение
COVID-19. Прежде всего к группе риска относятся по
жилые или лица с хроническими заболеваниями. Однако
и у молодых людей без хронических заболеваний также
могут возникнуть потенциально летальные осложнения,
такие как молниеносный миокардит и диссеминирован
ная внутрисосудистая коагулопатия (ДВС-синдром) [5]. Таким образом, риск инфицирования и тяжёлого течения
болезни или летального исхода затрагивает всех и имеет
ся во всех возрастных группах. В связи с этим возникает
необходимость анализа клинико-лабораторных особен
ностей течения новой коронавирусной инфекции у боль
ных различных возрастных категорий. M.M. Alimurzaeva1,2, S.M. Izudinova2, A.M. Dzhafarova1,2, R.A. Khalilov1 Dagestan State University, Makhachkala, Dagestan
2Medical and sanitary unit of the Ministry of Internal Affairs for the Republic of Dagestan, Makhachkala, Dagestan
Corresponding author: Albina M. Dzhafarova, albina19764@mail.ru Abstract. Objective: to study the effects of a new coronovirus infection on a number of standard biochemical blood
parameters in patients of different ages. Materials and methods: 514 patients of 3 age groups (group 1st — 25-35 years old,
group 2nd — 36-50 years old and group 3rd — over 51 years old) with a diagnosis of community-acquired pneumonia caused
by COVID-19 infection were studied. All biochemical parameters of the blood of patients were determined using an Indiko
biochemical analyzer. Results: the study of nitrogen metabolism indicators showed that in patients of all age groups, the content
of total protein and urea does not undergo significant changes, however, there is an increase in the concentration of creatinine
and uric acid, which is most pronounced in patients of the group 3rd. The study of the activities of AST, ALT in the blood of
patients showed a significant increase in all age categories, more significant in persons of the groups 2nd and 3rd. At the same time,
the activity of a-amylase, the concentration of bilirubin increase with age, and the levels of iron decrease, reaching pathological
values. The content of the inflammation marker — CRP shows the most pronounced dependence on the age of patients: in
patients of the group 1st, it increases by 6.9 times, in the 2nd group — by 12.3 times, in patients over 51 years old — by 17.25
once. Conclusion: with an increase in the age of patients, the deviations of many biochemical parameters from the control levels
corresponding to each group become more pronounced and reach values that deviate significantly from the reference
K
d
COVID 19 age of patients blood biochemical parameters Keywords: COVID-19, age of patients, blood, biochemical paramet Keywords: COVID-19, age of patients, blood, biochemical parameters. Finansing. The study did not have sponsorship. 90 INFECTIOUS DISEASES
3.1.22 Результаты У больных различных возрастных категорий были
исследованы биохимические показатели азотистого ме
таболизма, характеризующие синтетическую функцию
печени, распад белков и нуклеиновых кислот с образова
нием небелковых низкомолекулярных азотсодержащих
конечных продуктов. Исследования показало, что у всех
возрастных групп содержание общего белка и мочевины
не претерпевает существенных изменений (табл. 1). 91 М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов
ВОЗРАСТНЫЕ ОСОБЕННОСТИ ИЗМ ЕНЕНИЙ БИОХИМИЧЕСКИХ
ПАРАМЕТРОВ КРОВИ ПРИ COVID-19
ИНФЕКЦИ М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов
ВОЗРАСТНЫЕ ОСОБЕННОСТИ ИЗМ ЕНЕНИЙ БИОХИМИЧЕСКИХ
ПАРАМЕТРОВ КРОВИ ПРИ COVID-19 ИНФЕКЦИОННЫЕ БОЛЕЗНИ
3.1.22 Таблица / Table 1
Содержание маркеров азотистого метаболизма в крови больных COVID-19
The content of markers of nitrogen metabolism in the blood of patients with COVID-19
Возраст пациентов, лет
Age of patients
Общий белок (г/л)
Total protein (g/l)
Мочевина
(мМоль/л)
Urea (pmol/l)
Креатинин
(мкМ/л)
Creatinine (pmol/l)
Мочевая кислота
(мкМ/л)
Uric acid (pmol/l)
25-35
Контроль
Control
(n=32)
73,68±0,84
4,73±0,23
75,04±0,93
259,50±15,04
Больные
Sick
(n =83)
71,12±0,74
4,89±0,14
96,16±2,05*
358,0±23,67*
36-50
S
3
"
(n =36)
71,85±0,83
5,17±0,29
90,41±2,13
259,0±15,04
Б , Г “
(n=288)
69,83±0,65
4,91±0,15
100,82±3,89*
371,14±21,53*
51-75
Контроль
Control
(n =33)
75,66-±1,22
5,47±0,28
79,57±1,05
255,60±22,20
Больные
Sick
(n =66)
68,85±1,36
6,80±0,95
115,14±6,59*+
410,60±31,17*
Референтные интервалы
Reference intervals
65-85
3,2-7,3
74-110
210-420
Примечание: p<0,05 по сравнению * — с контрольной группой, +----I группой, # — II группой. Note: p<0,05 compared to * — the control group, +
group 1st, # — group 2nd. Содержание маркеров азотистого метаболизма в крови больных COVID-19
The content of markers of nitrogen metabolism in the blood of patients with COVID-19 Примечание: p<0,05 по сравнению * — с контрольной группой, +----I группой, # — II группой. Примечание: p<0,05 по сравнению * — с контрольной группой, +----I группой, # — II группой. Note: p<0,05 compared to * — the control group, +
group 1st, # — group 2nd. Note: p<0,05 compared to * — the control group, +
group 1st, # — group 2nd. Примечание: p<0,05 по сравнению * — с контрольной группой, +----I группой, # — Медицинский вестник Юга России
2023; 14(2):90-96 Медицинский вестник Юга России
2023; 14(2):90-96 Медицинский вестник Юга России Note: p<0,05 compared to * — the control group, +
group 1st, # — group 2nd. INFECTIOUS DISEASES
3.1.22 Из рисунка 1, видно, что у больных I воз
растной категории содержание СРБ увеличивается в
6,9 раз (p<0,05), у II — в 12,3 раза (p<0,05), у лиц старше
51 года — в 17,25 раз (p<0,05). Высокие значения СРБ у
исследованных нами больных с коронавирусной инфек
цией, особенно старшего возраста, указывают на про
грессирующее развитие острой воспалительной реакции. Во время инфекционных заболеваний помимо клас
сических маркеров воспаления также могут происхо
дить изменения и в обмене железа. Из рисунка 2 видно,
что содержание железа уменьшается у I группы на 54,2%
(p<0,05), а у II — на 74,2% (p<0,05), достигая уровня пата-
логически низких значений. Такое же выраженное сни
жение содержания железа пнаблюдается у пациентов из
III группы — 66,7%. Таким образом, у больных старших
возрастных категорий концентрации данного микроэле
мента становятся существенно ниже предела референт
ных значений. Коронавирусная инфекция инициирует у больных
всех возрастных категорий выраженную воспалитель
ную реакцию. Об этом свидетельствует повышение уров
ня С-реактивного белка (СРБ) — классического маркера
воспаления. Из рисунка 1, видно, что у больных I воз
растной категории содержание СРБ увеличивается в
6,9 раз (p<0,05), у II — в 12,3 раза (p<0,05), у лиц старше
51 года — в 17,25 раз (p<0,05). Высокие значения СРБ у
исследованных нами больных с коронавирусной инфек
цией, особенно старшего возраста, указывают на про
грессирующее развитие острой воспалительной реакции. Так у I группы активность АСТ выше в 1,9 раз, а
АЛТ — в 2,1 раз по сравнению со здоровыми донорами
того же возраста (p<0,05). У пациентов старших возраст
ных групп наблюдается ещё более выраженное повыше
ние активности данных ферментов: у II группы АСТ уве
личивается в 2,7 раз, а АЛТ — в 2,60 раз, а III — АСТ в
2,6 раза, АЛТ — в 2,5 раза (p<0,05). Анализ активности
АЛТ и АСТ у больных старших возрастных групп по
зволил предположить существенное нарушение прони
цаемости цитоплазматических (маркер — АЛТ) и мито
хондриальных мембран (маркер — АСТ) клеток печени. Интересно то, что содержание билирубина в I группе не
изменяется, что указывает на сохранение функциональ
ной активности гепатоцитов и эпителия желчевыводя
щих путей. Однако у II группы концентрация общего би
лирубина увеличивается в 3 раза, а прямого — в 2,4 раза Во время инфекционных заболеваний помимо клас
сических маркеров воспаления также могут происхо
дить изменения и в обмене железа. Результаты Note: p<0 05 compared to *
the controlgroup +
group 1st #
group 2nd Таблица / Table
Активность клеточных ферментов и содержание билирубина в крови больных COVID-19
The activity of cellular enzymes and the content of bilirubin in the blood of patients with COVID-19 Таблица / Table 2
Активность клеточных ферментов и содержание билирубина в крови больных COVID-19
The activity of cellular enzymes and the content of bilirubin in the blood of patients with COVID-19
Возраст пациентов, лет
Age of patients
АСТ
(Е/л)
AST
(U/l)
АЛТ
(Е/л)
ALT
(U/l)
Общий
билирубин
(мкмоль/л)
Total bilirubin
(pmol/l)
Прямой
билирубин
(мкмоль/л)
Direct bilirubin
(pmol/l)
а-Амилаза
(Е/л)
a-amylase
(U/l)
25-35
Контроль
Control
(n=32)
24,83
±1,83
24,72
±9,50
10,31
±4,08
4,90
±0,39
46,40
±3,69
Больные
Sick
(n =83)
39,72
±2,95 *
52,43
±5,98 *
11,13
±5,37
5,32
±0,32
69, 00
±6,80 *
36-50
Контроль
Control
(n =36)
26,58
±1,63
29,72
±1,62
10,52
±0,65
5,26
±0,47
57,71
±5,67
Больные
Sick
(n=288)
71,65
±5,03 *+
77,58
±4,30 *+
31,82
±2,24 *+
12,61
±0,84 *+
86,26
±5,78
51-75
Контроль
Control
(n =33)
28,85
±2,61
35,23
±3,49
9,21
±0,61
4,61
±0,40
60,20
±3,05
Больные
Sick
(n =66)
75,21
±8,86 *+
89,05
±13,09 *+
23,33
±1,56 *+
12,46
±2,06 *+
111,75
±4,09 *+ #
Референтные интервалы
Reference intervals
< 40 Е/л
< 40 Е/л
3,4-20,5
< 9,2
< 100 Табли
Активность клеточных ферментов и содержание билирубина в крови больных COVID-19
The activity of cellular enzymes and the content of bilirubin in the blood of patients with COVID-19 92 92 INFECTIOUS DISEASES
3.1.22 M.M. Alimurzaeva, S.M. Izudinova, A.M. Dzhafarova, R.A. Khalilov
AGE ASPECTS OF THE EFFECTS OF THE N E w CORONAVIRUS INFECTION (COVID 19)
ON CERTAIN BIOCHEMICAL BLOOD PARAMETERS Medical Herald of the South of Russia
2023; 14(2):90-96 INFECTIOUS DISEASES
3.1.22 M.M. Alimurzaeva, S.M. Izudinova, A.M. Dzhafarova, R.A. Khalilov
AGE ASPECTS OF THE EFFECTS OF THE N E w CORONAVIRUS INFECTION (COVID 19)
ON CERTAIN BIOCHEMICAL BLOOD PARAMETERS M.M. Alimurzaeva, S.M. Izudinova, A.M. Dzhafarova, R.A. Khalilov
AGE ASPECTS OF THE EFFECTS OF THE N E w CORONAVIRUS INFECTION (COVID 19)
ON CERTAIN BIOCHEMICAL BLOOD PARAMETERS При этом наблюдается повышение креатинина и мо
чевой кислоты, уровни которых зависят от возраста па
циентов. Так, у I группы концентрации данных продук
тов азотистого метаболизма увеличиваются на 28,2% и
38,2% (p<0,05), у I — на 50% и 43,3% (p<0,05), а у III —
на 44,3% и 61% (p<0,05) относительно соответствующих
контрольных групп. Интересно то, что сравнительный
анализ содержания креатинина и мочевой кислоты у
больных II и III возрастных групп не выявил статистиче
ски значимых различий. Следует отметить, что, несмо
тря на существенное повышение содержание креатини
на и мочевой кислоты у больных всех возрастных групп,
данные параметры всё же остается в пределах референс-
ных значений. (p<0,05), а у III — в 2,52 раз и в 2,7 раз соответственно
(p<0,05). Интересно то, что сравнительный анализ всех
указанных выше параметров (АСТ, АЛТ, билирубина)
между разными группами больных выявил, что стати
стически значимые различия имеют место только между
I группой и каждой из 2-х других групп, в то время как
между II и III группами различия несущественны. (p<0,05), а у III — в 2,52 раз и в 2,7 раз соответственно
(p<0,05). Интересно то, что сравнительный анализ всех
указанных выше параметров (АСТ, АЛТ, билирубина)
между разными группами больных выявил, что стати
стически значимые различия имеют место только между
I группой и каждой из 2-х других групп, в то время как
между II и III группами различия несущественны. У больных COVID-19 I и II групп наблюдается повы
шение (на 48,7% и 49,5 % (p<0,05)) активности а-амилазы,
однако оно укладывается в диапазон референсных значе
ний данного показателя. Значительное (на 85,6%) и вы
ходящие за пределы референсных значений увеличение
активности этого фермента наблюдается у III возрастной
категории (p<0,05). Исследование активности клеточных ферментов — ха
рактерных маркеров цитолиза различных клеток, в част
ности гепатоцитов (АСТ, АЛТ) и клеток поджелудочной
железы (а-амилазы) — показало, что во всех возрастных
группах содержание данных ферментов в сыворотке кро
ви резко возрастает (табл. 2). Коронавирусная инфекция инициирует у больных
всех возрастных категорий выраженную воспалитель
ную реакцию. Об этом свидетельствует повышение уров
ня С-реактивного белка (СРБ) — классического маркера
воспаления. INFECTIOUS DISEASES
3.1.22 Из рисунка 2 видно,
что содержание железа уменьшается у I группы на 54,2%
(p<0,05), а у II — на 74,2% (p<0,05), достигая уровня пата-
логически низких значений. Такое же выраженное сни
жение содержания железа пнаблюдается у пациентов из
III группы — 66,7%. Таким образом, у больных старших
возрастных категорий концентрации данного микроэле
мента становятся существенно ниже предела референт
ных значений. В,
О О
I — 25-35 лет
II — 36-50 лет
III — 51-75 лет
1st —
25-35 years old
2nd —
36-50 years old
3rd —
51-75 years old
контроль
control
больные
patients
p<0,05 по сравнению с
* — контрольной группой,
+ — I группой,
# — II группой. p<0,05 compared to
* —
the control group,
+ —
group 1st,
# — group 2nd. Рисунок 1. Содержание СРБ в сыворотке крови больных COVID-19 различных возрастных групп
Figure 1. The content of CRP in the blood serum of patients with COVID-19 of different age groups Рисунок 1. Содержание СРБ в сыворотке крови больных COVID-19 различных возрастных групп
Figure 1. The content of CRP in the blood serum of patients with COVID-19 of different age groups 93 М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов
ВОЗРАСТНЫЕ ОСОБЕННОСТИ ИЗМ ЕНЕНИЙ БИОХИМИЧЕСКИХ
ПАРАМЕТРОВ КРОВИ ПРИ COVID-19
ИНФЕКЦИОННЫЕ БОЛЕЗНИ
3.1.22
я
с;о
S
S
о"
5о
£а. сГ
I — 25-35 лет
II — 36-50 лет
III — 51-75 лет
1st —
25-35 years old
2nd —
36-50 years old
3rd —
51-75 years old
контроль
control
больные
patients
p<0,05 по сравнению
* — с контрольной группой,
+ — I группой,
# — II группой. p<0,05 compared to
* —
the control group,
+ —
group 1st,
# — group 2nd. Рисунок 2. Содержание железа в сыворотке крови больных COVID-19 различных возрастных групп
Figure 2. The content of iron in the blood serum of patients with COVID-19 of different age groups М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов
ВОЗРАСТНЫЕ ОСОБЕННОСТИ ИЗМ ЕНЕНИЙ БИОХИМИЧЕСКИХ
ПАРАМЕТРОВ КРОВИ ПРИ COVID-19 ИНФЕКЦИОННЫЕ БОЛЕЗНИ
3.1.22 Рисунок 2. Содержание железа в сыворотке крови больных COVID-19 различных возрастных групп
Figure 2. The content of iron in the blood serum of patients with COVID-19 of different age groups Сравнительный анализ содержания СРБ и железа в
крови больных разного возраста выявил, что статисти
чески значимые различия имеют место только между I
группой и каждой из других групп, в то время как между
II и II группой различия несущественны. INFECTIOUS DISEASES
3.1.22 скелетной мускулатуры и других органов. Повышение её
активности в сыворотке крови может быть связано с вы
ходом фермента при цитолизе клеток миокарда, печени
и др. Поскольку активности и АСТ и АЛТ у больных Co-
vid-19 увеличиваются в равной степени, то это указывает
на некроз гепатоцитов. Медицинский вестник Юга России
2023; 14(2):90-96 INFECTIOUS DISEASES
3.1.22 липазы у 1-2 и 17 % больных с нетяжёлым и тяжёлым те
чением соответственно. Хотя любое серьёзное заболева
ние может быть ассоциировано с гипергликемией, свя
занной со стрессом. Yang J. K. и соавт. ранее описали, что
пациенты с SARS (вызванным SARS-CoV, «двоюродным
братом» SARS-CoV-2), которые никогда не получали глю
кокортикоиды, имели значительно более высокий уро
вень глюкозы в плазме крови натощак по сравнению с
пациентами с пневмонией без SARS. В другом исследова
нии в качестве вероятного механизма развития «острого
диабета» у пациентов с SARS было предложено опосредо
ванное SARS-CoV повреждение ^-клеток поджелудоч
ной железы [10]. старшей возрастной группы. Предполагается, что сниже
ние уровня железа происходит за счёт поглощения и де
понирования его активированными гистиоцитами (ма
крофагами). Железо накапливается в макрофагах в виде
ферритина, перенос которого от ферритина к трансфер-
рину нарушается, в результате концентрация ферритина
в крови увеличивается в 3 и более раз, что мы и наблюда
ем у пациентов больных COVID-19 [3]. Medical Herald of the South of Russia
2023; 14(2):90-96 ЛИТЕРАТУРА / REFERENCES 1. Lan J, Ge J, Yu J, Shan S, Zhou H, et al. Structure of the SARS-
CoV-2 spike receptor-binding domain bound to the ACE2
receptor. Nature. 2020;581(7807):215-220. https://doi.org/10.1038/s41586-020-2180-5 cell in culture. Int J Biol Macromol. 2011;49(1):79-84. doi: 10.1016/j.ijbiomac.2011.03.018 cell in culture. Int J Biol Macromol. 2011;49(1):79-84. doi: 10.1016/j.ijbiomac.2011.03.018 cell in culture. Int J Biol Macromol. 2011;49(1):79-84. doi: 10.1016/j.ijbiomac.2011.03.018 cell in culture. Int J Biol Macromol. 2011;49(1):79- doi: 10.1016/j.ijbiomac.2011.03.018 7. Zheng KI, Gao F, Wang XB, Sun QF, Pan KH, et al. Letter
to the Editor: Obesity as a risk factor for greater severity of
COVID-19 in patients with metabolic associated fatty liver
disease. Metabolism. 2020;108:154244. doi: 10.1016/j.metabol.2020.154244 2. Perrella A, Trama U, Bernardi FF, Russo G, Monastra L, et al. Editorial - COVID-19, more than a viral pneumonia. Eur Rev
Med Pharmacol Sci. 2020;24(9):5183-5185. https://doi.org/10.26355/eurrev_202005_21216 8. Liu PP, Blet A, Smyth D, Li H. The Science Underlying
COVID-19: Implications for the Cardiovascular System. Circulation. 2020;142(1):68-78. doi: 10.1161/CIRCULATIONAHA.120.047549 p
g
3. Полушин Ю.С., Шлык И.В., Гаврилова Е.Г., Паршин Е.В.,
Гинзбург А.М. Роль ферритина в оценке тяжести
COVID-19. Вестник анестезиологии и реаниматологии. 2021;18(4):20-28. Polushin Yu.S., Shlyk I.V, Gavrilova E.G., Parshin E.V,
Ginzburg A.M. The Role of Ferritin in Assessing COVID-19
Severity. Messenger of anesthesiology and resuscitation. 2021;18(4):20-28. (In Russ.)
https://doi.org/10.21292/2078-5658-2021-18-4-20-28 9. Liu F, Long X, Zhang B, Zhang W, Chen X, Zhang Z. ACE2
Expression in Pancreas May Cause Pancreatic Damage
After SARS-CoV-2 Infection. Clin Gastroenterol Hepatol. 2020;18(9):2128-2130.e2. doi: 10.1016/j.cgh.2020.04.040 https://doi.org/10.21292/2078-5658-2021-18-4-20-28 10. Yang JK, Feng Y, Yuan MY, Yuan SY, Fu HJ, et al. Plasma
glucose levels and diabetes are independent predictors for
mortality and morbidity in patients with SARS. Diabet Med. 2006;23(6):623-8. doi: 10.1111/j.1464-5491.2006.01861.x 4. Chan JF, Zhang AJ, Yuan S, Poon VK, Chan CC, et al. Simulation of the Clinical and Pathological Manifestations of
Coronavirus Disease 2019 (COVID-19) in a Golden Syrian
Hamster Model: Implications for Disease Pathogenesis and
Transmissibility. Clin Infect Dis. 2020;71(9):2428-2446. doi: 10.1093/cid/ciaa325 11. Андреева Е.А. С-реактивный белок в оценке пациентов
с респираторными симптомами до и в период пандемии
COVID-19. РМЖ. 2021;6:14-17. Andreeva E.A. C-reactive protein in the assessment of
patients with respiratory symptoms before and during the
COVID-19 pandemic. RMJ. 2021;6:14-17. (in Russ.). eLIBRARY ID: 46433896 5. Madjid M, Safavi-Naeini P, Solomon SD, Vardeny O. Potential
Effects of Coronaviruses on the Cardiovascular System: A
Review. JAMA Cardiol. 2020;5(7):831-840. doi: 10.1001/jamacardio.2020.1286 6. Aragao DS, Cunha TS, Arita DY, Andrade MC, Fernandes
AB, et al. Выводы Коронавирусная инфекция, вызванная SARS-CoV2,
приводит к существенным изменениям ряда стандарт
ных биохимических параметров крови (креатинина, мо
чевой кислоты, АСТ, АЛТ, а- амилазы) даже у людей мо
лодого возраста (25-35 лет). С повышением возраста
больных отклонения многих биохимических показателей
от соответствующих каждой группе контрольных уров
ней становятся более выраженными и достигают значе
ний, существенно отклоняющихся от референсных. При
этом сравнительные межгрупповые исследования пока
зали, что обнаруженные различия между многими био
химическими показателями больных II (36-50 лет) и III
(51-75 лет) возрастной групп не являются статистически
достоверными. Таким образом, статистический анализ
лабораторных параметров крови больных из различных
возрастных групп показал, что возраст является значи
мым предиктором тяжёлого течения COVID-19, но не аб
солютным, поскольку риски неблагоприятного развития
заболевания могут затрагивать людей разного возраста. В соответствии с результатами нашего исследования
в крови больных короновирусной инфекцией существен
но повышается содержание СРБ и снижается уровень
железа. СРБ — острофазный белок, уровни которого в
крови резко повышаются при острых респираторных
заболеваниях и используются для их диагностики. Мо
ниторинг маркеров воспаления, в том числе СРБ, в рам
ках биохимического анализа крови при COVID-19 вклю
чён в национальные рекомендации многих стран, в том
числе в России. При этом, по данным ресурса UpToDate,
концентрация СРБ, ассоциированная с тяжёлым течени
ем COVID-19, превышает 100 мг/л [11]. Таким образом,
больные старшей возрастной группы, содержание СРБ в
крови которых является критическим, являются наибо
лее подверженной рискам тяжёлого течения COVID-19. Исследование указывает на значительное снижение
уровня железа в сыворотке крови, особенно у больных Обсуждение Khalilov
AGE ASPECTS OF THE EFFECTS OF THE N E w CORONAVIRUS INFECTION (COVID 19)
ON CERTAIN BIOCHEMICAL BLOOD PARAMETERS 1. Lan J, Ge J, Yu J, Shan S, Zhou H, et al. Structure of the SARS-
CoV-2 spike receptor-binding domain bound to the ACE2
receptor. Nature. 2020;581(7807):215-220.
https://doi.org/10.1038/s41586-020-2180-5 Обсуждение Можно предположить разные механизмы поврежде
ния печени: в результате прямого цитопатического дей
ствия вируса, токсических эффектов комплексной те
рапии COVID-19-инфекции. Важное воздействие на
тяжесть течения заболевания оказывают также хрониче
ские гепатиты разной этиологии, жировой гепатоз, ток
сическое и аутоиммунное повреждение, а также цирроз
печени. В печени также выявляются признаки, характер
ные для вирусного поражения (апоптоз и регенерацию
гепатоцитов) и дистрофию (жировую и баллонную) ге-
патоцитов [7]. Причём, чем более тяжёлое течение забо
левания было отмечено, тем чаще выявлялись признаки
поражения печени. Исследование стандартных биохимических параме
тров крови, характеризующих состояние различных ор
ганов и систем организма, свидетельствует о том, что ко
ронавирусная инфекция, вызванная Sars-Cov-2, приводит
к развитию целого ряда структурно-функциональных на
рушений. При этом наблюдается следующая закономер
ность: чем старше возраст больных, тем более выражены
патологические эффекты вирусной экспансии. Анализ конечных продуктов азотистого метаболизма
показал повышение содержания креатинина и мочевой
кислоты в сыворотке крови больных COVID-19. это мо
жет указывать на нарушение клиренса данных низкомо
лекулярных веществ почками, вызванными структурно
функциональными изменениями нефронов. Известно, что
одним из важнейших органов-мишеней COVID-19 могут
быть почки, что, вероятно, связано с широким представи
тельством АСЕ-2 в организме человека, который является
точкой входа SARS-CoV-2. В почках АСЕ-2 присутствует
в подоцитах, мезангиальных клетках, париетальном эпи
телии капсулы Боумена, нефроцитах проксимальных ка
нальцев и клетках собирательных трубочек [6]. Повышение активности АСТ может быть также и мар
кером повреждения кардиомиоцитов. По данным обще
мировой статистики, у 19 % пациентов, госпитализиру
емых с COVID-19, наблюдаются признаки поражения
сердца (боли в грудной клетке, гипотензия, аритмия, при
знаки сердечной недостаточности), также отмечаются слу
чаи брадиаритмии, острого коронарного синдрома [8]. Более высокая активность амилазы в сыворотке крови
больных COVID-19 может быть обусловлена нарушени
ями проницаемости клеток поджелудочной железы. Из
вестно, что ACE-2 усиленно экспрессируется в поджелу
дочной железе, причем уровень мРНК в поджелудочной
железе выше, чем в лёгких. экспрессия ACE2 наблюдается
как в клетках экзокринной части поджелудочной железе,
так и эндокринных клетках островков [9]. Повреждение
экзокринной части поджелудочной железы проявляет
ся повышением уровня сывороточной амилазы и/или Исследование показало, что у всех групп больных ко
роновирусной инфекцией происходит в разной степени
выраженное повышенные уровни активности фермен
тов АСТ и АЛТ. Оба фермента распространены во всех
тканях. Поскольку наибольшая концентрация АЛТ от
мечается в печени, то данный фермент является специ
фическим маркёром заболеваний этого органа. АСТ со
держится в тканях сердца, печени, почек, нервной ткани, 94 INFECTIOUS DISEASES
3.1.22 INFECTIOUS DISEASES INFECTIOUS DISEASES
3.1.22 M.M. Alimurzaeva, S.M. Izudinova, A.M. Dzhafarova, R.A. M.M. Alimurzaeva, S.M. Izudinova, A.M. Dzhafarova, R.A. Khalilov
AGE ASPECTS OF THE EFFECTS OF THE N E w CORONAVIRUS INFECTION (COVID 19)
ON CERTAIN BIOCHEMICAL BLOOD PARAMETERS Медицинский вестник Юга России
2023; 14(2):90-96 Information about the authors ф р
ц
р
Алимурзаева Марьям Мурадовна, магистр 2-го
года обучения Дагестанского государственного уни
верситета,
фельдшер-лаборант
Медико-санитарная
часть МВД по Республике Дагестан, Махачкала, Россия;
alimurzayeva95@mail.ru. ф р
р
Алимурзаева Марьям Мурадовна, магистр 2-го
года обучения Дагестанского государственного уни
верситета,
фельдшер-лаборант
Медико-санитарная
часть МВД по Республике Дагестан, Махачкала, Россия;
alimurzayeva95@mail.ru. Maryam M. Alimurzaeva, master of the 2nd year of study
of the Dagestan State University, medical assistant-laboratory
assistant of the Medical and Sanitary Department of the
Ministry of Internal Affairs for the Republic of Dagestan,
Makhachkala, Russia; alimurzayeva95@mail.ru. Sagibat M. Izudinova, laboratory assistant of Medical and
sanitary unit of the Ministry of Internal Affairs for the Republic
of Dagestan, Makhachkala, Russia; izudinovas@gmail.com. Изудинова Сагибат Магомедовна, врач-лаборант,
Медико-санитарная часть МВД по Республике Дагестан,
Махачкала, Россия; izudinovas@gmail.com. Sagibat M. Izudinova, laboratory assistant of Medical and
sanitary unit of the Ministry of Internal Affairs for the Republic
of Dagestan, Makhachkala, Russia; izudinovas@gmail.com. Изудинова Сагибат Магомедовна, врач-лаборант,
Медико-санитарная часть МВД по Республике Дагестан,
Махачкала, Россия; izudinovas@gmail.com. джафарова Альбина Мехьядиновна, доцент кафедры
биохимии и биофизики Дагестанского государственного
университета, врач-лаборант, Медико-санитарная часть
МВД по Республике Дагестан, Махачкала, Россия; albi-
na19764@mail.ru; https://orcid.org/0000-0001-7744-859x Albina M. Dzhafarova, Associate Professor of the
Department of Biochemistry and Biophysics of the Dagestan
State University, laboratory assistant at the Medical and
Sanitary Unit of the Ministry of Internal Affairs for the
Republic of Dagestan, Makhachkala, Russia; albina19764@
mail.ru; https://orcid.org/0000-0001-7744-859x Халилов Рустам Абдуразакович, заведующий ка
федрой биохимии и биофизики Дагестанского государ
ственного университета, Махачкала, Россия; dagbiofak@
mail.ru; https://orcid.org/0000-0001-9369-1645 Rustam A. Khalilov, Head of the Department of
Biochemistry and Biophysics, Dagestan State University,
Makhachkala, Russia. E-mail: dagbiofak@mail.ru; https://
orcid.org/0000-0001-9369-1645 Rustam A. Khalilov, Head of the Department of
Biochemistry and Biophysics, Dagestan State University,
Makhachkala, Russia. E-mail: dagbiofak@mail.ru; https://
orcid.org/0000-0001-9369-1645 Authors' contribution Алимурзаева Марьям Мурадовна — получение и ана
лиз данных; Alimurzaeva Maryam Muradovna — data acquisition and
analysis; Izudinova Sagibat Magomedovna — review of publications
on the topic of the article; Izudinova Sagibat Magomedovna — review of publications
on the topic of the article; Изудинова Сагибат Магомедовна — обзор публикаций
по теме статьи; Джафарова Альбина Мехьядиновна — интерпретация
данных, написание текста рукописи; Dzhafarova Albina Mekhyadinovna — interpretation of
data, writing the text of the manuscript; Dzhafarova Albina Mekhyadinovna — interpretation of
data, writing the text of the manuscript; Халилов Рустам Абдуразакович — разработка дизай
на исследования, окончательное утверждение версии для
публикации. Khalilov Rustam Abdurazakovich — study design
development, final approval of the version for publication. Khalilov Rustam Abdurazakovich — study design
development, final approval of the version for publication. ЛИТЕРАТУРА / REFERENCES Purification and characterization of angiotensin
converting enzyme 2 (ACE2) from murine model of mesangial 95 95 Medical Herald of the South of Russia
2023; 14(2):90-96 М.М. Алимурзаева, С.М. Изудинова, А.М. Джафарова, Р.А. Халилов
ВОЗРАСТНЫЕ ОСОБЕННОСТИ И ЗМ ЕН ЕН И й БИОХИМИЧЕСКИХ
ПАРАМЕТРОВ КРОВИ ПРИ COVID-19 ИНФЕКЦИОННЫЕ БОЛЕЗНИ
3.1.22 ИНФЕКЦИОННЫЕ БОЛЕЗНИ
3.1.22 Conflict of interest Авторы заявляют об отсутствии конфликта интересов. Authors declares no conflict of interest. Поступила в редакцию / Received: 16.02.2023
Доработана после рецензирования / Revised: 28.03.2023
Принята к публикации / Accepted: 11.04.2023 Поступила в редакцию / Received: 16.02.2023
Доработана после рецензирования / Revised: 28.03.2023
Принята к публикации / Accepted: 11.04.2023 96 96
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https://europepmc.org/articles/pmc6529697?pdf=render
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English
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NUPR1 acts as a pro-survival factor in human bone marrow-derived mesenchymal stem cells and is induced by the hypoxia mimetic reagent deferoxamine
|
Journal of clinical biochemistry and nutrition
| 2,019
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cc-by
| 5,737
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Kazuhito Matsunaga,1,# Koichi Fujisawa,1,2,# Taro Takami,1,* Guzel Burganova,1,3 Nanami Sasai,1,4
Toshihiko Matsumoto,1,5 Naoki Yamamoto1 and Isao Sakaida1,2 Kazuhito Matsunaga,1,# Koichi Fujisawa,1,2,# Taro Takami,1,* Guzel Burganova,1,3 Nanami Sa
Toshihiko Matsumoto,1,5 Naoki Yamamoto1 and Isao Sakaida1,2 1Department of Gastroenterology and Hepatology, 2Center for Regenerative Medicine, 4Department of Laboratory Science and 5Department of Oncology
and Laboratory Medicine, Yamaguchi University School of Medicine, Minami Kogushi 111, Ube, Yamaguchi 7558505, Japan
3Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, Kazan, Russia 5
(Received 19 December, 2018; Accepted 25 December, 2018; Published online 23 March, 2019) requires a specific equipment and cells may be temporarily exposed
to non-hypoxia states during medium exchange. Thus, the possi-
bility to employ hypoxia-mimetic reagents, such as deferoxamine
(DFO), an iron chelator, has drawn much attention.(6) Iron deple-
tion by DFO mimics hypoxia by inhibiting iron-dependent prolyl-4
hydroxylase activity and, thereby, HIF1a hydroxylation and
degradation. DFO preconditioning in Wharton’s jelly-derived
neural-like cells has been shown to improve tolerance and thera-
peutic potential, and enhance the effectiveness of cell therapy.(7) It
has been reported that MSC preconditioning with DFO, before
transplantation, increases the homing capacity of MSCs and endo-
thelial progenitor cells(8) and improves cell therapy.(9) Moreover,
hypoxic preconditioning potentiates the trophic and anti-apoptotic
effects of MSCs on hepatocytes(10) or brain cells.(11) On the other
hand, additional investigation is necessary to clarify the molecular
basis of DFO effects. We have compared the changes in MSC
metabolism induced by DFO and hypoxia, and found that low
concentrations of DFO may be appropriate for MSC precondi-
tioning.(12) Copyright © 2019 JCBN
2019
This is an open access article distributed under the terms of the
Creative Commons Attribution License, which permits unre-
stricted use, distribution, and reproduction in any medium, pro-
vided the original work is properly cited. Differences in the culturing conditions of mesenchymal stem cells
used in regenerative medicine may affect their differentiation
ability, genome instability, and therapeutic effects. In particular,
bone marrowderived mesenchymal stem cells cultured under
hypoxia are known to proliferate while maintaining an undiffer
entiated state and the use of deferoxamine, a hypoxia mimetic
reagent, has proven to be a suitable strategy to maintain the cells
under hypoxic metabolic state. Here, the deferoxamine effects
were investigated in mesenchymal stem cells to gain insights into
the mechanisms regulating stem cell survival. A 12h deferox
amine treatment reduced proliferation, oxygen consumption,
mitochondrial activity, and ATP production. Microarray analysis
revealed that deferoxamine enhanced the transcription of genes
involved in glycolysis and the HIF1α pathway. #Equally contributed.
*To whom correspondence should be addressed.
Email: ttakami@yamaguchiu.ac.jp Kazuhito Matsunaga,1,# Koichi Fujisawa,1,2,# Taro Takami,1,* Guzel Burganova,1,3 Nanami Sasai,1,4
Toshihiko Matsumoto,1,5 Naoki Yamamoto1 and Isao Sakaida1,2 Among the earliest
changes, transcriptional variations were observed in HIF1α,
NUPR1, and EGLN, in line with previous reports showing that
short deferoxamine treatments induce substantial changes in
mesenchymal stem cells glycolysis pathway. NUPR1, which is
induced by stress and involved in autophagymediated survival,
was upregulated by deferoxamine in a concentrationdependent
manner. Consistently, NUPR1 knockdown was found to reduce
cell proliferation and increase the proapoptotic effect of stauro
sporine, suggesting that deferoxamineinduced NUPR1 promotes
mesenchymal stem cell survival and cytoprotective autophagy. Our findings may substantially contribute to improve the effec
tiveness of mesenchymal stem cellbased regenerative medicine. Nuclear protein 1 (NUPR1) (also known as p8) is a molecule
that has recently caught much attention owing to its association
with chemotherapy resistance in a wide range of cancer types. Its
suppression of cell cycle via p53 and cytoprotective autophagy
induction have been previously reported.(13) To date, NUPR1 has
mainly been studied in cancer cells, where it supports drug resis-
tance. However, the underlying mechanisms have not yet been
clarified in detail. Moreover, although an involvement of NUPR1
in the survival of mesenchymal stem cells has been speculated,
no clues are available, to date, regarding its function in MSCs. Original Article Original Article J. Clin. Biochem. Nutr. | May 2019 | vol. 64 | no. 3 | 209–216 doi: 10.3164/jcbn.18112
©2019 JCBN Results Furthermore, fluorescent immunohistochemical staining
of cells treated with 10 mM DFO and CQ showed an increase in
the LC3-positive puncta, suggesting DFO-induced autophagy
enhancement (Fig. 3B). An analysis of NUPR1 expression revealed
a DFO concentration-dependent increase 48 h after DFO treat-
ment (Fig. 3C). In light of the observed DFO-induced NUPR1 up-
regulation, we next verified whether similar expression changes
could be induced by hypoxia and found that these conditions also
increased NUPR1 expression (Fig. 3D). In addition, enhanced
NUPR1 expression was detected after a 2-h cell incubation
with HBSS, an amino acid starvation medium known to enhance
autophagy (Fig. 3E). y
y
g
y
y
y
(
)
Oxygen consumption rate (OCR) measurements. OCR
measurements were performed using a Seahorse Biosciences XF96
Extracellular Flux Analyzer. Cells were seeded at 6,000/well in
XF96 microplates (Seahorse Biosciences, Santa Clara, CA). After
a 24-h incubation, the growth medium was replaced with XF
Assay Medium (Seahorse Biosciences) supplemented with 25 mM
glucose (Sigma-Aldrich, Darmstadt, Germany). OCR measurements
were made over 5-min periods following a 3-min mix period. The following components were sequentially added to the cells:
1 mg/mL oligomycin (Sigma-Aldrich), 300 nM carbonylcyanide-p-
trifluoromethoxyphenylhydrazone (FCCP; Sigma-Aldrich), and
2 mM rotenone (MP Biomedicals, Tokyo, Japan). The spare
respiratory capacity and coupling efficiency were calculated
according to the Seahorse Bioscience instructions and the basal
OCR was normalized to the cell number. Immunofluorescence Analysis. MSCs were cultured in
chamber slides, and then fixed in 4% paraformaldehyde. Immuno-
fluorescence analysis was performed by using the Opal Multiplex
Immunohistochemistry Kit (Perkin Elmer, Norwalk, CT) as per
manufacturer’s instructions. NUPR1 knockdown reduces MSC proliferation and
decreases drug resistance. Next, to directly explore the role
of NUPR1 in MSC proliferation, its expression was suppressed by
siRNA knockdown. The knockdown was efficient and resulted in
clear LC3-II upregulation (Fig. 4A). Cell proliferation was
slightly reduced in NUPR1-knocked down cells, but in the pres- NUPR1 knockdown reduces MSC proliferation and
decreases drug resistance. Next, to directly explore the role
of NUPR1 in MSC proliferation, its expression was suppressed by
siRNA knockdown. The knockdown was efficient and resulted in
clear LC3-II upregulation (Fig. 4A). Cell proliferation was
slightly reduced in NUPR1-knocked down cells, but in the pres- Statistical analysis. The homogeneity of variance was
assessed by the F-test. Results DFO suppresses mitochondrial oxygen consumption and
lowers the level of intracellular ATP. In order to investigate
the effects of DFO on cell proliferation, BMSCs were treated
with different drug concentrations, for 12 and 48 h, followed by
cell culturing in DFO-free medium. Proliferation was then
evaluated by IncuCyte HD imaging system. We found that treat-
ment for 48 h with 3 mM DFO, a concentration that is effective
for medium preconditioning, and for 12 h with 50 mM DFO,
resulted in comparable effects on cell proliferation (Fig. 1A). Next, the amount of ATP per cell was measured and found to
be substantially reduced at DFO concentrations above 100 mM
(Fig. 1B). To investigate the reason behind the ATP depletion,
the OCR (oxygen consumption rate) was measured with a flux
analyzer, revealing a detectable decrease of both basal and
maximal respiration with 10 mM DFO, and a drastic reduction
with 100 mM and 300 mM (Fig. 1C). Cell and cell culture. Bone marrow cells were purchased
from (Lonza, Basel, Switzerland) and MSCs were cultured in
DMEM medium, and cells at passages 3–8 were used. The MSC
phenotype, CD11b(-), CD90(+), CD73(+), and CD105(+), was
confirmed by flow cytometry. y
y
y
Proliferation assay. To measure proliferation, 1,000 MSCs
were seeded in each well of a 96-well plate, DFO was then added
at different concentrations, and the cellular area was evaluated
with an IncuCyte HD imaging system (Essen BioScience, London,
UK). DFO was purchased from Novartis Pharma K.K. (Tokyo,
Japan). Apoptosis assay. After siRNA treatment, 3,000 cells were
seeded in each well of a 96-well plate and cultured for 3 days. Next, cells were treated with staurosporine (0.3 mM) and 6 h
later the cell number was measured with a CyQUANT® cell pro-
liferation assay (Thermo Fisher Scientific, Waltham, MA). Cells
were then evaluated with a Caspase-3 Assay Kit (Promega,
Tokyo, Japan) and caspase-3 activity per cell was measured. The
results were obtained from three independent experiments with 6
replicates each. m
m
(
g
)
DFO treatment alters the expression of genes for energy
metabolism. After treatment of MSCs with 50 mM DFO for
12 h, RNA was extracted for microarray analysis. Functional
classification of genes was performed using the IPA software,
and pathways involved in sugar metabolism, such as glycolysis
and gluconeogenesis, HIF1a-mediated and VEGF-mediated
signaling, were classified as Canonical pathways (Fig. 2A). Results Exam-
ination of the glycolysis pathway revealed an upregulation of the
enzymes glucose 6 phosphate isomerase, 6-phosphofructokinase,
fructose-bisphosphate
aldolase,
triose-phosphate
isomerase,
glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate
kinase, phosphoglycerate mutase, and phosphopyruvate hydratase
(Fig. 2B). In the HIF1a pathway, due to HIF1a degradation
inhibition by DFO, HIF1a was downregulated, and HIF1a target
genes, namely GLUT, LDH and VEGF, were upregulated (Fig. 2C). Among the upstream DFO-induced transcriptional changes,
HIF1a was mostly activated, and hypoxia-induced EPAS1 and
PDGF were also upregulated. Notably, NUPR1, which is known
to be involved in mitochondrial stress, was activated. On the other
hand, EGLN (hypoxia-inducible factor prolyl hydroxylase) and
curcumin were among the suppressed upstream factors (Fig. 2D). Western blot analysis. Western blotting was performed
according to a standard method.(14) In brief, cell lysis buffer
contained 62.5 mM Tris-HCl (pH 6.8), 4% SDS, and 200 mM
dithiothreitol. Cell lysates were electrophoresed on 12% acryl-
amide gels. Anti b-actin antibody (Sigma, Darmstadt, Germany),
anti-p62 antibody (Abcam, Tokyo, Japan), anti-LC3 antibody
(Life Technologies, Waltham, MA), and anti-NUPR1 antibodies
(ProteinT, Chicago, IL) were used. Total RNA isolation. Total RNA was isolated from cells
using TRIzol Reagent (Life Technologies) and purified using SV
Total RNA Isolation System (Promega) according to the manufac-
turer’s instructions. RNA samples were quantified by an ND-1000
spectrophotometer (NanoDrop Technologies, Wilmington, DE) and
RNA quality was confirmed with an Experion System (Bio-Rad
Laboratories, Hercules, CA). )
SAGE. Ion Ampliseq Transcriptome Human Gene Expression
Kit (Life Technologies) was used to create an Ion Proton next-
generation sequencer library of analysis beads. An Ion PI IC 200
Kit (Life Technologies) and an Ion PI Chip Kit v2 BC were used
for sequencing, using an Ion Proton next-generation sequencer. SAGE results were analyzed by Ingenuity Pathways Analysis (IPA). O
ti
t
(OCR)
t
OCR DFO treatment enhances NUPR1 expression and auto
phagy. In a previously reported microarray analysis, we found
that NUPR1 expression was increased in MSCs treated with 3 mM
DFO for 48 h.(12) Moreover, NUPR1 was found to be involved in
cytoprotective autophagy. Thus, we examined the impact of DFO
on autophagy. Interestingly, LC3-II was upregulated and p62 was
slightly downregulated, and the amplitude of these effects was
dependent on DFO concentration. Furthermore, both LC3-II and
p62 were upregulated by DFO, in a concentration-dependent
manner, after a 16-h treatment with chloroquine (CQ), an
autophagy inhibitor, indicating the activation of autophagy
(Fig. 3A). Key Words:
mesenchymal stem cells, transcriptome, hypoxia,
iron chelator, autophagy This study demonstrated the survival-promoting effect of treat-
ments with low DFO concentrations for relatively short times
(as low as 12 h) and investigated DFO-induced gene expression
changes in MSCs. In addition, DFO-induced stress resistance
mechanisms mediated by NUPR1 were explored. We expect that
our findings will substantially contribute to the improvement of
MSC survival and the advancement of MSC-based regenerative
medicine. Introduction
Human bone marrow-derived mesenchymal stem cells (BMSCs)
possess the ability to differentiate into adipocytes, chondro-
cytes, and osteocytes, and their application in regenerative medicine
is highly anticipated. However, the culturing conditions may affect
the quality of BMSCs and, thereby, their therapeutic performance. As the oxygen concentration in the bone marrow is about 1–7%,
BMSCs cultured under 21% oxygen undergo stress, which may
result in genetic instability. Repeated culturing may also result in
genetic instability, cell aging, and decreased stemness mainte-
nance. Interestingly, it has been reported that these problems can
be overcome by hypoxic culturing.(1–3) Furthermore, it is known
that hypoxia preconditioning promotes mesenchymal cell pro-
liferation and is utilized to increase MSC survival after transplan-
tation.(4,5) Although hypoxic culturing can be advantageous, it
H H doi: 10.3164/jcbn.18112
©2019 JCBN Results DFO was administered for 12 h (brown profile) or 48 h (blue
ndicate the termination time of DFO treatments (brown arrow,
d cells were seeded in 96well plates and, on the next day, ATP
ed based on the cell number deduced from CyQUANT® cell pro
t
t
i
th
Fl
A
l
Si th
d
ll
d d Fig. 1. Evaluation of DFO effects on proliferation, ATP, and oxygen consumption of BMSCs. (A) A comparison of the effects of different DFO
concentrations and treatment times on cell proliferation. In each well one thousand cells were seeded, DFO was added at different concentrations
on the next day, and proliferation was evaluated every two hours with IncuCyte. DFO was administered for 12 h (brown profile) or 48 h (blue
profile), and then the medium was replaced with DFOfree medium. The arrows indicate the termination time of DFO treatments (brown arrow,
12 h; blue arrow, 48 h). (B) The effects of DFO on ATP production. Three thousand cells were seeded in 96well plates and, on the next day, ATP
production was measured 12 h after DFO treatment. The ATP level was normalized based on the cell number deduced from CyQUANT® cell pro
liferation assay (*p<0.05). (C) An evaluation of the OCR of cells after a 12h DFO treatment using the Flux Analyzer. Six thousand cells were seeded
in 96well plates and OCR was measured on the next day, 12 h after DFO treatment. OCR was measured using the Flux Analyzer before and after the
administration of oligomycin, FCCP, antimycin, and rotenone, as indicated. It is thought that DFO suppression of cell proliferation occurs
because HIF1a accumulates as a result of reduced PDH activity,
required for HIF1a degradation. In the pathway analysis of gene
expression, glycolysis was ranked first, because of the overexpres-
sion of 5 of the 6 enzyme-encoding genes involved in glycolysis. Moreover, sirtuin, HIF1a, and VEGF signaling pathways, involved
in glycolytic and mitochondrial metabolism, were also ranked,
thus confirming the presence of DFO-induced changes even after
a 12-h treatment. DFO-induced variations occurred in a few
upstream regulators. HIF1a and NUPR1 were upregulated, while
EGLN and curcumin related genes were downregulated. As
EGLN requires iron for enzyme activities, its inhibition upon
DFO-induced iron deprivation was expected. Results It has been reported
that curcumin possesses iron chelation ability(15) and reduces iron
in mice and rats.(16) Moreover, its application as a suppressor of
oxidative stress has been proposed.(15) Although NUPR1 upregula-
tion was an upstream effect of the DFO treatment, it was
maintained after cell treatment with 10 mM DFO for 48 h.(12)
NUPR1 is thought to be involved in chemotherapy resistance in
cancer cells and is induced by environmental stresses such as
nutrient starvation and hypoxia. Moreover, NUPR1 is known to be ence of DFO the decline became greater (Fig. 4B). Next, as
NUPR1 has been reported to be associated with drug resistance in
cancer cells, staurosporine-induced apoptosis was evaluated. NUPR1 knockdown increased caspase-3 activity in MSCs treated
with staurosporine (Fig. 4C). J. Clin. Biochem. Nutr. | May 2019 | vol. 64 | no. 3 | 211
©2019 JCBN K. Matsunaga et al. Results The results were analyzed by either the
Student’s t test or Welsh’s two-factor t tests, and the data are
presented as the mean ± SD, with the significance threshold
established at p<0.05. doi: 10.3164/jcbn.18112
©2019 JCBN 210 Fig. 1. Evaluation of DFO effects on proliferation, ATP, and oxygen consumption of BMSCs. (A) A comparison of the effects of different DFO
concentrations and treatment times on cell proliferation. In each well one thousand cells were seeded, DFO was added at different concentrations
on the next day, and proliferation was evaluated every two hours with IncuCyte. DFO was administered for 12 h (brown profile) or 48 h (blue
profile), and then the medium was replaced with DFOfree medium. The arrows indicate the termination time of DFO treatments (brown arrow,
12 h; blue arrow, 48 h). (B) The effects of DFO on ATP production. Three thousand cells were seeded in 96well plates and, on the next day, ATP
production was measured 12 h after DFO treatment. The ATP level was normalized based on the cell number deduced from CyQUANT® cell pro
liferation assay (*p<0.05). (C) An evaluation of the OCR of cells after a 12h DFO treatment using the Flux Analyzer. Six thousand cells were seeded
in 96well plates and OCR was measured on the next day, 12 h after DFO treatment. OCR was measured using the Flux Analyzer before and after the
administration of oligomycin, FCCP, antimycin, and rotenone, as indicated. ption of BMSCs. (A) A comparison of the effects of different DFO
sand cells were seeded, DFO was added at different concentrations
yte. DFO was administered for 12 h (brown profile) or 48 h (blue
ws indicate the termination time of DFO treatments (brown arrow,
and cells were seeded in 96well plates and, on the next day, ATP
ized based on the cell number deduced from CyQUANT® cell pro
treatment using the Flux Analyzer. Six thousand cells were seeded
nt. OCR was measured using the Flux Analyzer before and after the on of BMSCs. (A) A comparison of the effects of different DFO
d cells were seeded, DFO was added at different concentrations
e. doi: 10.3164/jcbn.18112
©2019 JCBN Discussion Red arrows indicate genes with a statistically significant upregulation in known
nalysis (no downregulated genes were present in this data set). Rectangles indicate enzymeencoding genes, while ovals indicate met
C) HIF1 signaling pathway analysis using IPA. The upstream and downstream effects of the mapped genes on HIF1 signaling pathway are in
ed and green symbols indicate genes up and downregulated in DFO treated MSC, respectively. The color intensity is proportional to
h
(D) T
5
d b
5
l
B
i di
l
di
d
b
i
d
hil
bl
i di Fig. 2. Analysis of DFOinduced gene expression changes. (A) Known pathway ranking through IPA. The bar graph shows the reciprocal display of
p value calculated with the IPA software, while the green line indicates the proportion of genes included in each pathway. (B) Functional classifica
tion of gene clusters with altered expression (glycolysis). Red arrows indicate genes with a statistically significant upregulation in known pathway
analysis (no downregulated genes were present in this data set). Rectangles indicate enzymeencoding genes, while ovals indicate metabolites. (C) HIF1 signaling pathway analysis using IPA. The upstream and downstream effects of the mapped genes on HIF1 signaling pathway are indicated. Red and green symbols indicate genes up and downregulated in DFO treated MSC, respectively. The color intensity is proportional to the fold
change. (D) Top 5 and bottom 5 upstream regulators. Brown indicates upstream regulators predicted to be activated, while blue indicates upstream
regulators predicted to be inhibited. Fig. 2. Analysis of DFOinduced gene expression changes. (A) Known pathway ranking through IPA. The bar graph shows the reciprocal display of
p value calculated with the IPA software, while the green line indicates the proportion of genes included in each pathway. (B) Functional classifica
tion of gene clusters with altered expression (glycolysis). Red arrows indicate genes with a statistically significant upregulation in known pathway
analysis (no downregulated genes were present in this data set). Rectangles indicate enzymeencoding genes, while ovals indicate metabolites. (C) HIF1 signaling pathway analysis using IPA. The upstream and downstream effects of the mapped genes on HIF1 signaling pathway are indicated. Red and green symbols indicate genes up and downregulated in DFO treated MSC, respectively. The color intensity is proportional to the fold
change. (D) Top 5 and bottom 5 upstream regulators. Brown indicates upstream regulators predicted to be activated, while blue indicates upstream
regulators predicted to be inhibited. Discussion Previous reports have demonstrated that hypoxia precondi-
tioning enhances MSC retention during intramuscular injec-
tion.(7,9) We have also shown that DFO, a highly safe drug that
has long been used to treat iron overload, can be an alternative to
oxygen depletion as it mimics hypoxia.(12) We found that relatively
short incubations (12 h) with 50 mM DFO resulted in cell pro-
liferation rates comparable to those observed after 48-h treatments
with 3 mM DFO, i.e., the concentration normally used for pre-
conditioning. Furthermore, ATP depletion, OCR decrease, inhibi-
tion of mitochondrial oxidative phosphorylation, as well as
changes in glycolytic metabolism, were also observed after 12-h
treatments. Therefore, we concluded that shorter preconditioning
could be as effective as the conventional 48-h treatments. J. Clin. Biochem. Nutr. | May 2019 | vol. 64 | no. 3 | 211
©2019 JCBN 212 doi: 10.3164/jcbn.18112
©2019 JCBN involved in cytoprotective autophagy(13) and possesses cell cycle
suppression ability via p53. Therefore, the effects of DFO on
functions under stress, and prevents cell death through organelle
turnover.(17) Although it has been reported that DFO treatment
Fig. 2. Analysis of DFOinduced gene expression changes. (A) Known pathway ranking through IPA. The bar graph shows the reciprocal display of
p value calculated with the IPA software, while the green line indicates the proportion of genes included in each pathway. (B) Functional classifica
tion of gene clusters with altered expression (glycolysis). Red arrows indicate genes with a statistically significant upregulation in known pathway
analysis (no downregulated genes were present in this data set). Rectangles indicate enzymeencoding genes, while ovals indicate metabolites. (C) HIF1 signaling pathway analysis using IPA. The upstream and downstream effects of the mapped genes on HIF1 signaling pathway are indicated. Red and green symbols indicate genes up and downregulated in DFO treated MSC, respectively. The color intensity is proportional to the fold
change. (D) Top 5 and bottom 5 upstream regulators. Brown indicates upstream regulators predicted to be activated, while blue indicates upstream
regulators predicted to be inhibited. g. 2. Analysis of DFOinduced gene expression changes. (A) Known pathway ranking through IPA. The bar graph shows the reciprocal d
value calculated with the IPA software, while the green line indicates the proportion of genes included in each pathway. (B) Functional c
on of gene clusters with altered expression (glycolysis). J. Clin. Biochem. Nutr. | May 2019 | vol. 64 | no. 3 | 213
©2019 JCBN K. Matsunaga et al. Discussion involved in cytoprotective autophagy(13) and possesses cell cycle
suppression ability via p53. Therefore, the effects of DFO on
autophagy were explored in MSCs. In fact, it was found that
combined cell treatments with DFO and CQ increased the intra-
cellular levels of LC3-II and p62, indicating autophagy enhance-
ment. Moreover, NUPR1 was upregulated by DFO treatment,
hypoxia, and amino acid starvation. By regenerating metabolic
precursors and eliminating unwanted substances in the cell,
autophagy functions as a survival mechanism that maintains cell functions under stress, and prevents cell death through organelle
turnover.(17) Although it has been reported that DFO treatment
induces protein aggregation and aggresome in NIH3T3 fibro-
blasts, as well as the autophagic gene p62/SQSTM1,(18) a report
has indicated that DFO may suppress autophagy caused by iron
overload.(19) Thus, autophagy may result in different outcomes
depending on environmental and/or physiopathological cell condi-
tions. The possible involvement of NUPR1 in cytoprotective
autophagy has drawn much attention.(13) Here, its function was J. Clin. Biochem. Nutr. | May 2019 | vol. 64 | no. 3 | 213
©2019 JCBN K. Matsunaga et al. Fig. 3. DFOinduced changes in NUPR1 expression and autophagy. (A) DFOinduced expression of autophagyrelated proteins. Proteins were
collected 16 h after the addition of chloroquine (CQ), and evaluated by western blotting. (B) DFOinduced autophagy visualized by immuno
histochemical staining. The white bar indicates 50 mm. (C) DFOinduced changes in NUPR1 expression. (D) Hypoxiainduced changes in NUPR1
expression. (E) NUPR1 expression changes induced by cell culturing in amino aciddeficient medium (HBSS). Fig. 3. DFOinduced changes in NUPR1 expression and autophagy. (A) DFOinduced expression of autophagyrelated proteins. Proteins were
collected 16 h after the addition of chloroquine (CQ), and evaluated by western blotting. (B) DFOinduced autophagy visualized by immuno
histochemical staining. The white bar indicates 50 mm. (C) DFOinduced changes in NUPR1 expression. (D) Hypoxiainduced changes in NUPR1
expression. (E) NUPR1 expression changes induced by cell culturing in amino aciddeficient medium (HBSS). investigated by gene knockdown, resulting in inhibition of cell
proliferation. This finding was consistent with the function of
NUPR1 in MSCs as a pro-survival factor. Author Contributions Conceived and designed the experiments: KM, KF, TT and IS. Performed the experiments: KM, KF, TT, GB and NS. Analyzed
the data: KM, KF and TT. Contributed reagents/materials/analysis
tools: TM and NY. Wrote the paper: KM, KF and TT. In this study, NUPR1 depletion enhanced staurosporine-
induced apoptosis, indicating the importance of NUPR1 in drug
resistance, in line with previously reported results.(22) However, its
role in drug resistance is yet to be clarified in detail. Pancreatic
cancer cells have been reported to be resistant to starvation and
gemcitabine treatment owing to the NUPR1/RELB/IER3 survival
pathway, and CDKN1A/p21 phosphorylation and relocalization
from the nucleus to the cytoplasm are important for breast cancer
cell resistance to chemotherapeutic agents such as doxorubicin.(23)
In this study, the phosphorylation and localization of p21 were Discussion However, the degree of
this proliferation block was lower than the reported effects in
cancer cells.(20) The increase in LC3-II upon NUPR1 knockdown
was consistent with a previous study reporting that NUPR1 is
involved in the maintenance of the autolysosomal efflux in A549
cells and that NUPR1 depletion deregulates autophagic flux and
impairs autolysosomal clearance.(21) In addition, it has been reported
that NUPR1 depletion leads to massive cytoplasmic vacuolization
and premature senescence. However, the latter events were not
observed in MSCs in this study (data not shown). As in these
cells the block of proliferation induced by NUPR1 knockdown is
less pronounced compared to cancer cells, MSCs might have a
lower dependency on NUPR1 and phenotypes such as premature
senescence might be less likely to occur. Further work will be
necessary to solve these issues. also evaluated but no clear changes were observed (data not
shown). Recently, the usefulness of NUPR1 in cells other than
cancer cells has also been studied, and it has been reported that,
in keratinocytes, NUPR1 is involved in UV-induced stress
response.(24) Our findings, including the observation of glycolysis-dominant
metabolic changes in MSCs treated with DFO for 12 h, suggested
that it is possible to apply shorter times of hypoxic precondi-
tioning, compared to traditional protocols. Furthermore, as DFO
treatment, similarly to hypoxia and starvation, upregulates
NUPR1, and this may lead to improved cell survival, our results
may contribute to the design of optimized methods of MSC pre-
paration for regenerative medicine. Acknowledgments We would like to thank Ms. Mariko Yamada, Ms. Risa
Mochizuki and Ms. Kumie Ota for their technical assistance. doi: 10.3164/jcbn.18112
©2019 JCBN 214 g. 4. Changes induced by NUPR1 knockdown. (A) Western blot analysis in MSCs after siRNAinduced NUPR1 knockdown. (B) Effects of NUPR1
nockdown on MSC proliferation. Proliferation was measured every 2 h with IncuCyte (n = 6). (C) Effects of NUPR1 knockdown on staurosporine
nduced apoptosis. Caspase3 activity is shown 6 h after treatment with staurosporine was shown. Three thousand cells were seeded in 96well
lates and Caspase3 activity was measured 6 h after staurosporine treatment. Caspase activity was normalized based on the cell number deduced
rom analysis with CyQUANT® cell proliferation assay. * indicates statistically significant difference compared with siCONTROL #1, p<0.01. Fig. 4. Changes induced by NUPR1 knockdown. (A) Western blot analysis in MSCs after siRNAinduced NUPR1 knockdown. (B) Effects of NUPR1
knockdown on MSC proliferation. Proliferation was measured every 2 h with IncuCyte (n = 6). (C) Effects of NUPR1 knockdown on staurosporine
induced apoptosis. Caspase3 activity is shown 6 h after treatment with staurosporine was shown. Three thousand cells were seeded in 96well
plates and Caspase3 activity was measured 6 h after staurosporine treatment. Caspase activity was normalized based on the cell number deduced
from analysis with CyQUANT® cell proliferation assay. * indicates statistically significant difference compared with siCONTROL #1, p<0.01. Fig. 4. Changes induced by NUPR1 knockdown. (A) Western blot analysis in MSCs after siRNAinduced NUPR1 knockdown. (B) Effects of NUPR1
knockdown on MSC proliferation. Proliferation was measured every 2 h with IncuCyte (n = 6). (C) Effects of NUPR1 knockdown on staurosporine
induced apoptosis. Caspase3 activity is shown 6 h after treatment with staurosporine was shown. Three thousand cells were seeded in 96well
plates and Caspase3 activity was measured 6 h after staurosporine treatment. Caspase activity was normalized based on the cell number deduced
from analysis with CyQUANT® cell proliferation assay. * indicates statistically significant difference compared with siCONTROL #1, p<0.01. in-Aid for Challenging Exploratory Research (24659369) from the
Japan Society for the Promotion of Science. Financial Support in-Aid for Challenging Exploratory Research (24659369) from the
Japan Society for the Promotion of Science. This study was supported by The Project Promoting The
Research and Development (R&D) Center on Regenerative
Medicine in Yamaguchi Prefecture. This study was also supported
by Grant-in-Aid for Young Scientists (B) (18K15815), and Grant- Conflict of Interest No potential conflicts of interest were disclosed. J. Clin. Biochem. Nutr. | May 2019 | vol. 64 | no. 3 | 215
©2019 JCBN e0178011. 1 Dos Santos F, Andrade PZ, Boura JS, Abecasis MM, da Silva CL, Cabral
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©2019 JCBN K. Matsunaga et al. 19 Zheng Q, Zhao Y, Guo J, et al. Iron overload promotes mitochondrial frag-
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Inclusive Financing’s Development and Urban-rural Income Gap—Empirical analysis based on provincial panel data
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Inclusive Financing's Development and Urban-rural Income Gap
—Empirical analysis based on provincial panel data Xu Jing1 a*, Sun Mingyue2 b, Wang Hening3c Xu Jing1 a*, Sun Mingyue2 b, Wang Hening3c 123Shandong University of Science and Technology, Shandong, Jinan, 250031 123Shandong University of Science and Technology, Shandong, Jinan, 250031 123Shandong University of Science and Technology, Shandong, Jinan, 250031 Abstract: With the further development of China's economy, the income of urban and rural residents has
increased. At present, China's economy has changed from a stage of high-speed growth to a stage of
high-quality development. However, uneven and insufficient regional development is still a problem that
requires great attention. Inclusive Financing's development provides residents with more access to financial
services and is one of the important ways to build a well-off society and develop the economy in recent
years. Based on the Peking University Digital Inclusive Financing Index, this paper studies the relationship
between the development level of Inclusive Financing and the income gap between urban and rural
residents by constructing panel data of 31 provinces, cities and regions in China and establishing a model
for empirical analysis. The study found that the development of Inclusive Financing has a significant impact
on the income gap between urban and rural areas, among which the improvement of the level of Inclusive
Financing in the economically developed eastern region has a very obvious effect on narrowing the income
gap between urban and rural areas. However, blindly developing Inclusive Financing in the underdeveloped
central and western regions will also cause the further widening of the income gap between urban and rural
areas. The conclusion of this paper has certain positive significance for narrowing the income gap between
urban and rural areas and promoting the development of Inclusive Financing. The Third Plenary Session of the 18th CPC Central
Committee in 2013 established "developing Inclusive
Financing" as a national strategy. In 2015, the State
Council issued the Plan for Promoting the Development
of Inclusive Financing (2016-2020). In recent years, the
development of Inclusive Financing in our country has
been supported by the government and the public. Vigorously developing Inclusive Financing is an
important embodiment of the financial industry's support
for the construction of a modern economic system and
the enhancement of its ability to serve the real economy. It is also an important way to ease the contradiction
between the people's growing demand for financial
services and the inadequate and unbalanced financial
supply. *a698766474@qq.com
b2819252853@qq.com, c1145720083@qq.com
© 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/). Inclusive Financing's Development and Urban-rural Income Gap
—Empirical analysis based on provincial panel data It is also an inevitable requirement for China to
build a well-off society in an all-round way. E3S Web of Conferences 275, 03015 (2021)
EILCD 2021 E3S Web of Conferences 275, 03015 (2021)
EILCD 2021 https://doi.org/10.1051/e3sconf/202127503015 EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
icenses/by/4.0/). 1
Introduction How to further narrow the income gap between urban
and rural areas is a hot issue studied by experts in the
economic field in recent years. Although the income of
urban and rural residents has been rising since the reform
and opening up, the gap has been gradually widening,
and the dual economic structure of urban and rural
division has not changed. The report of the 19th National
Congress of the Communist Party of China in 2017
pointed out that the gap between urban and rural regional
development and income distribution is still large, some
outstanding problems of insufficient development
imbalance have not yet been solved, and the quality and
efficiency of development are still not high, all of which
need us to focus on solving. With the rapid development of science and
technology, Digital Inclusive Financing has reduced the
transaction costs of financial services through the
advantages of digital technology, which is a new idea and
new way for Inclusive Financing to develop innovation. With the use of information technology, the coverage of
basic financial services continues to expand, so that
low-income people can have more opportunities and
more easy access to financial services, which is of great
significance for the income growth of low-income people
and thus narrowing the income gap between urban and
rural areas. The concept of Inclusive Financing first appeared in
the UN's vocabulary of promoting micro-credit in 2005. It is a financial system that can effectively and
comprehensively serve all social strata and groups. The
inclusive financial system believes that everyone should
have access to financial services and everyone should
participate in the economic development process, so as to
realize the common prosperity of the society. And the
inclusive financial system provides opportunities for
low-income people, which is also of great significance in
narrowing the income gap between urban and rural areas. However, the inclusive financial system is still facing E3S Web of Conferences 275, 03015 (2021) E3S Web of Conferences 275, 03015 (2021)
EILCD 2021 https://doi.org/10.1051/e3sconf/202127503015 many practical difficulties and challenges in its
development. The uneven development in regions with
economic levels has also affected the development of
Inclusive Financing in some regions. However, the
concept of digital Inclusive Financing, which has arisen
in recent years, is facing greater controversy and
restriction. role in promoting the narrowing of the urban-rural
income gap at a high level, but it is not statistically
significant. 2
Literature review and theoretical
analysis In the existing literature, scholars have different views on
whether Inclusive Financing and the digital Inclusive
Financing developed in recent years can narrow the
income gap between urban and rural areas. Some scholars build panel data and models for
empirical analysis, and think that the development of
Inclusive Financing can effectively narrow the income
gap between urban and rural areas. Allen(2016) found
that developing Inclusive Financing can expand
employment and raise income level [1]. Zhang Xiaoyan
(2016) measured the level of Inclusive Financing in
China by using the relevant data of China's Internet
finance, Inclusive Financing and urban-rural income gap
from 2004 to 2014, and based on this, established a
VECM model to empirically analyze the impact of
Inclusive Financing's development on urban-rural income
gap under the background of Internet finance. The
research shows that there is a long-term equilibrium
relationship between the development of Inclusive
Financing and the income gap between urban and rural
areas. Improving the development level of Inclusive
Financing can significantly reduce the income gap
between urban and rural areas, and the effect is lasting. There is also a mutual promotion effect between the two
[2]. Zhang Zihao (2018) constructed provincial spatial
panel data from 2013 to 2015, and used spatial panel
econometric model to empirically analyze the impact of
digital Inclusive Financing on China's urban-rural
income gap. The results show that the income gap
between urban and rural areas in all provinces,
autonomous regions and municipalities in China shows a
significant spatial dependence on the whole, and shows
"high
spatial
agglomeration"
and
"low
spatial
agglomeration" locally. Digital Inclusive Financing can
significantly promote the narrowing of the income gap
between urban and rural areas, improve the welfare level
of low-income groups and help underdeveloped areas
accelerate their development. And the decomposition
indicators of digital Inclusive Financing can significantly
reduce the urban-rural income gap [3]. By combing the literature, some scholars believe that
the impact of Inclusive Financing on residents' income is
mainly due to the following reasons. First, the inclusive
financial system covers a wider range. Especially after
the development of Digital Inclusive Financing, groups
with different income levels, especially low-income
groups, have more access to financial services and have
improved their awareness of financial products. Secondly,
the development of Inclusive Financing has provided
more people with financial services suitable for them. Many financial institutions have also innovated their
products and businesses. 1
Introduction When the level of digital Inclusive Financing
is greater than the threshold value, the income gap
between urban and rural areas in the regions with low
economic development level will effectively narrow with
the improvement of the level of digital Inclusive
Financing [4]. Xiong Deping (2020) selected the data and
information of 31 provinces (autonomous regions and
municipalities) in China from 2011 to 2018, constructed
a fixed and random effect double regression model, and
tested the impact of digital Inclusive Financing
development on urban-rural income gap by using the
empirical method of non-equilibrium effect test. According to the analysis of the results, the infrastructure
construction in rural areas is relatively backward in less
developed areas, the primitive accumulation of capital in
urban areas is significantly higher than that in rural areas,
and financial resources are concentrated in cities. On the
whole, the income gap between urban and rural areas has
not narrowed, but expanded [5]. 3.1 Data sources (1) Urbanization Level (PURB) In recent years, urbanization has expanded rapidly,
and the urban-rural dual economic structure has widened
the income gap between urban and rural areas. The
urbanization level is measured by selecting the ratio of
urban population to total population. This paper selects the panel data of 31 provinces,
municipalities and autonomous regions in China from
2010 to 2019. Among them, the data related to digital
Inclusive Financing comes from "Digital Inclusive
Financing Index of Peking University (2011-2018)",
which is compiled by using the massive data of Ant
Financial Service on digital Inclusive Financing, and
mainly consists of indicators such as account coverage,
payment business, credit business and insurance business. Other data such as urban and rural residents' income,
economic growth and per capita GDP mainly come from
China Statistical Yearbook, website of National Bureau
of Statistics, website of local bureau of statistics and
Wind database. (2) The level of economic development (RGDP) The level of economic development in different
regions will have an impact on the income of urban and
rural residents. In this paper, the per capita GDP is
selected to measure the level of economic development,
and considering the dimension problem, the index is
treated logarithmically. (3) fiscal expenditure (TAX) Fiscal expenditure can promote the development of
different industries, thus affecting the income of urban
and rural residents. When government expenditure tends
to be urban, the income gap between urban and rural
residents is likely to widen further. This paper measures
this index by the proportion of fiscal expenditure to
regional GDP. 2
Literature review and theoretical
analysis Compared with the traditional
bank savings income, residents are more inclined to
invest in financial products. Third, with the development
of digital Inclusive Financing, information technology
has improved the efficiency and level of financial
services. Through big data analysis and other means, the
scheme analysis can be provided for different types of
users, so as to reduce investment risks and financing
costs for low-income groups, and thus encourage more
residents to participate in the development of Inclusive
Financing. To sum up, the narrowing of the income gap between
urban and rural areas needs to be driven by economic
development, but there are still differences on whether
inclusive financial system can play a role in narrowing
the income gap between urban and rural areas. Moreover,
due to the imbalance of regional economic development
in our country, whether the impact of Inclusive
Financing's development is always positive or negative in
regions with different economic levels needs to be tested
and proved. At present, Digital Inclusive Financing has
opened up an innovative way for the development of
Inclusive Financing, but how to graft relevant
technologies with different regions so as to have a
positive impact on residents' income and further narrow
the income gap is still a problem that needs to be focused
on.. Some scholars believe that the level and stage of
development in Inclusive Financing will have different
effects on the income gap between urban and rural areas. Zhao Bingqi and others (2020) constructed the provincial
panel data from 2011 to 2018, and selected a single
threshold model to conduct an empirical analysis and
research on the relationship between the two. They
proposed that the development of digital Inclusive
Financing will further widen the urban-rural income gap
at a low level of digital Inclusive Financing, and play a 2 2 E3S Web of Conferences 275, 03015 (2021)
EILCD 2021 E3S Web of Conferences 275, 03015 (2021) https://doi.org/10.1051/e3sconf/202127503015 3
Data sources and variable selection dimensions:
coverage
breadth,
use
depth
and
digitalization degree. 3. Control variables: 3. Control variables: 3. Control variables: (5) Education level (EDU)
A
Chi
'
b
d (5) Education level (EDU)
A
Chi
'
b
d At present, China's urban education level and teachers
have obvious advantages compared with rural areas, so
the education level also has an impact on urban and rural
income. This paper selects the average number of
students in colleges and universities per 100,000
population to measure the education level. 2. Core explanatory variable: Inclusive Financing
Index (IFI) According to the report of Digital Inclusive Financing
Index of Peking University (Phase II, 2011-2018)
released by Digital Finance Research Center of Peking
University in April 2019, the provincial digital Inclusive
Financing index calculated by this method from 2011 to
2018 is selected as the explanatory variable of the model. And some missing data are supplemented by difference
method. The index includes three first-level index (6) Employment status (EMP) (6) Employment status (EMP) The employment situation has a high correlation with
the income of urban and rural residents. The high
unemployment rate in rural areas and the low
unemployment rate in urban areas will further widen the
income gap between urban and rural areas. This paper
selects the proportion of employed population to the total
population to measure the employment situation. Table 1 Descriptive statistics of each variable index
Variable
Obs
Mean
Std. Dev. Min
Max
IG
310
84466
27677
10475
190064
IFI
310
184.6
103.4
6.800
419.2
PURB
310
0.561
0.134
0.227
0.896
RGDP
310
4.641
0.207
3.843
5.216
TAX
310
0.293
0.209
0.113
1.354
IS
310
0.900
0.0526
0.742
0.997
EDU
310
2535
830.5
1082
6196
EMP
310
0.254
0.116
0.125
0.761
3.3 Possible problems in the model
(1) the endogenous problem
This paper discusses the influence of the development
level of Inclusive Financing on the income gap between
urban and rural areas. The endogenous problem of the
model mainly comes from the measurement error. Measurement error refers to the error between the values Table 1 Descriptive statistics of each variable index level of Inclusive Financing on the income gap between
urban and rural areas. The endogenous problem of the
model mainly comes from the measurement error. Measurement error refers to the error between the values 3.2 variable selection 1. Explained variable: urban-rural income gap (IG)
This paper chooses the ratio of urban per capita
disposable income to rural per capita disposable income
to reflect the urban-rural income gap. The smaller the
ratio, the smaller the income gap between urban and rural
areas, whereas the more serious the income gap between
urban and rural areas. According to the relevant data in
China Statistical Yearbook, there were no statistics on per
capita disposable income of rural residents before 2013,
so the per capita net income of rural residents from 2010
to 2012 was selected for analysis. g
(4) Industrial structure (IS) The industrial structure is measured by the ratio of
the added value of the secondary and tertiary industries
to GDP in various provinces and cities. 3.3 Possible problems in the model 3 E3S Web of Conferences 275, 03015 (2021)
EILCD 2021 https://doi.org/10.1051/e3sconf/202127503015 random disturbance, IG for income gap between urban
and rural residents, IFI for development level of
Inclusive Financing, PURB for urbanization level, RGDP
for economic development level, TAX for fiscal
expenditure, IS for industrial structure, EDU for
education level and EMP for employment situation. of the explanatory variables used in the model and the
true values. There are some errors in the measurement of
Inclusive Financing level with Inclusive Financing index
as an index. (2) Robustness When considering the impact of Inclusive Financing's
development level on the urban-rural income gap, we
should also consider the heterogeneity of different
regions. For example, whether it is located in the eastern
or central and western regions, and the economic
development level of the region. In this paper, the
robustness is tested by sub-sample regression. 4.2 Regression analysis of benchmark model In this paper, the panel data model is used for regression
analysis, so we should judge whether to use mixed model,
fixed effect model or random effect model. Therefore,
this paper uses Hausmann test method to judge the model. The judging steps are as follows: firstly, Hausmann test
method is used to judge whether to choose fixed effect
model or random effect model. When P value is greater
than 0.05, the original hypothesis is accepted and the
random effect model is established; If the p value is less
than 0.05, the original hypothesis is rejected and the
fixed effect model is adopted. 4.1 model setting IGi,t=α1 + β1IFIi,t + β2PURBi,t + β3RGDPi,
t + β4TAXi,t + β5ISi,t+β6EDUi,t+β7EMPi,t+εi,t t + β4TAXi,t + β5ISi,t+β6EDUi,t+β7EMPi,t+εi,t
Among them, I stands for province, T for year, ε for Among them, I stands for province, T for year, ε for Table 2 Stepwise regression results of benchmark model
(1)
(2)
(3)
(4)
(5)
(6)
m1
m2
m3
m4
m5
m6
VARIABLES
IG
IG
IG
IG
IG
IG
IFI
-0.0847
***
-0.0909
***
-0.0726
***
-0.0806
***
-0.0317
***
-0.0327
***
(0.008)
(0.008)
(0.008)
(0.009)
(0.006)
(0.006)
RGDP
2.4711
***
2.4628
***
2.6012
***
2.6167
***
3.0202
***
3.0355
***
(0.063)
(0.062)
(0.060)
(0.061)
(0.040)
(0.040)
EMP
0.4419***
0.2441*
0.2540*
-0.2456
***
-0.2387
***
(0.148)
(0.138)
(0.138)
(0.085)
(0.084)
EDU
-0.3951
***
-0.3666
***
-0.0147
-0.0213
(0.054)
(0.056)
(0.037)
(0.036)
TAX
0.3057*
0.1409
0.1333
(0.164)
(0.098)
(0.096)
PURB
-3.3371
***
-3.1879
***
(0.150)
(0.154)
IS
-0.7279
***
(0.220)
Constant
11.7108***
11.7413
***
11.6514
***
11.6904
***
11.4494
***
11.4544
*** Table 2 Stepwise regression results of benchmark model 4 E3S Web of Conferences 275, 03015 (2021) E3S Web of Conferences 275, 03015 (2021)
EILCD 2021 https://doi.org/10.1051/e3sconf/202127503015 (0.039)
(0.039)
(0.038)
(0.043)
(0.028)
(0.028)
Observations
310
310
310
310
310
310
R-squared
0.93
0.93
0.94
0.94
0.98
0.98
Number of pro1
31
31
31
31
31
31
Adj. R-squared
0.98
0.98
0.98
0.98
0.98
0.98
Standard errors in parentheses
*** p<0.01, ** p<0.05, * p<0.1 economy and pull in the distance between rural and
urban areas, thus effectively reducing the income gap
between urban and rural areas. Industrial structure has a
significant impact on narrowing the income gap between
urban and rural areas. It shows that the development of
secondary and tertiary industries can effectively improve
the transformation and development of urban and rural
industrial structure, especially in rural areas, where the
industrial structure is weaker than that in cities. By
focusing on the development of secondary and tertiary
industries, the efficiency of industrial adjustment and
optimization can be improved, which is closer to the
urban industrial development and structure, thus raising
the income level and narrowing the income gap. According to the regression results, the coefficient
sign and significance level of the core explanatory
variable
are
generally
consistent,
especially
the
coefficient sign and significance level of the core
explanatory variable number Hewlett-Packard Finance
are basically the same, indicating that the regression
results are relatively robust. 4.1 model setting By establishing a panel data model, this paper studies
and analyzes the direct relationship between the
development level of Inclusive Financing and the income
gap between urban and rural residents in 31 provinces,
cities and regions of China. According to Hausman test results, the P value is less
than 0.05, and the fixed effect model is adopted for solid
selection. Table 2 shows the regression results of
gradually adding control variables. In addition to Inclusive Financing's development
level, urbanization level, economic development level,
fiscal expenditure, industrial structure, education level
and employment status will also affect the income gap
between urban and rural areas. As shown in the table, the first to sixth columns are
the regression results obtained by gradually adding the
control variables of economic development level,
employment status, education level, fiscal expenditure,
urbanization level and industrial institutions according to
the optimal significance. In order to study the relationship between the
development level of Inclusive Financing and the income
gap between urban and rural areas, the following model
is established: The last column shows the most significant set of
regression results, that is, the regression results after
considering all the above-mentioned core variables and
control variables. 4.1 model setting There is an obvious negative
correlation between Inclusive Financing's development
level and urban-rural income gap, which means that the
improvement of Inclusive Financing's development level
can narrow the urban-rural income gap, and the impact is
very significant. By promoting the development of
Inclusive Financing, more township residents can learn
about various financial products and services, and
increase their participation in investment and financial
management, thereby increasing their income and further
reducing the income gap with urban residents. At the same time, we can see that education level and
government expenditure have little effect on reducing
urban and rural income. Education level negatively
affects the income gap between urban and rural areas,
while government expenditure positively affects the
income gap between urban and rural areas, but it is not
statistically significant. Therefore, these two factors have
no obvious influence on the income gap between urban
and rural areas. Among the control variables, economic development
level, employment status, urbanization level and
industrial structure also have a very significant impact on
the income gap between urban and rural areas. There is a
positive relationship between the level of economic
development and the income gap between urban and
rural areas, which indicates that the improvement of
economic level in different regions will lead to the
further widening of the gap between urban and rural
areas. For urban residents, the income growth brought by
the improvement of economic level may be greater than
that of rural residents, so it will increase the income gap
between urban and rural areas. Employment status,
urbanization level, industrial structure and urban-rural
income gap are negatively correlated. From the results,
the higher the employment coverage, the higher the
income level of urban and rural residents will be. For
cities, the employment rate is close to saturation most of
the time, but for rural areas, many jobs are vacant, and
the employment coverage is far less optimistic than that
of cities. The increase of rural employment rate can
effectively increase the income of rural residents, thus
helping to narrow the income gap between urban and
rural areas. The improvement of urbanization level can
also narrow the income gap between urban and rural
areas, which is consistent with the conclusion of Zhang
Yiming et al. (2018), which shows that the improvement
of urbanization level can drive the development of rural 5.1 Endogenous problems Although
the
regression
results
show
that
the
development level of Inclusive Financing has a
significant effect on the income gap between urban and
rural areas, its endogenous problems may still have an
impact on the significance, so this paper chooses
endogenous test. Using the explanatory variable of the lag period as
the instrumental variable to test the endogeneity, that is,
the development level of Inclusive Financing in the lag
period. On the one hand, there is a high correlation
between lagging indicators and current indicators; on the
other hand, lagging indicators affect urban-rural income
gap through current digital Inclusive Financing indicators,
so
lagging indicators
meet
the
correlation
and
exclusiveness, which is a more reasonable tool variable. Table 3 Estimation of tool variables
(1)
(2)
(3)
(4)
(5)
(6)
m1
m2
m3
m4
m5
m6
VARIABLES
IG
IG
IG
IG
IG
IG
IFI
-0.1286***
-0.1361
***
-0.1106
***
-0.1194
***
-0.0423
***
-0.0429
*** Table 3 Estimation of tool variables IFI 5 5 E3S Web of Conferences 275, 03015 (2021) E3S Web of Conferences 275, 03015 (2021)
EILCD 2021 https://doi.org/10.1051/e3sconf/202127503015 (0.010)
(0.010)
(0.010)
(0.011)
(0.007)
(0.007)
RGDP
2.6389
***
2.6413
***
2.7683
***
2.7853
***
3.0929
***
3.1023
***
(0.067)
(0.066)
(0.061)
(0.062)
(0.038)
(0.038)
EMP
0.5617***
0.4570***
0.4669***
-0.1803**
-0.1771**
(0.170)
(0.152)
(0.152)
(0.091)
(0.090)
EDU
-0.4337
***
-0.3981
***
-0.0467
-0.0564
(0.060)
(0.062)
(0.038)
(0.038)
TAX
0.3844**
0.3707***
0.3474***
(0.176)
(0.099)
(0.098)
PURB
-3.3705
***
-3.2475
***
(0.156)
(0.165)
IS
-0.5236**
(0.241)
Constant
11.9368***
11.9725
***
11.8449
***
11.8880
***
11.5024
***
11.5055
***
(0.051)
(0.052)
(0.050)
(0.054)
(0.035)
(0.035)
Observations
279
279
279
279
279
279
Number of pro1
31
31
31
31
31
31
Adj. R-squared
0.98
0.98
0.98
0.98
0.98
0.98
Standard errors in parentheses
*** p<0.01, ** p<0.05, * p<0.1 According to the test results, it can be seen that the
significance of the core explanatory variables is
consistent with the benchmark regression, and the
improvement of Inclusive Financing level plays a very
significant role in narrowing the income gap between
urban and rural areas. Moreover, the most significant
model selected from the benchmark regression is still the
best in the endogeneity test. In this group, the
development level of Inclusive Financing has more
obvious influence on narrowing the income gap between
urban and rural areas. 5.1 Endogenous problems Among the control variables, the
economic development level and urbanization level are
still
significant,
showing
positive
and
negative
correlation respectively. Although the significance of
employment status and industrial institutions has
declined, the overall results are consistent with the results
of the benchmark regression model. The effect of
education level on narrowing the income gap between
urban and rural areas is still not obvious. However, in the
test, government expenditure has played a significant role in narrowing the income gap between urban and
rural areas. It can be seen that under the influence of
some missing variables, the significance of government
expenditure has changed. To sum up, the test situation basically coincides with
the significant results of the benchmark regression model,
so this paper controls the endogenous problem well. 5.2 Robustness test This paper considers the regional heterogeneity of
Inclusive Financing's development level, that is, the
development of Inclusive Financing in different regions
will have a significant impact on narrowing the income
gap between urban and rural areas. In this paper, we set
up sub-samples of the whole country, the eastern region
and the central and western regions for grouping
regression to test the robustness. Table 4 Test results of different regional samples
(1)
(2)
(3)
M8
M9
M10
VARIABLES
IG
IG
IG
IFI
-0.0327***
-0.0663***
0.0199***
(0.006)
(0.011)
(0.005)
RGDP
3.0355***
3.2424***
2.0248***
(0.040)
(0.046)
(0.070)
EMP
-0.2387***
-0.2433**
-0.3358***
(0.084)
(0.105)
(0.109)
EDU
-0.0213
-0.0439
0.0010
(0.036)
(0.078)
(0.026)
TAX
0.1333
0.0164
-0.2622*** Table 4 Test results of different regional samples 6 https://doi.org/10.1051/e3sconf/202127503015 E3S Web of Conferences 275, 03015 (2021)
EILCD 2021 E3S Web of Conferences 275, 03015 (2021) (0.096)
(0.226)
(0.071)
PURB
-3.1879***
-2.0068***
-1.5933***
(0.154)
(0.366)
(0.162)
IS
-0.7279***
-3.6483***
0.0169
(0.220)
(1.032)
(0.147)
Constant
11.4544***
11.6545***
11.1810***
(0.028)
(0.049)
(0.027)
Observations
310
110
200
R-squared
0.98
0.99
0.99
Number of pro1
31
11
20
Adj. R-squared
0.99
0.99
0.99
Standard errors in parentheses
*** p<0.01, ** p<0.05, * p<0.1 quite special. The impact of fiscal expenditure changes
from insignificant to significant, while the impact of
industrial
structure
changes
from
significant
to
insignificant. The results in the eastern region and the
whole country are opposite. This shows that the impact
of fiscal expenditure in the central and western regions is
very direct and effective. Because the economic
development is at a low level, if there is some rural bias
in fiscal expenditure, it will have a very obvious impact
on narrowing the income gap between urban and rural
areas. However, in the eastern region, where the
economic level is developing at a high level, the impact
of fiscal expenditure cannot produce such an effect in the
central and western regions. By the same token, the
transformation of industrial structure is more urgent and
crucial for the eastern region with better economic
development, and the eastern region has more
preparation and resources for the development of
industrial structure, so the upgrading of industrial
structure can play a positive role in narrowing the income
gap between urban and rural areas. However, the
industrial structure in the central and western regions is
unreasonable. 5.2 Robustness test For the central and western regions, the
time for development and further transformation is not
yet ripe, which will increase the pressure of economic
development and lead to more problems, and may reduce
the stability of rural economic development, which is not
conducive to the improvement of rural residents' income,
thus further widening the income gap between urban and
rural areas. From the regression results, it can be seen that the
development level of Inclusive Financing has a very
significant impact on the urban-rural income gap in the
whole country, the east and the central and western
regions. For the whole country and the eastern region,
they are negatively correlated, but in the central and
western regions, they have different results and are
positively correlated. This shows that from the
perspective of the whole country and the eastern region,
the overall economic development of Inclusive Financing
is relatively moderate. With the improvement of
Inclusive Financing level, the income gap between urban
and rural areas can be effectively narrowed. According to
the absolute value of the regression coefficient of
variables, Inclusive Financing has the most significant
effect in the eastern region, that is, developing Inclusive
Financing in the eastern region can effectively narrow the
income gap between urban and rural areas. The central
and western regions are relatively low-income regions
with low economic development level. Developing
Inclusive Financing here may further widen the income
gap between urban residents and rural residents. For the
central and western regions, the economic structure is
imperfect, the development is insufficient, and the
conditions for contacting with financial services are
lacking. If Inclusive Financing is developed hard, it is
likely to lead urban and rural residents to participate in
financial activities that do not match their own income
level and bear higher financial risks. Moreover, the
ability to resist risks in the central and western regions is
weaker than that in the eastern regions, which makes the
development of Inclusive Financing have an opposite
impact. We judge that Inclusive Financing can effectively
narrow the income gap between urban and rural areas on
the basis of economic development to a certain extent,
otherwise it will further widen the gap. 6.2 Relevant suggestions (1) Improve the development system of Inclusive
Financing and further promote the digital Inclusive
Financing. At present, Inclusive Financing is still in the
development stage, and many urban and rural residents
don't understand the true meaning and function of
Inclusive Financing, which leads to the difficulty of
promoting Inclusive Financing, especially in rural areas. Relevant departments should actively popularize the
knowledge of Inclusive Financing and do a good job of
top-down
information
transmission. With
the
development of information technology, the importance
of the development of digital Inclusive Financing is
increasing,
and
digital
Inclusive
Financing
can
effectively improve the coverage, expand the coverage
and make it easier for residents to understand digital
Inclusive Financing. The diversified financial products
and services developed through digital Inclusive
Financing have carried out risk management and control
more rationally, reduced the financial investment risks
faced by low-income people, helped more residents to
invest in the construction of Inclusive Financing,
experienced
financial
services,
promoted
the
development of Inclusive Financing through personal
experience, promoted the economic level of urban and
rural areas, and further narrowed the income gap between
urban and rural areas. (3) Pay attention to the innovation of financial
services and promote the diversification of products. Innovative design of financial services and products can
reduce the cost of financial services and products. Encourage relevant institutions to open business
departments or outlets in rural areas, design and provide
corresponding services for rural areas and residents,
provide more diversified and personalized products, and
set up functional institutions such as self-service banks. Increase the scope of rural collateral, and explore
innovations such as rural forest rights, housing property
rights and land contractual management rights as
collateral. Transfer financial services through digital
technology, explore low-cost and high-efficiency digital
financial services, improve coverage breadth and depth
through
financial
innovation,
and
increase
agriculture-related financial business. (2) Optimize the development environment of rural
Inclusive Financing. Compared with urban areas, rural
areas have a lower level of economic development, and
their education level and industrial development are in a
relatively weak position. We should pay attention to the
environmental construction of Inclusive Financing in
rural areas, and open up residents' understanding of
Inclusive Financing through various publicity and
promotion channels. Table 5 Basic conclusions of empirical research Development level of Inclusive
Financing
Income gap between urban and rural
areas
whole country
outstanding
Zoom out (negative)
eastern region
outstanding
Zoom out (negative)
Midwest
outstanding
Expand (forward) departments
to
strengthen
residents'
confidence. Attention should be paid to the development of Internet
information technology in rural areas. Technology is an
important driving force and a support for the
development of Inclusive Financing. Providing better
Internet technology environment for rural residents can
accelerate the popularization of Inclusive Financing in
rural areas. The government should gradually change the
intensity of fiscal expenditure from urban to rural areas,
and increase financial support. Encourage and guide
private capital to establish new financial institutions in
rural areas. Encourage and help small and micro
enterprises to obtain financial services, help residents in
rural areas who do not need education to choose
appropriate financial services or products, promote
economic development in rural areas, and further narrow
the income gap between urban and rural areas. 6.1 Conclusion From the control variables, the effects of regional
economic development level, employment status and
urbanization level on narrowing the urban-rural income
gap are still very significant, while the effects of
education level and industrial structure on narrowing the
urban-rural income gap are still not obvious. With the continuous improvement of economic level,
China's economy has turned into a high-quality
development stage, but narrowing the income gap
between urban and rural areas has always been the
direction that needs efforts. In recent years, the rise of
Inclusive Financing has provided new ideas for
narrowing the income gap between urban and rural areas. Compared with the eastern region and the whole
country, the results in the central and western regions are 7 E3S Web of Conferences 275, 03015 (2021)
EILCD 2021 https://doi.org/10.1051/e3sconf/202127503015 With the development and wide application of digital
information technology, digital Inclusive Financing has
gradually entered the public's field of vision and played
an important role in narrowing the income gap between
urban and rural areas. Based on the panel data of 31
provinces and cities in China, this paper establishes a
model for empirical analysis and studies, and draws the
following conclusions: with access to financial products and services, improve
the income level and narrow the income gap between
urban and rural areas. (2) From the regional situation, the development of
Inclusive Financing plays the most significant role in
narrowing the gap between the rich and the poor in the
economically developed eastern region. (3) In the less developed central and western regions,
the improvement of Inclusive Financing level may
further widen the income gap between urban and rural
areas, which is not conducive to the development of rural
economy. (1) The development level of Inclusive Financing has
a significant effect on narrowing the income gap between
urban and rural areas, and developing Inclusive
Financing can effectively narrow the income gap
between urban and rural areas. To provide more residents 6.2 Relevant suggestions Establish relevant units or carry out
structural grafting in relevant rural departments, and
carry out publicity and promotion work by authoritative (4) Promote the economic development of the
central and western regions and narrow the regional
development gap. Because the economic level of the
central and western regions is weaker than that of the
eastern regions, it is necessary to further improve the
economic level and lay a good economic foundation for 8 E3S Web of Conferences 275, 03015 (2021)
EILCD 2021 E3S Web of Conferences 275, 03015 (2021) https://doi.org/10.1051/e3sconf/202127503015 the expansion and development of Inclusive Financing. Pay attention to the rational distribution of educational
resources and fiscal expenditure, pay attention to the
infrastructure construction and the distribution of
educational resources in the central and western regions,
and ensure the development of the central and western
regions. Through policy implementation and publicity
and guidance, we will provide more employment
opportunities for the central and western regions and
create more possibilities for capital inflow. For the
central and western regions, foreign funds can be
introduced to improve the development level of regional
enterprises, so that they have certain policy support in the
development process. At the same time, actively promote
and implement digital Inclusive Financing, do a good job
in the early stage of promotion, and improve the degree
of digital development in the central and western regions. the expansion and development of Inclusive Financing. Pay attention to the rational distribution of educational
resources and fiscal expenditure, pay attention to the
infrastructure construction and the distribution of
educational resources in the central and western regions,
and ensure the development of the central and western
regions. Through policy implementation and publicity
and guidance, we will provide more employment
opportunities for the central and western regions and
create more possibilities for capital inflow. For the
central and western regions, foreign funds can be
introduced to improve the development level of regional
enterprises, so that they have certain policy support in the
development process. At the same time, actively promote
and implement digital Inclusive Financing, do a good job
in the early stage of promotion, and improve the degree
of digital development in the central and western regions. (5) Strengthen the supervision system to prevent
financial risks. The development of Internet finance is
accompanied by corresponding problems and risks,
which to a great extent hinders and affects the further
development of digital Inclusive Financing. 6.2 Relevant suggestions The
government should improve the information disclosure
mechanism and relevant laws and regulations to provide
legal guarantee for the development of Inclusive
Financing. Improve the credit information system, enrich
the information and situation of financial participants,
reduce the probability of financial risks, improve the
security
of
inclusive
finance,
enhance
residents'
resistance to risks, maintain financial market stability,
promote economic growth, and narrow the income gap
between urban and rural areas. References: 1. Allen F,Demirguc-Kunt A,Klapper L,et al. The
foundations of financial inclusion:Understanding
ownership and use of formal accounts[J]. Journal of
Financial Intermediation,2016,27: 2. Zhang Xiaoyan. The impact of Inclusive Financing's
development on urban-rural income gap under the
background of Internet finance [J]. Accounting
Monthly, 2016(17):94-97. 3. Zhang Zihao, Tan Yanzhi. Digital Inclusive
Financing and Urban-Rural Income Gap in China:
An
Empirical
Analysis
Based
on
Spatial
Econometric Model [J]. Financial Theory and
Practice, 2018(06):1-7. 4. Zhao Bingqi. Digital Inclusive Financing and
Urban-Rural Income Gap in China: An Empirical
Study Based on Panel Threshold Model [J]. Journal
of Social Sciences, 2020(01):196-205. 5. Xiong Deping, Chen Yuran. The impact of the
development of digital Inclusive Financing on the
income gap between urban and rural areas —— An
empirical analysis based on unbalanced effect and
threshold effect [J]. Changbai Journal, 2020(05):
99-106. 9 9
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http://www.foliamalacologica.com/pdf-117829-46888?filename=First records of.pdf
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First records of Viviparus acerosus (Bourguignat, 1862) (Gastropoda: Viviparidae) from the Czech Republic outside its native range
|
Folia Malacologica
| 2,019
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cc-by
| 2,006
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Folia Malacol. 27(3): 223–229
https://doi.org/10.12657/folmal.027.021
FIRST RECORDS OF VIVIPARUS ACEROSUS
(BOURGUIGNAT, 1862) (GASTROPODA: VIVIPARIDAE)
FROM THE CZECH REPUBLIC OUTSIDE ITS
NATIVE RANGE
Luboš Beran1*, Michal Horsák2, Sebastian Hofman3
Nature Conservation Agency of the Czech Republic, Regional Office Kokořínsko – Máchův kraj Protected
Landscape Area Administration, Česká 149, CZ-27601 Mělnik, Czech Republic
(e-mail: lubos.beran@nature.cz); https://orcid.org/0000-0002-5851-6048
2
Department of Botany and Zoology, Masaryk University, Kotlářská 2, CZ-611 37 Brno, Czech Republic;
https://orcid.org/0000-0003-2742-2740
3
Department of Comparative Anatomy, Institute of Zoology and Biomedical Research, Jagiellonian
University, Gronostajowa 9, 30-387 Cracow, Poland;
https://orcid.org/0000-0001-6044-3055
*corresponding author
1
Abstract: Three native species of the Viviparidae are known in the Czech Republic, namely Viviparus
acerosus, V. contectus and V. viviparus. The native range of V. acerosus includes the Danube and probably
Dnieper and Dvina river basins. In the Czech Republic it inhabits slowly flowing rivers and canals in the
floodplain of the Dyje and Morava rivers in South Moravia (Danube River basin). Recently, this species was
found outside its native range in four sites in Bohemia (Labe River basin) and in one site in Silesia (Odra
River basin). The species identity of the abundant population from the Švihov dam reservoir (Labe River
basin) was verified using COI sequences. These finds represent the first records of the species in the Labe
and Odra river basins. The possible origin of these populations is discussed.
Key words: Viviparus acerosus, Czech Republic, non-native occurrence, new records, recent spread
INTRODUCTION
There are three native species of the Viviparidae
reported from the Czech Republic (Beran 2002,
Horsák et al. 2013). Viviparus contectus (Millet, 1813)
inhabits stagnant waters in the floodplains of large
lowland rivers in the Labe, Odra and Danube river basins, while V. viviparus (Linnaeus, 1758) was known
in the Czech Republic only from rivers of the Labe
River basin (Beran 2002). Recently, probably native
populations of the latter species have been discovered
also in the upper section of the Dyje River (Danube
River basin) at the boundary between Austria and
the Czech Republic (Beran 2013). The third spe-
cies, V. acerosus Bourguignat, 1862, typically inhabits
slow-flowing rivers and canals in the floodplain of
the Dyje and Morava rivers in South Moravia, also
parts of the Danube River basin (Beran 2002, 2013).
The species is considered endemic to the Danube
and probably also Dnieper and Dvina river basins
(Vavrova & Van Damme 2011, Welter-Schultes
2012, Vinarski & Kantor 2016) (Fig. 1). However,
it has been recently found outside its native range, for
example in the Netherlands (Soes et al. 2009). In this
paper, we present and discuss the first records of this
species in the Czech Republic outside its native range.
Folia Malacologica is funded by the Ministry of Science and Higher Education, Republic of Poland, under
agreement no 534/P-DUN/2018 of April 4th, 2018 allocated to the activities for disseminating science:
Task 1: Preparation of English versions of publications (sum funded by DUN 12,000 PLN) and
Task 2: Digitalisation of publications and scientific monographs to enable their open access in the Internet
(sum funded by DUN 11,070 PLN).
224
Luboš Beran, Michal Horsák, Sebastian Hofman
Fig. 1. Map of the Czech Republic with the known occurrence of Viviparus acerosus. Red dots – native occurrence, black
triangles – records outside native range. Drawn by J. Vrba
MATERIAL AND METHODS
The material used in this study was collected by
hand with searching of sediments, vegetation and
surfaces of stones, wood and artificial materials. The
snails for DNA analysis were fixed in 80% pure ethanol and DNA was extracted from foot tissue. The tissue was hydrated in TE buffer (3 × 10 min.); then total genomic DNA was extracted with the SHERLOCK
extracting kit (A&A Biotechnology) and the final
product was dissolved in 20 μl TE buffer. Details of
PCR conditions, primers used and sequencing as well
as of phylogenetic analysis were given in Szarowska
et al. (2016) and Rysiewska et al. (2019). The following COI sequences from GenBank were used as references: KY781182 (deposited by David et al. 2017)
and MK517417, MK517418, MK517420, MK517421,
MK517423–MK5174427 (Rysiewska et al. 2019).
Two new sequences MN272426–MN272427 obtained from specimens of the site Tvrdonice were
also added to GenBank. We used COI sequences only,
because other loci were monomorphic for Viviparus.
Selected material of shells and/or specimens fixed in
80% ethanol is deposited in the authors’ collections.
RESULTS AND DISCUSSION
The first locality of V. acerosus outside the Danube
River basin was found in the Švihov (Želivka) dam
reservoir in 2015 (Figs 2, 4, 6). An abundant population was recorded in one site (49°37'25.2"N,
15°14'52.5"E, 2.8.2015, 15.9.2018, L. Beran leg.).
The collected material was originally identified as V.
viviparus (Beran 2017). Both species are morphologically similar and their populations are very variable
in size. The pointed apex of V. acerosus, which in the
field is the most useful feature distinguishing it from
V. viviparus with blunt apex, is in the case of adult
individuals very often difficult to use due to corrosion. Later revision and also COI sequences showed
that this population actually belonged to V. acerosus.
Our analysis indicated that snails from Švihov and
Tvrdonice (V. acerosus from the native range in the
Danube River basin) were distinct from other the
Viviparus species (Fig. 3), including V. contectus (p-distance 0.115) and V. viviparus (p-distance also 0.115).
The sequences from Švihov and Tvrdonice were almost identical (1% differences) and represented one
species. Another two populations of this species were
found also in two ponds (Sýkora pond, 49°29'32.4"N,
15°23'12.7"E, 4.6.2016, L. Beran leg., Tuksa pond,
49°29'15.9"N, 15°23'52.3"E, 4.6.2016, 15.9.2018, L.
Beran leg., Fig. 7), only about 18 km away from
Records of Viviparus acerosus outside of its native range
225
Fig 2. Viviparus acerosus from the Švihov dam reservoir. Photo: Aleksandra Rysiewska
Fig. 3. Cytochrome oxidase subunit I (COI) maximum likelihood tree; bootstrap supports and Bayesian probabilities and
GenBank numbers were given
Fig. 4. Live specimens of V. acerosus collected at the Švihov dam reservoir. Photo: Luboš Beran
226
Luboš Beran, Michal Horsák, Sebastian Hofman
Fig. 5. Accumulation of fresh empty shells of V. acerosus at the Horní Bartošovický rybník pond (Silesia). Photo: Iva
Pospíšilová
Fig. 6. Švihov dam reservoir. Photo: Luboš Beran
Records of Viviparus acerosus outside of its native range
Fig. 7. Tuksa Pond. Photo: Luboš Beran
Fig. 8. Horní Bartošovický rybník Pond. Photo: Iva Pospíšilová
227
228
Luboš Beran, Michal Horsák, Sebastian Hofman
the first mentioned locality. In the case of the extensive Švihov dam reservoir (1,602 ha) other six sites
were surveyed between 2015 and 2019 and V. acero
sus was found in one other site only (49°36'51.5"N,
15°15'10"E, 25.7.2019, L. Beran leg.). Suprisingly,
an abundant population of this species was also
found in the pond of Horní Bartošovický rybník in
the Poodří Protected Landscape Area in the Odra
River basin in Silesia (49°40'38.6"N, 18°01'44.9"E,
5.9.2018, M. Horsák leg., Figs 5 and 8). These new
records outside the native range represent the first
records of this species from the Labe and Odra river
basins (Fig. 1).
The origin of these populations can be only
deduced. This species (and other species of the
Viviparidae) are often sold in garden centres and pet
shops, so the first possible way might be an accidental escape from garden pools or a deliberate release.
This way is mentioned as the most likely explanation for the occurrence of V. acerosus in Dordrecht in
the Netherlands (Soes et al. 2009). As gastropods
are capable to passive dispersal by birds over long
distances, natural dispersal cannot be ruled out either. The nearest sites with the native populations
of this gastropod are ca. 123 km and 105 km away
from the sites in Bohemia in the Labe River basin
and ca. 110 km away from the Horní Bartošovický
rybník pond in Silesia. This site is near the boundary with Poland, but V. acerosus has not been reported yet from Poland (Piechocki & WawrzyniakWydrowska 2016). Thus, the nearest populations
are known also only from South Moravia. The future
population dynamic and even the species presence at
these newly colonised sites are hard to predict. In the
case of ponds, populations are affected adversely by
water level manipulation. This is probably the case
of a large extinction recorded in the Tuksa Pond. In
2016, a very abundant population was found there,
while during the visit in 2018 only empty shells were
recorded. Eutrophication may also have a negative
effect on pond populations. However the situation in
the Švihov dam reservoir is probably different. The
reservoir serves as a source of drinking water and
thus the quality of the environment is relatively well
secured and stable. The reservoir is also inhabited by
an abundant population of the invasive Dreissena poly
morpha (Pallas, 1771), however no effect on the population of V. acerosus was observed (sessile specimens
of D. polymorpha were found only on empty shells of
V. acerosus) and the changes in the water level have
caused extinction of only a part of the population. The
question is whether the species will spread because
it has been found in the reservoir only in two sites
out of the seven sites surveyed between 2015 and
2019. From another point of view, a possible effect of
these non-native populations should be considered.
The impact of this species outside its native range
is, however, difficult to predict; the most probable
is only its potential impact on the two native species of Viviparus. All three species differ somewhat in
their habitat preferences in the Czech Republic, but V.
acerosus ecologically overlaps with the other two species. V. viviparus is known mostly from rivers, while
V. contectus prefers stagnant and overgrown habitats.
However, V. acerosus was found mostly in transitional habitats between stagnant and flowing waters and
often occupied muddy and slow-flowing canals and
rivers in floodplains.
ACKNOWLEDGEMENTS
The authors would like to thank Jan Vrba
for drawing maps, Aleksandra Rysiewska, Iva
Pospíšilová for photos and two anonymous reviewers for their valuable comments.
REFERENCES
Beran L. 2002. Vodní měkkýši České republiky – rozšíření a jeho změny, stanoviště, šíření, ohrožení a ochrana, červený seznam. Sborník přírodovědného klubu
v Uh. Hradišti, Supplementum 10. Přírodovědný klub v
Uherském Hradišti, Uherské Hradiště.
Beran L. 2013. Freshwater molluscs of the Dyje (Thaya)
river and its tributaries – the role of these water bodies
in expansion of alien species and as a refuge for endangered gastropods and bivalves. Folia Malacologica 21:
143–160. http://doi.org/10.12657/folmal.021.018
Beran L. 2017. Vodní měkkýši Vysočiny. Malacologica
Bohemoslovaca 16: 44–76.
David A. A., Zhou H., Lewis A., Yhann A., Verra S.
2017. DNA Barcoding of the Banded Mystery Snail,
Viviparus georgianus in the Adirondacks with quantifi-
cation of parasitic infection in the species. American
Malacological Bulletin 35: 175–180. https://doi.
org/10.4003/006.035.0211
Horsák M., Juřičková L., Picka J. 2013. Měkkýši České a
Slovenské republiky. Molluscs of the Czech and Slovak
Republics. Kabourek, Zlín.
Piechocki A., Wawrzyniak-Wydrowska B. 2016. Guide
to freshwater and marine Mollusca of Poland. Bogucki
Wydawnictwo Naukove, Poznań.
Rysiewska A., Hofman S., Osikowski A., Beran L.,
Pešić. V., Falniowski A. 2019. Viviparus mamilla
tus (Küster, 1852), and partial congruence between
the morphology- allozyme- and DNA-based phylogeny in European Viviparidae (Caenogastropoda:
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Architaenioglossa). Folia Malacologica 27: 43–51,
https://doi.org/10.12657/folmal.027.004
e.T23040A9408473.
http://doi.org/10.2305/IUCN.
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Soes D. M., Glöer P., Winter A. J. de 2009. Viviparus ace
rosus (Bourguignat, 1862) (Gastropoda: Viviparidae), a
new exotic snail species for the Dutch fauna. Aquatic
Invasions 4: 373–375.
Vinarski M. V., Kantor Y. I. 2016. Analytical catalogue of
fresh and brackish water molluscs of Russia and adjacent countries. A. N. Severtsov Institute of Ecology and
Evolution of RAS, Moscow.
Szarowska M., Osikowski A., Hofman S., Falniowski A.
2016. Pseudamnicola Paulucci, 1878 (Caenogastropoda:
Truncatelloidea) from the Aegean Islands: a long or
short story? Organisms Diversity & Evolution 16: 121–
139. https://doi.org/10.1007/s13127-015-0235-5
Welter-Schultes F. W. 2012. European non-marine molluscs, a guide for species identification. Planet Poster
Editions, Göttingen
Vavrova L., Van Damme D. 2011. Viviparus aceros
us. The IUCN Red List of Threatened Species 2011:
Received: July 31st, 2019
Revised: August 6th, 2019
Accepted: August 7th, 2019
Published on-line: September 14th, 2019
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Electrodeposition of Nickel onto 12K Carbon Fibre Tow in a Continuous Manner
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Croatica chemica acta
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INTRODUCTION homogeneous throughout the tow, it is therefore very
likely that numerous (present) active sites might require
different levels of activation energy for this process. The described voltage levels are between 10 and 50 V,
and electric charge in the amount of 600 C/1 000 fibre
strands, at 30 V. The single plating cell of the Cyanamid
system consists of a cylindrical electrolyte tank, a series
of contact and non-contact rollers, and several anode
baskets, containing electrolytic nickel pellets. The report-
ed operational line speed is 1.5 m min–1 and thickness of
suchproduced Ni deposit (for 7 µm in diameter 12K fibre
tow) about 0.5 µm. It has to be stated, how- ever, that the
description of the Cyanamid process (given in Refs. 14
through 19) is quite superficial and does concern a large-
scale (80 tow), commercial plating process. Carbon fibre (CF) and nickel-coated carbon fibre
(NiCCF) materials are of primary interest to a number
of industry sectors. One of the most important applica-
tions for such materials is their utilization in electrostat-
ic dissipation (ESD) and electromagnetic shielding
interference (EMI) technologies. These technologies are
especially important for automotive, cell-phone, laptop
computer and military industry markets.1–5 Other report-
ed applications for carbon fibre-based materials involve
electrochemical corrosion processes and lithium-ion
battery technologies.6–8 Nickel-coated carbon fibre composites can be pro-
duced by means of chemical vapour deposition (CVD)
method,9 electroless10–13 or by electrochemical deposi-
tion of nickel layer onto the surface of carbon fibre tow
material. Originally, the commercial process of Ni
electrodeposition on 12K carbon fibre tow was exten-
sively studied by L. Morin of American Cyanamid
company. The Cyanamid technology (protected by the
US patent law)14–19 is a several-step, Watts bath-based
Ni deposition process. According to this invention,14 an
initial stage of metal electrodeposition (nucleation
phase) is realized through application of high external
voltages, in order to provide uniform nucleation with
the fibrils. As the surface of CF filaments is not very On the other hand, current study reports a small
(single tow) laboratory-size CF electroplating system,
which in principal was set to study the effect of basic
CF pretreatments, working parameters of the plating
setup and the plating bath composition on the quality of
such-produced NiCCF composite material. CROATICA CHEMICA ACTA
CCACAA, ISSN 0011-1643, e-ISSN 1334-417X
Croat. Chem. Acta 85 (1) (2012) 1–8. http://dx.doi.org/10.5562/cca1743 Original Scientific Article RECEIVED AUGUST 9, 2010; ACCEPTED DECEMBER 17, 2010 Abstract. Nickel-coated carbon fibre (NiCCF) is an important composite material, which finds nu-
merous engineering applications, primarily within: computer, telecommunication, automotive and mili-
tary industries. In general, NiCCF can be produced by one of the three methods, namely: chemical va-
pour deposition (CVD), electroless, and electrochemical (galvanic) deposition of Ni on a carbon fibre
tow material. The present paper reports a study of the process of nickel electrodeposition (at ultrathin
layers of ≈ 0.3–0.5 μm) onto the surface of 12K (12 000 single filaments) carbon fibre (CF) tow,
carried-out in a continuous way. In addition, the effect of selected pre-treatments applied to carbon
fibre, as well as that of operational parameters of the process, on the quality of the NiCCF product
were investigated. (doi: 10.5562/cca1743) Keywords: nickel-coated carbon fibre, NiCCF, nickel electrodeposition, CF pre-treatments Electrodeposition of Nickel onto 12K Carbon Fibre Tow
in a Continuous Manner Boguslaw Pierozynski Department of Chemistry, Faculty of Environmental Management and Agriculture,
University of Warmia and Mazury in Olsztyn, Plac Lodzki 4, 10-957 Olsztyn, Poland
(E-mail: bogpierozynski@yahoo.ca) EXPERIMENTAL Figure 2. A PVC-made carbon fibre electroplating unit, where
the following components are: spring-loaded, adjustable pinch
type cathodic CR assembly (1); electrolyte sprinkler (2); elec-
trolyte overflow compartment (3); electrolyte circulation (in)
(4); electrolyte circulation (out) (5); DSA anode assembly (6);
adjustable wall (7): adjustable, wrap type cathodic CR assem-
bly, mounted on electroplater’s top cover (8); electrolyte drain
(9); CF tow’s passage (10); distance between an initial cathod-
ic contact point and entrance of the nucleation plating com-
partment (d). d A laboratory system for electrodeposition of Ni on a
single tow of 12K carbon fibre material (see Figure 1)
consists of the following elements: d d
fibre pay-out assembly,
PVC-made fibre electroplating unit (including two
dc 10 A / 20 V power supplies, with current resolu-
tion of 10 mA),
fibre rinse station (run on distilled H2O),
fibre drying station (hair dryer-based chamber) and Figure 2. A PVC-made carbon fibre electroplating unit, where
the following components are: spring-loaded, adjustable pinch
type cathodic CR assembly (1); electrolyte sprinkler (2); elec-
trolyte overflow compartment (3); electrolyte circulation (in)
(4); electrolyte circulation (out) (5); DSA anode assembly (6);
adjustable wall (7): adjustable, wrap type cathodic CR assem-
bly, mounted on electroplater’s top cover (8); electrolyte drain
(9); CF tow’s passage (10); distance between an initial cathod-
ic contact point and entrance of the nucleation plating com-
partment (d).
motorized spool take-up assembly. The PVC electroplating unit (see Figure 2) was
built for the purpose of this work. The plating unit al-
lowed to study Ni electrodeposition process in single
and double-cell configurations, with adjustable effective
cell lengths of 2.5, 5 and 8 cm (first cell), and 5 and 10
cm for the second cell. The electroplater was designed
to operate at current-densities ranging from 5 to 50 mA
cm–2, resulting voltages ≈ 2–10 V (for the first cell) and
the plating line speed between 10 and 100 cm min–1. Two DSA (Nb clad Pt) anodes per cell (20×20 and
45×20 mm strip type, with adjustable separation gap of
10 to 20 mm) were used. Two sets of highly-polished
(20 mm in diam.), stainless-steel (SS) and/or Cu cathod-
ic contact rollers (pinch or wrap configuration, see Fig-
ure 3) per each deposition cell were employed, with
flexible adjustment of the distance d between the ca-
thodic contact points and an entrance (or exit) of the
plating chamber. INTRODUCTION Some of the
most important parameters that have been addressed in
this work are the following:
quality and initial condition of carbon fibre precursor,
fibre passage system: fibre unwinding, cell passage
and take-up assemblies, 2 B. Pierozynski, Electrodeposition of Nickel onto 12K Carbon Fibre Tow
fibre spreading (optimization of the CF tow
spreading within an electroplater),
fibre spreading (optimization of the CF tow
spreading within an electroplater),
electrochemical and other parameters, and cell
design (current-density and voltage, design of fibre
contact and non-contact roller assemblies, internal
cathode-to-anode distances, bath composition, op-
erational pH and temperature parameters, electro-
lyte circulation, etc.). Figure 1. Schematic diagram of a laboratory-size carbon fibre
electroplating line, where the following elements are: fibre
pay-out and spreading assembly (1); electroplating cell assem-
bly (2); fibre rinse station (3); fibre drying station (hair dryer-
based) (4); and fibre take-up assembly (5). EXPERIMENTAL In addition, the electroplater was
equipped with four sets of electrolyte sprinklers, cou-
pled with an original design of electrolyte overflow
compartments, aimed both at minimizing the d parame-
ter, as well as the effect of electrolyte splashing outside
the plating cells. The fibre pay-out assembly consisted
of a regular CF 12K spool (Hexcel 12K AS4 product),
several 10 cm in diam. PTFE-made fibre lead rollers
and three, 5 cm in diam. Cu (or SS)-made fibre spread-
ing contact rollers. The tension for the CF tow was
arbitrarily set through adjustment of the clutch on the
pay-out spool assembly. Similar design was employed
on the motorized, cross-winding take-up end of the fibre
plating system. Figure 3. Cathodic contact rollers, shown in pinch (a) and
wrap (b) configurations. Figure 3. Cathodic contact rollers, shown in pinch (a) and
wrap (b) configurations. A typical Watts20,21 nickel plating bath was select-
ed for the purpose of this work. Base electrolyte con-
tained 300 g L–1 NiSO4 · 6H2O, 50 g L–1 NiCl2 · 6H2O
and 40 g L–1 H3BO3. In addition, two bath additives
(wetting agent: Na lauryl sulphate and Ni grain refiner:
Na saccharine, in the amount of 0.1–0.5 g L–1 and 0.5–1.0
g L–1, correspondingly) were also tested. Plating tempera-
ture varied between 45 and 60 °C, and pH was kept be-
tween 3.0 and 5.0. As pH of the plating bath continued to
decline upon Ni electrodeposition, this parameter was
constantly monitored during the process and readjusted Croat. Chem. Acta 85 (2012) 1. B. Pierozynski, Electrodeposition of Nickel onto 12K Carbon Fibre Tow 3 verified through tow side viewing, as well as by cross-
section examination of the composites, at magnifica-
tions between 25 and 3 000. For the cross-sectional
examination, tow samples were secured in a holder by
epoxy resin. Then, they were ground flat on fine-grade
silicon carbide abrasive paper of 2 500 grade
(Metalogis, Poland). The above procedure was followed
by polishing the surface, using 3 and 1 micron polycrys-
talline diamond suspensions (also from Metalogis). Finally, powder XRD (x-ray diffraction) technique was
employed to determine average sizes of crystallite do-
mains27 for chosen NiCCF specimens. (when necessary) through addition of small quantities of
NaOH solution (w = 10 %). Based on the deposited mass
of Ni, the electrolyte was also replenished (at regular
intervals) with nickel and chloride ions. EXPERIMENTAL )
Hexcel 12K AS4 carbon fibre22 is polyacrylonitrile
(PAN) based product that is delivered to the market as a
continuous 12K ribbon, in ≈ 3.6 kg spools. The tow is
sized by the manufacturer with an epoxy resin (0.21 % on
average), in order to ease fibre handling and thus to re-
duce damage to the material. This organic sizing was
removed from the fibre (prior to Ni deposition) by heat-
treatment, carried-out in a muffle (tube) furnace at low
oxygen atmosphere (with fibre residence time of 4 hours
at 350 °C). Another, alternative CF pre-treatment in-
volved subjection of "as received" Hexcel CF tow to
desizing in acetone, which was followed by electrochem-
ical oxidation in 0.5 mol dm–3 H2SO4 (performed at an
anodic current-density, ja = 0.5 mA cm–2 for 5 min, at
room temperature with a Ti/Pt auxiliary electrode). The
latter (anodic oxidation) treatment leads to functionaliza-
tion23–26 of the CF surface, which process could potential-
ly have a strong impact on both mechanical and electro-
chemical properties of such-treated carbon fibre tow. In addition, a simple, SEM-supported knot test
was used to qualitatively evaluate (and compare) the
strength of interfacial adhesion of Ni (potentially, also
its ductility) to the surface of CF filaments. The knot
test28,29 is a mechanical bending experiment, in which a
piece of NiCCF tow is bent sharply enough (a constant
load of 500 g for 5 s was applied to tie the NiCCF knots
in this work) to break the metal coating on the tension
side of the bend. Then, the knots are qualitatively exam-
ined and compared by the SEM method. Examination of the NiCCF product started with
randomly carried-out selection of NiCCF samples (20
cm long, collected every 10 m of the product). Firstly,
these fibre specimens were (again) carefully washed in
distilled water. Then, they were placed in an excicator
for 48 hours, followed by weighing on a precision bal-
ance (Sartorius CP224-OCE) to 0.1 mg. The above
procedure allowed precise evaluation of characteristic
nickel loading levels by means of a typical "weighing
method". In addition, resistance measurements were
performed on selected samples of the 12K NiCCF tow,
by measuring the voltage drop at constant applied cur-
rent (100 mA), over a distance of 20 cm. Both electrical
contact points on the NiCCF samples were silver-
painted in order to reduce the electrical contact re-
sistance. RESULTS AND DISCUSSION Table 1 presents an optimized configuration of the car-
bon fibre electroplating system, which includes electro-
chemical and all operational parameters that happened
to play a key role in the plating process. EXPERIMENTAL Fibre resistance was then calculated from
equation 1: Croat. Chem. Acta 85 (2012) 1. Operating Parameters and the Plating Cells'
Configuration Tow position Vertical
Electrolyte
Watts (SO4
2– + Cl–) based
Bath temperature/°C
48–50
Bath pH/adjustment
4.0–5.0 (10 % NaOH solution)
Bath additives
Sodium lauryl sulphate: 0.2 g L–1
Sodium saccharine: 0.5 g L–1
12K50 NiCCF product
wNi ≈ 50 %; Ni coating ≈ 0.4 µm;
Ni grain size ≈ 25 nm (PXRD-
calculated) Table 1. An optimized configuration for a laboratory-size
carbon fibre electroplating system, employed during deposi-
tion of nickel onto 12K carbon fibre tow
Carbon Fibre Electroplating Cell Assembly
Plating cells
2 cells; 5 cm long each
Contact rollers (CR)
Highly-polished SS, 20 mm diam. Anodes (A)
4.5 × 2.0 cm DSA strips
(two A per each cell)
Distances
First cell: 1st CR-A: 2 cm, pinch
contact; 2nd CR-A: 3.5 cm, wrap
contact
Second cell: 2nd CR-A: 2 cm, pinch
contact
Both cells: CF-A: 0.5 cm
Voltage / V
First cell: 7.0–10.5
Second cell: 3.0–5.5
Current / A
(current ranges de-
pendent on the set Ni
loading level)
First cell: 4.5–6.0
Second cell: 1.0–3.0
Line speed / m min–1
0.5
Tow spread / cm
0.8
Spreading method
Mechanical; Cu rollers, 50 mm diam. Carbon fibre
Hexcel 12K AS4 tow
CF pre-treatment
Heat-treated at 350 °C for 4 hours. Tow position Vertical
Electrolyte
Watts (SO4
2– + Cl–) based
Bath temperature/°C
48–50
Bath pH/adjustment
4.0–5.0 (10 % NaOH solution)
Bath additives
Sodium lauryl sulphate: 0.2 g L–1
Sodium saccharine: 0.5 g L–1
12K50 NiCCF product
wNi ≈ 50 %; Ni coating ≈ 0.4 µm;
Ni grain size ≈ 25 nm (PXRD-
calculated) With respect to configuration of the plating system,
superior quality NiCCF (see later Figure 9a) was pro-
duced with the two-cell (5 cm each) plating arrangement,
equipped with three (or four) sets of highly-polished, SS-
made contact rollers (CR). The foremost, spring-loaded
pinch CR set, was positioned just over 2 cm (distance d in
Figure 2) away from the DSA anode of the first cell. The
above was only possible due to the unique design of the
electrolyte overflow compartment, which allowed to
minimize an effective, cathode-to-anode voltage (and
thus prevented fibre from severe overheating and burn-
ing) of the first cell. Then, two (or a single) wrap-type
fibre contact roller assemblies were installed, both were
mounted on the plater's cover (see also Figure 2 for more
details). Another pinchtype CR assembly was fastened at
the exit of the second plating cell. Operating Parameters and the Plating Cells'
Configuration Thus, pH control of the electrolyte was found to be a
crucial element of the plating process. As acidity of the
bath kept continuously rising during the course of metal
deposition, frequent adjustment of pH was necessary,
where optimum pH range was found to be around 4.5–
5.0 (for most favorable plating temperature range of 48–
50 °C). For more acidic solution, hydrogen evolution
reaction (HER) becomes more competitive with reduc-
tion of Ni2+ ions at low overvoltages (see the two possi-
ble cathodic reactions in equations 2 and 3, and the
corresponding anodic reaction in equation 4). As a con-
sequence, the product (especially at pH around 3.0)
appears to have much less homogeneous distribution of
nickel throughout the tow than that produced at higher pH
values (see Figures 4a and 4b, correspondingly). At pH =
3.0, it is the outer tow sections which primarily become
metal-coated (Figure 4a), in relation to the fact that diffu-
sion of evolved (and likely surface-covering) hydrogen
bubbles from the centre part of the CF tow is much more
difficult than the corresponding process, but proceeding
at the outer tow parts. V
R
I l
(1) (1) where: R / Ω cm–1 is calculated resistance of the NiCCF
tow sample, V / mV is measured voltage, V / mV is measured voltage, V / mV is measured voltage, I / mA is applied current, l / cm is sample's length. Subsequently, selected NiCCF samples were sent
for SEM analysis, where quality of nickel deposits was Croat. Chem. Acta 85 (2012) 1. 4 B. Pierozynski, Electrodeposition of Nickel onto 12K Carbon Fibre Tow Ni2+(aq) + 2e–
Ni0(s)
(2)
2H+(aq) + 2e–
H2(g)
(3)
2Cl–(aq) + 2e–
Cl2(g)
(4)
(insoluble DSA anodes) Ni2+(aq) + 2e–
Ni0(s)
(2)
2H+(aq) + 2e–
H2(g)
(3)
2Cl–(aq) + 2e–
Cl2(g)
(4)
(insoluble DSA anodes)
With respect to configuration of the plating system,
superior quality NiCCF (see later Figure 9a) was pro-
duced with the two-cell (5 cm each) plating arrangement,
equipped with three (or four) sets of highly-polished, SS-
made contact rollers (CR). The foremost, spring-loaded
pinch CR set, was positioned just over 2 cm (distance d in
Figure 2) away from the DSA anode of the first cell. Operating Parameters and the Plating Cells'
Configuration The
above was only possible due to the unique design of the
electrolyte overflow compartment, which allowed to
minimize an effective, cathode-to-anode voltage (and
thus prevented fibre from severe overheating and burn-
ing) of the first cell. Then, two (or a single) wrap-type
fibre contact roller assemblies were installed, both were
mounted on the plater's cover (see also Figure 2 for more
details). Another pinchtype CR assembly was fastened at
the exit of the second plating cell. The carbon fibre tow
was vertically positioned within the plater, giving on
optimum cathode-to-anode distance of ≈ 0.5 cm in both
cells. The above configuration of the electroplater al-
lowed to maintain (for the CF line speed of 50 cm min–1)
voltage ranges between 7.0–10.5 V within the first (nu-
cleation) chamber and ≈ 3.0–5.5 V within the second
nickel deposition cell. The resulting current-densities
came to 32.0–42.4 mA cm–2 (for the nucleation cell) and
7.1–21.3 mA cm–2 for the second plating cell (current
ranges varied along with the set loading level of Ni). In
addition, it should be stressed here that employment of
much lower current densities (on the order of 20 mA cm–2)
for the primary deposition cell resulted in inferior Ni
deposit homogeneity. Table 1. An optimized configuration for a laboratory-size
carbon fibre electroplating system, employed during deposi-
tion of nickel onto 12K carbon fibre tow
Carbon Fibre Electroplating Cell Assembly
Plating cells
2 cells; 5 cm long each
Contact rollers (CR)
Highly-polished SS, 20 mm diam. Anodes (A)
4.5 × 2.0 cm DSA strips
(two A per each cell)
Distances
First cell: 1st CR-A: 2 cm, pinch
contact; 2nd CR-A: 3.5 cm, wrap
contact
Second cell: 2nd CR-A: 2 cm, pinch
contact
Both cells: CF-A: 0.5 cm
Voltage / V
First cell: 7.0–10.5
Second cell: 3.0–5.5
Current / A
(current ranges de-
pendent on the set Ni
loading level)
First cell: 4.5–6.0
Second cell: 1.0–3.0
Line speed / m min–1
0.5
Tow spread / cm
0.8
Spreading method
Mechanical; Cu rollers, 50 mm diam. Carbon fibre
Hexcel 12K AS4 tow
CF pre-treatment
Heat-treated at 350 °C for 4 hours. Operating Parameters and the Plating Cells'
Configuration (a) (a) In this work, experimentally optimized CF tow
spreading (investigated for ribbon widths between 0.2
and 1.2 cm) came to about 0.8 cm (see the effect of the
CF tow spreading on the quality of the NiCCF product
in Figures 5a and 5b), which for the springloaded pinch
CR assembly (two cathode rollers) means the presence
of ≈ 11 carbon fibre layers between the two cathode
rollers (see Figure 6). Thus, for the tow's spread of only
0.2 cm, most of the CF filaments were not in direct
contact with the contact roller(s) and as such they did
experience dramatically reduced current-densities, as
compared to those which were concentrated at the out-
side of the CF tow. As a result, the tow was primarily
plated with nickel at its external regions (see Figure 5a
again and contrast this behaviour with that observed in
Figure 5b). The above behaviour is in principal similar
(although for different reasons) to that observed in Fig-
ure 4a for the plating carried-out at pH = 3.0. On the
other hand, for the tow's spread over 0.8 cm, a consider-
able increase of fibre nesting within the electroplating
unit was observed over time of metal deposition. The
above resulted in gradual deterioration of mechanical
properties, as well as the plating quality for the NiCCF
composite. (b) (b) Figure 5. The effect of CF tow’s spread on the quality of la-
boratory-electrodeposited NiCCF tow samples, shown in SEM
cross-sectional micrograph pictures for the tow’s spread of 0.2
cm at wNi ≈ 36 % (a) and 0.8 cm at wNi ≈ 40 % (b), taken at a
given magnification. On the other hand, the quality of electrodeposited
NiCCF significantly depended on the extent of CF tow
spreading within the electroplater. As the fibre tow is
spread, it forms a ribbon comprised of layers of individ-
ual carbon filaments. Then, as the ribbon moves over
the contact rollers, each individual carbon fibre changes
position and its distance from the cathode roller, leading
to variation of contact resistance. For the 12K fibre tow
and ideal spreading, each carbon filament should be in
intimate contact with the cathode roller. Thus, assuming
the 12K tow of 7 µm in diam. Operating Parameters and the Plating Cells'
Configuration The carbon fibre tow
was vertically positioned within the plater, giving on
optimum cathode-to-anode distance of ≈ 0.5 cm in both
cells. The above configuration of the electroplater al-
lowed to maintain (for the CF line speed of 50 cm min–1)
voltage ranges between 7.0–10.5 V within the first (nu-
cleation) chamber and ≈ 3.0–5.5 V within the second
nickel deposition cell. The resulting current-densities
came to 32.0–42.4 mA cm–2 (for the nucleation cell) and
7.1–21.3 mA cm–2 for the second plating cell (current
ranges varied along with the set loading level of Ni). In
addition, it should be stressed here that employment of
much lower current densities (on the order of 20 mA cm–2)
for the primary deposition cell resulted in inferior Ni
deposit homogeneity. Figure 4. The effect of pH on the quality of laboratory-electrodeposited NiCCF tow samples (at wNi ≈ 30 %), shown in SEM
cross-sectional micrograph pictures for pH = 3.0 (a) and pH = 5.0 (b), taken at a given magnification. (a) (b) (a) (b) (b) (a) Figure 4. The effect of pH on the quality of laboratory-electrodeposited NiCCF tow samples (at wNi ≈ 30 %), shown in SEM
cross-sectional micrograph pictures for pH = 3.0 (a) and pH = 5.0 (b), taken at a given magnification. Croat. Chem. Acta 85 (2012) 1. 5 B. Pierozynski, Electrodeposition of Nickel onto 12K Carbon Fibre Tow Figure 5. The effect of CF tow’s spread on the quality of la-
boratory-electrodeposited NiCCF tow samples, shown in SEM
cross-sectional micrograph pictures for the tow’s spread of 0.2
cm at wNi ≈ 36 % (a) and 0.8 cm at wNi ≈ 40 % (b), taken at a
given magnification. (a)
(b) minimize the electrical contact resistance between the
fibres, in order to provide more uniform current distri-
bution within the plating CF tow. However, an idea
behind a practical configuration of the CF tow spreading
within the electroplater is always to compromise be-
tween the contact resistance parameter and the extent of
fibre damage (the latter effect being directly proportion-
al to the width of the fibre ribbon, in relation to the fibre
tension parameter). Significant increase of fibre tension
leads to extensive formation of fibre nests inside the
plating cells and as a consequence to further increase of
fibre tension, and its damage. Operating Parameters and the Plating Cells'
Configuration fibre (and a single cath-
ode contact roller), for perfect tow spreading (each
filament is in contact with the CR) the tow spreading
should provide about 8.4 cm (12 000 × 7 × 10–4 cm)
wide ribbon. In addition, it should be mentioned that
any fibre that is not in direct contact with the cathode
roller will have to overcome the radial resistivity of the
fibre (or fibres) that shield it from the roller. These non-
contact fibres will exhibit significantly reduced current-
densities upon plating, as compared to those being in
direct contact with the cathode roller(s). Therefore, the
cathode roller configuration should primarily attempt to Croat. Chem. Acta 85 (2012) 1. CF Pre-treatments and Bath Additives An optimized, pinch-type cathodic CR assembly,
with residing 11 layers of carbon fibre filaments. Figure 7. SEM micrograph pictures of knot-tied NiCCF sam-
ples (at wNi ≈ 40 %) for NiCCF obtained with surface
electrooxidized CF (a) and heat-treated CF tow (b), taken at
300 magnification. (b) (b) Figure 6. An optimized, pinch-type cathodic CR assembly,
with residing 11 layers of carbon fibre filaments. as compared to that produced with simply heat-treated
CF tow (see Figure 7b). These effects are likely to re-
flect improved adhesion between the Ni coating and the
CF substrate, in relation to surface functionalization and
roughening of electrooxidized CF tow filaments. It has
to be stated here that surface-electrooxidized carbon
fibre (see an earlier work from this laboratory in Ref. 30) exhibited significant increase of the electrochemi-
cally accessible surface area. Based on the calculated
double-layer capacitance (Cdl) parameter in Ref. 30, the
surface area of H2SO4-electrooxidized Hexcel 12K CF
tow was ≈ 6.6 times greater than that of the heat-
pretreated carbon fibre tow. as compared to that produced with simply heat-treated
CF tow (see Figure 7b). These effects are likely to re-
flect improved adhesion between the Ni coating and the
CF substrate, in relation to surface functionalization and
roughening of electrooxidized CF tow filaments. It has
to be stated here that surface-electrooxidized carbon
fibre (see an earlier work from this laboratory in Ref. 30) exhibited significant increase of the electrochemi-
cally accessible surface area. Based on the calculated
double-layer capacitance (Cdl) parameter in Ref. 30, the
surface area of H2SO4-electrooxidized Hexcel 12K CF
tow was ≈ 6.6 times greater than that of the heat-
pretreated carbon fibre tow. Figure 7. SEM micrograph pictures of knot-tied NiCCF sam-
ples (at wNi ≈ 40 %) for NiCCF obtained with surface
electrooxidized CF (a) and heat-treated CF tow (b), taken at
300 magnification. Figure 8. A resistance vs. nickel loading plot for laboratory-
electrodeposited (and optimized) NiCCF tow samples. Nevertheless, it has to be stressed that the knot test
is itself somewhat ambiguous in character, as tension
and shape of the knot may vary from one sample to
another. Thus, the knot method enables only qualitative
evaluations and comparisons, and its results have to be
discussed with caution. CF Pre-treatments and Bath Additives Interestingly, neither selected bath additives nor an
electrooxidation pre-treatment applied to the CF tow
(see Experimental part above for details) had any signif-
icant effect on the quality of such-produced NiCCF
composite (including Ni coverage on the CF tow). However, it is important to maintain a relatively low
level of sodium lauryl sulphate (e.g. at 0.1–0.2 g L–1) in
the plating bath. Excessive amounts of the wetting agent
tend to induce severe bath foaming (accelerated by the
presence of hydrogen bubbles), which leads to
nonuniform Ni electrodeposits. On the other hand, SEM-supported knot tests
showed some improvement in the composite mechanical
performance for the NiCCF produced with surface-
electrooxidized 12K carbon fibre tow. Here (see Figure
7a), somewhat less extensive delamination and Ni frac-
turing effects are observed for a knot-tied tow sample, Croat. Chem. Acta 85 (2012) 1. 6 B. Pierozynski, Electrodeposition of Nickel onto 12K Carbon Fibre Tow Figure 7. SEM micrograph pictures of knot-tied NiCCF sam-
ples (at wNi ≈ 40 %) for NiCCF obtained with surface
electrooxidized CF (a) and heat-treated CF tow (b), taken at
300 magnification. (a)
(b) as compared to that produced with simply heat-treated
CF tow (see Figure 7b). These effects are likely to re-
flect improved adhesion between the Ni coating and the
CF substrate, in relation to surface functionalization and
roughening of electrooxidized CF tow filaments. It has
to be stated here that surface-electrooxidized carbon
fibre (see an earlier work from this laboratory in Ref. 30) exhibited significant increase of the electrochemi-
cally accessible surface area. Based on the calculated
double-layer capacitance (Cdl) parameter in Ref. 30, the
surface area of H2SO4-electrooxidized Hexcel 12K CF
tow was ≈ 6.6 times greater than that of the heat-
pretreated carbon fibre tow. Nevertheless, it has to be stressed that the knot test
is itself somewhat ambiguous in character, as tension
and shape of the knot may vary from one sample to
Figure 7. SEM micrograph pictures of knot-tied NiCCF sam-
ples (at wNi ≈ 40 %) for NiCCF obtained with surface
electrooxidized CF (a) and heat-treated CF tow (b), taken at
300 magnification. Figure 6. An optimized, pinch-type cathodic CR assembly,
with residing 11 layers of carbon fibre filaments. (a)
(b) Figure 6. An optimized, pinch-type cathodic CR assembly,
with residing 11 layers of carbon fibre filaments. (a) Figure 6. CONCLUSION Fairly uniform, ultra-thin deposits of Ni on 12 000-
filament CF tow can suitably be produced through a
continuous metal electrodeposition process. However,
technological aspects of this process are quite complex,
in addition to the required advanced knowledge of fibre
handling. The most important conclusions that can be
drawn from this work are the following:
in-line resistance measurement could be utilized
for direct monitoring of electrodeposited lev-
el/thickness of Ni on the CF tow
in-line resistance measurement could be utilized
for direct monitoring of electrodeposited lev-
el/thickness of Ni on the CF tow
applied bath additives and basic CF tow pre-
treatments had rather insignificant effect on the
quality (homogeneity) of such-produced NiCCF
composites; however surface electrooxidation
treatment applied to CF led to somewhat improved
(qualitatively) adhesion between Ni coating and
the carbon fibre surface. The thickness of the Ni coating was estimated by
measurements conducted on high magnification, cross-
sectional SEM images. Thus, 100 sample filaments
were representatively selected in 10 different areas of
the tow (10 × 10 samples). Such estimated thickness of
the Ni coating for wNi ≈ 50 % composite came to 0.39 ±
0.02 μm. Acknowledgements. Grateful acknowledgement is made by the
author of this work to the Commission of the European Com-
munities for financial support of the research described in this
paper. This work was financed by the European Union, Marie
Curie International Reintegration Grant, acronym "Fibres",
contract No.: 031073. In-line Monitoring of Ni Loading Levels Figure 8 shows the influence of Ni loading on the
NiCCF's electrical resistance parameter. The fibre re-
sistance parameter was derived (see again the Exper-
imental part above for details) in reference to the Ni
loading levels, estimated by means of the weighing
method. The resistance measurements were performed
for selected run tests, all carried-out with optimized
plating setup and bath parameters. It is strongly believed
that the abovediscussed correlation could be found suit-
able for continuous, in-line monitoring of the nickel
loading level. Thus, any unwanted change in the re- Figure 8. A resistance vs. nickel loading plot for laboratory-
electrodeposited (and optimized) NiCCF tow samples. sistance parameter (caused by variation in the mass
fraction, w/% of the metal deposit) could immediately
be compensated through an appropriate re-adjustment of
preset electrical parameters, or the plating line speed. Croat. Chem. Acta 85 (2012) 1. 7 B. Pierozynski, Electrodeposition of Nickel onto 12K Carbon Fibre Tow Figure 9. The effect of Ni loading on the quality of laborato-
ry-electrodeposited NiCCF tow samples, shown in SEM mi-
crograph pictures for the Ni loadings of wNi ≈ 40 % (a) and
wNi ≈ 10 % (b), taken at 500 and 3000 magnifications. (a)
(b)
(a)
(b) the grain growth phase takes place in the following
plating chamber (both cells together constitute a
CF tow electroplating unit) (a) (a) (a) (a) superior/homogeneous Ni deposits require: superior/homogeneous Ni deposits require: superior/homogeneous Ni deposits require: (i) electrolyte pH within 4.0 to 5.0 (with proper
bath circulation and agitation) (i) electrolyte pH within 4.0 to 5.0 (with proper
bath circulation and agitation) (ii) electrolyte temperature about 50 °C (b) (b) (b) (b) (iii) CF tow's spread (optimized in this work at
0.8 cm for 12K CF tow) should be set to
compromise between the electrical contact
resistance parameter and the extent of actual
fibre damage within the plater (more advanced
laboratory designs could capitalize on the ap-
plication of a professional fibre tension con-
trolling device, e.g. Helman tensioner unit) Figure 9. The effect of Ni loading on the quality of laborato-
ry-electrodeposited NiCCF tow samples, shown in SEM mi-
crograph pictures for the Ni loadings of wNi ≈ 40 % (a) and
wNi ≈ 10 % (b), taken at 500 and 3000 magnifications. (iv) minimized distances between cathodic CRs
and DSA anode assemblies (to reduce opera-
tional voltages and thus to limit fibre over-
heating) (v) electroplater's line speeds between 20 and
100 cm min–1 [with cathodic current-densities
of ≈ 30–40 and 10–20 mA cm–2 for the nu-
cleation and the grain growth cells, corre-
spondingly (for the line speed of 50 cm min–1)] However, only NiCCF composites with Ni depos-
its on the order of wNi = 30 % (or higher) exhibited
fairly continuous (but non-uniform) metal coverage of
individual carbon fibre filaments (see Figure 9a) within
the 12K CF tow. Conversely, all NiCCF samples with
Ni loadings below wNi ≈ 20 % were completely non-
homogeneous, with respect to the surface distribution of
the metal deposit (Figure 9b). The above-observed phe-
nomenon strongly supports the idea of severe incon-
sistency of the applied "electrode potential" (and as a
consequence also the Gibbs free energy of activation for
the process) within the complex, 12 000-filament CF
tow entity. REFERENCES 1. D. D. L. Chung, J. Mater. Sci. 39 (2004) 2645–2661. 2. D. Markham, Mater. Design 21 (2000) 45–50. 3. S. S. Tzeng and F. Y. Chang, Mater. Sci. Eng. A302 (2001)
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Part A 31 (2000) 1117–1125.
Ni deposition should be realized in two steps;
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(1994). Croat. Chem. Acta 85 (2012) 1. 8 B. Pierozynski, Electrodeposition of Nickel onto 12K Carbon Fibre Tow 8. J. K. Lee, K. W. An, J. B. Ju, B. W. Cho, W. I. Cho, D. Park, and
K. S. Yun, Carbon 39 (2001) 1299–1305. 21. G. Di Bari, Metal Finishing 100(1) (2002) 257–274. 22. HexTowTM
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Version of Record: A version of this preprint was published at Scienti¦c Reports on July 17th, 2023. See
the published version at https://doi.org/10.1038/s41598-023-38658-w. Conservation Laws, Solitary Wave Solutions, and Lie
Analysis for the Nonlinear Chains of Atoms
Muhammad Junaid U Rehman ( muhammad-junaid.u-rehman@dokt.p.lodz.pl )
Lodz University of Technology
Jan Awrejcewicz
Lodz University of Technology
Grzegorz Kudra
Lodz University of Technology
Article
Keywords: Nonlinear chains of atoms, Lie symmetry approach, Multiplier scheme, Conserved vectors,
New auxiliary method
Posted Date: June 13th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-3026335/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
Additional Declarations: No competing interests reported.
Version of Record: A version of this preprint was published at Scienti¦c Reports on July 17th, 2023. See
the published version at https://doi.org/10.1038/s41598-023-38658-w. Conservation Laws, Solitary Wave Solutions, and Lie
Analysis for the Nonlinear Chains of Atoms
Muhammad Junaid U Rehman ( muhammad-junaid.u-rehman@dokt.p.lodz.pl )
Lodz University of Technology
Jan Awrejcewicz
Lodz University of Technology
Grzegorz Kudra
Lodz University of Technology
Article
Keywords: Nonlinear chains of atoms, Lie symmetry approach, Multiplier scheme, Conserved vectors,
New auxiliary method
Posted Date: June 13th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-3026335/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Scienti¦c Reports on July 17th, 2023. See
the published version at https://doi.org/10.1038/s41598-023-38658-w. Article License: This work is licensed under a Creative Commons Attribution 4.0 International License. d
ll License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Scienti¦c Reports on July 17th, 2023. See
the published version at https://doi.org/10.1038/s41598-023-38658-w. Abstract Nonlinear chains of atoms(NCA) are complex systems with rich dynamics, influ-
encing various scientific disciplines. Lie symmetry approach is considered to analyze
the NCA. The Lie symmetry method is a powerful mathematical tool for analyz-
ing and solving differential equations with symmetries, facilitating the reduction of
complexity and obtaining solutions. After getting the entire vector field by using the
Lie scheme, we find the optimal system of symmetries. Using the optimal system
we have converted assumed PDE into nonlinear ODE. The new auxiliary scheme in-
troduces novel approaches to complement existing techniques, enhancing accuracy
and simplifying computations. Travelling wave solutions describe wave-like prop-
agation in systems, while graphical behavior visually represents relationships and
patterns in data or mathematical models. The multiplier method enables the iden-
tification of conservation laws, fundamental principles in physics that assert certain
quantities remain constant over time. Understanding these concepts contributes to
a deeper comprehension of nonlinear chains of atoms and their dynamics, fostering
advancements in related fields. Keywords: Nonlinear chains of atoms, Lie symmetry approach, Multiplier scheme,
Conserved vectors, New auxiliary method. 1Department of Automation, Biomechanics, and Mechatronics, Lodz University of
Technology, 1/15 Stefanowski st. (Building A22), Lodz, 90-924, Poland 1Department of Automation, Biomechanics, and Mechatronics, Lodz University o
Technology, 1/15 Stefanowski st. (Building A22), Lodz, 90-924, Poland E-mail address: ∗muhammad-junaid.u-rehman@dokt.p.lodz.pl (Corresponding author),
grzgorz.kudra@p.lodz.pl, jan.awrjcewicz@p.lodz.pl E-mail address: ∗muhammad-junaid.u-rehman@dokt.p.lodz.pl (Corresponding author),
grzgorz.kudra@p.lodz.pl, jan.awrjcewicz@p.lodz.pl Conservation Laws, Solitary Wave
Solutions, and Lie Analysis for the
Nonlinear Chains of Atoms June 11, 2023 June 11, 2023 Muhammad Junaid-U-Rehman1,∗, Grzegorz Kudra1, Jan Awrejcewicz1 1
Introduction The Lie symmetry analysis approach [1–8] has many applications in different fields, in-
cluding physics, engineering, and mathematical modeling. It can be used to study a wide
range of nonlinear PDEs, including those that are difficult to solve using other meth-
ods. Additionally, this approach provides a powerful tool for developing new theories
and models that improve our understanding of complex physical systems. Overall, the 1 Lie symmetry analysis approach is a valuable tool for studying nonlinear PDEs and has
many significant applications in different branches of science and engineering. The Lie symmetry analysis approach [12–22] is a powerful method used in the study
of nonlinear PDEs. It is based on the concept of Lie groups and Lie algebras, which
are mathematical structures that describe the symmetries of a system. The Lie symme-
try analysis approach involves transforming a given PDE into an equivalent system of
ODEs using a Lie group transformation. This transformation is constructed from a set
of symmetry generators that preserve the form of the original PDE. Once the PDE is
transformed into an equivalent system of ODEs, it is possible to use various analytical
and numerical methods to solve the system and obtain the solution to the original PDE. Additionally, the Lie symmetry analysis approach can be used to identify the conservation
laws that govern the physical behavior of the system under study. These conservation
laws provide important insights into the underlying physical mechanisms responsible for
the observed behavior of the system. Nonlinear PDEs [23–26] play a critical role in mechanical engineering by modeling
complex phenomena such as stress and deformation in materials, fluid flow, and heat
transfer. Unlike linear PDEs, which can be solved analytically in many cases, nonlinear
PDEs require numerical or approximate methods to solve due to their complex nature. The use of nonlinear PDEs is essential in the design and optimization of mechanical
systems such as turbines, engines, and aircraft. They also provide a framework for pre-
dicting the behavior of materials under different conditions, such as high temperatures,
high pressure, and rapid deformation. By incorporating nonlinear PDEs into mechanical
engineering models, engineers can improve the accuracy of their designs and ensure that
their systems are safe, reliable, and efficient. Nonlinear chains of atoms [27–33] have a wide range of applications in mechanical
engineering, particularly in the study of materials science and solid mechanics. 1
Introduction These
models provide valuable insights into the behavior of materials at the atomic level, en-
abling the design of high-performance materials for various applications. Applications
of nonlinear chains of atoms include the study of thermal conductivity in materials, in-
vestigating the deformation and fracture mechanisms of materials under various loading
conditions, and studying the dynamics of crystals, such as the propagation of waves and
the formation of defects. Nonlinear chains of atoms models are crucial in understanding
the behavior of materials under extreme conditions and can inform the design of materi-
als for high-performance applications, leading to the development of new materials with
enhanced mechanical and thermal properties. The new auxiliary method [10,11] is a recently proposed method for solving challenging
nonlinear PDEs. This method involves introducing an auxiliary variable and constructing
a system of coupled equations involving both the original variables and the auxiliary
variable. The resulting system of equations can be solved using numerical methods to
obtain the solution to the original PDE. The new auxiliary method can handle highly
nonlinear PDEs that are difficult to solve using other numerical methods, such as the finite
difference approach or the finite element scheme. Additionally, this method can be used
to obtain exact solutions to certain types of nonlinear PDEs, reducing the computational
cost required to solve some types of nonlinear PDEs. Overall, the new auxiliary method
is a promising tool for solving challenging nonlinear PDEs in various fields, including
physics, engineering, and mathematical modeling. Conservation laws of nonlinear PDEs [36–39] are essential concepts that relate to the
principle of conservation of physical quantities like mass, energy, and momentum. These 2 laws are expressed in terms of PDEs and have crucial importance in various fields, in-
cluding engineering, physics, and mathematical modeling. They provide a mathematical
framework to predict the behavior of complex physical systems accurately and develop
new theories and models to improve our understanding of the underlying physical mech-
anisms. Furthermore, conservation laws play a vital role in the design and analysis of
physical systems and the development of numerical methods for solving challenging non-
linear PDEs, making them fundamental concepts in the study of nonlinear PDEs. 2
Formation of model The Hamiltonian of the system is Foroutan et al. [34], The Hamiltonian of the system is Foroutan et al. [34], H =
X
n
( X
l̸=n
V(|Un −Ul|) + 1
2mU .2
n
)
,
(1) (1) where m is the mass of the atom, V(|Un −Ul|) stands for nonlinear potential and dot
indicates for derivative w.r.t time. We consider l = 1 ± n and the subsequent potential: where m is the mass of the atom, V(|Un −Ul|) stands for nonlinear potential and dot
indicates for derivative w.r.t time. We consider l = 1 ± n and the subsequent potential: V(hnl) = 1
4βih4
nl + 1
3αih3
nl + 1
2γih2
nl,
(2) (2) where hnl is relative displacement among l −th atom and n −th. The index i shows
the distinct interactions via the particles. We omit our focus on the first and second
neighbors. From Eqs. (1) and (2) through Hamiltonian equations which are where hnl is relative displacement among l −th atom and n −th. The index i shows
the distinct interactions via the particles. We omit our focus on the first and second
neighbors. From Eqs. (1) and (2) through Hamiltonian equations which are ∂H
∂Un
= −P . n,
∂H
∂P . n
= ∂Un
∂τ = U . n, ∂H
∂Un
= −P . n,
∂H
∂P . n
= ∂Un
∂τ = U . n, which gives us the equation of motion; d2Un
dτ 2 =γ1
Un+1 −2Un + Un−1
+ γ2
Un+2 −2Un + Un−2
+ α1
Un+1 −Un
2 −
Un −Un−1
2
+ α2
Un+2 −Un
2 −
Un −Un−2
2
+ β1
Un+1 −Un
2 −
Un −Un−1
2
+ β2
Un+2 −Un
2 −
Un −Un−2
2
. (3) (3) In Hamiltonian’s equations, P . n stands for generalized momentum. Assuming that the
δ(inter-atom spacing) is small enough so that the continuum limit is reached, we substi-
tute δn →χ. Then Un±1 = U ± δUχ + 1
2δ2Uχχ ± 1
6δ3Uχχχ + 1
24δ4Uχχχχ + . . . (4)
Un±2 = U ± 2δUχ + 4
2δ2Uχχ ± 8
6δ3Uχχχ + 16
24δ4Uχχχχ + . . . ,
(5) Un±1 = U ± δUχ + 1
2δ2Uχχ ± 1
6δ3Uχχχ + 1
24δ4Uχχχχ + . . . 2
Formation of model (4) (4) and and Un±2 = U ± 2δUχ + 4
2δ2Uχχ ± 8
6δ3Uχχχ + 16
24δ4Uχχχχ + . . . ,
(5) (5) 3 hence, Eq.(3) can be supposed as Foroutan et al. [35] hence, Eq.(3) can be supposed as Foroutan et al. [35] ∂2U
∂τ 2 = δ2
o
∂2U
∂χ2 + po
∂U
∂χ
∂2U
∂χ2 + qo
∂U
∂χ
2∂2U
∂χ2 + r∂4U
∂χ4 ,
(6) (6) with the subsequent constants; δ2
o = δ2
m
γ1 + 4γ2
, po = 2δ3
m
γ1 + 8α2
, qo = 3δ4
m
β1 + 16β2
, r =
δ4
12m
γ1 + 16γ2
. (7) (7) Here in this paper, we will find out ...... for nonlinear Eq.(6) with the use of appropriate
transformation method. 3.1
New auxiliary approach (14)
Case 2: When B1
2 −B2B3 > 0 and B3 ̸= 0
Fq(ϱ) = −B1
B3
+
p
(B1
2 −B2B3)
B3
tanh
p
(B1
2 −B2B3)
2
ϱ
,
(15)
Fq(ϱ) = −B1
B3
−
p
(B1
2 −B2B3)
B3
coth
p
(B1
2 −B2B3)
2
ϱ
. (16)
Case 3: When B1
2 + B2B3 > 0 and B3 ̸= 0 and B3 ̸= −B2
Fq(ϱ) = B1
B3
+
p
(B1
2 + B2
2)
B3
tanh
p
(B1
2 + B2
2)
2
ϱ
,
(17)
Fq(ϱ) = B1
B3
+
p
(B1
2 + B2
2)
B3
coth
p
(B1
2 + B2
2)
2
ϱ
. (18)
Case 4: When B1
2 + B2B3 < 0, B3 ̸= 0 and B3 ̸= −B2
Fq(ϱ) = B1
B3
+
p
−(B1
2 + B2
2)
B3
tan
p
−(B1
2 + B2
2)
2
ϱ
,
(19)
Fq(ϱ) = B1
B3
+
p
−(B1
2 + B2
2)
B3
cot
p
−(B1
2 + B2
2)
2
ϱ
. (20)
Case 5: When B1
2 −B2
2 < 0 and B3 ̸= −B2
Fq(ϱ) = −B1
B3
+
p
−(B1
2 −B2
2)
B3
tan
p
−(B1
2 −B2
2)
2
ϱ
,
(21)
Fq(ϱ) = −B1
B3
+
p
−(B1
2 −B2
2)
B3
cot
p
−(B1
2 −B2
2)
2
ϱ
. (22)
Case 6: When B1
2 −B2
2 > 0 and B3 ̸= −B2
Fq(ϱ) = −B1
B3
+
p
(B1
2 −B2
2)
B3
tanh
p
(B1
2 −B2
2)
2
ϱ
,
(23)
Fq(ϱ) = −B1
B3
+
p
(B1
2 −B2
2)
B3
coth
p
(B1
2 −B2
2)
2
ϱ
. 3.1
New auxiliary approach (16) (15) (16) Fq(ϱ) = B1
B3
+
p
(B1
2 + B2
2)
B3
tanh
p
(B1
2 + B2
2)
2
ϱ
,
(17)
Fq(ϱ) = B1
B3
+
p
(B1
2 + B2
2)
B3
coth
p
(B1
2 + B2
2)
2
ϱ
. (18)
2 (17) (18) B3
B3
2
(
)
Case 4: When B1
2 + B2B3 < 0, B3 ̸= 0 and B3 ̸= −B2
Fq(ϱ) = B1
B3
+
p
−(B1
2 + B2
2)
B3
tan
p
−(B1
2 + B2
2)
2
ϱ
,
(19)
Fq(ϱ) = B1
B3
+
p
−(B1
2 + B2
2)
B3
cot
p
−(B1
2 + B2
2)
2
ϱ
. (20)
Case 5: When B1
2 −B2
2 < 0 and B3 ̸= −B2
Fq(ϱ) = −B1
B3
+
p
−(B1
2 −B2
2)
B3
tan
p
−(B1
2 −B2
2)
2
ϱ
,
(21)
Fq(ϱ) = −B1
B3
+
p
−(B1
2 −B2
2)
B3
cot
p
−(B1
2 −B2
2)
2
ϱ
. (22)
Case 6: When B1
2 −B2
2 > 0 and B3 ̸= −B2
Fq(ϱ) = −B1
B3
+
p
(B1
2 −B2
2)
B3
tanh
p
(B1
2 −B2
2)
2
ϱ
,
(23)
Fq(ϱ) = −B1
B3
+
p
(B1
2 −B2
2)
B3
coth
p
(B1
2 −B2
2)
2
ϱ
. (24)
Case 7: When B2B3 > 0, B3 ̸= 0 and B1 = 0 Case 4: When B1
2 + B2B3 < 0, B3 ̸= 0 and B3 ̸= −B2
Fq(ϱ) = B1
B3
+
p
−(B1
2 + B2
2)
B3
tan
p
−(B1
2 + B2
2)
2
ϱ
,
(19)
Fq(ϱ) = B1
B3
+
p
−(B1
2 + B2
2)
B3
cot
p
−(B1
2 + B2
2)
2
ϱ
. 3.1
New auxiliary approach Assuming the general form of partial PDE is of the form: Assuming the general form of partial PDE is of the form: (8) F(U, Uτ, Uχ, Uχχ, ...) = 0,
(8) where τ is the time part and χ is the spatial part and U = U(χ, τ) is the dependent
variable. We will follow the following steps. g
Step 1: Suppose the new similarity variables or transformation is of the form U(χ, τ) = H(ϱ),
where
ϱ = k(χ + cτ),
(9) (9) where k and c both are actual parameters for equation (8). Putting the Eq.(9) into
Eq. (8) and we get the new ODE below. P(H, H′, H′′, ...) = 0. (10) (10) Step 2: Assume the general solution for Eq.(10) is of the form Step 2: Assume the general solution for Eq.(10) is of the form H(ϱ) =
N
X
i=0
CiFiq(ϱ),
(11) (11) in the above solution, the Ci’s are constants and we will fine later and the 1st ODE
satisfied q(ϱ). in the above solution, the Ci’s are constants and we will fine later and the 1st ODE
satisfied q(ϱ). q′(ϱ) =
1
ln(F){B2F−q(ϱ) + B1 + B3Fq(ϱ)},
F > 0,
F ̸= 1. (12) (12) Step 3: In this step, we will use the balancing scheme to execute the value of N. For
this, we have to compare the highest order linear and nonlinear terms to find the value
of N. Step 4: Getting the coefficients of the powers of Fq(ϱ) (i = 0, 1, 2, 3..) by Eqs.(11), (12),
and (8). Then collecting the same powers terms and put it equal to zero which gives us
system of algebraic equations. After solving these system of euations by Maple. 4 Step 5: Finally we will get the different family of solutions for Eq.(12) of the form:
Case 1: When B1
2 −B2B3 < 0 and B3 ̸= 0
Fq(ϱ) = −B1
B3
+
p
−(B1
2 −B2B3)
B3
tan
p
−(B1
2 −B2B3)
2
ϱ
,
(13)
Fq(ϱ) = −B1
B3
+
p
−(B1
2 −B2B3)
B3
cot
p
−(B1
2 −B2B3)
2
ϱ
. 3.1
New auxiliary approach (24)
Case 7: When B2B3 > 0, B3 ̸= 0 and B1 = 0 Step 5: Finally we will get the different family of solutions for Eq.(12) of the form:
Case 1: When B 2
B B < 0 and B ̸
0 Step 5: Finally we will get the different family of solutions for Eq.(12) of the form:
Case 1: When B1
2 −B2B3 < 0 and B3 ̸= 0 p
y
g
Case 1: When B1
2 −B2B3 < 0 and B3 ̸= 0 Fq(ϱ) = −B1
B3
+
p
−(B1
2 −B2B3)
B3
tan
p
−(B1
2 −B2B3)
2
ϱ
,
(13)
Fq(ϱ) = −B1
B3
+
p
−(B1
2 −B2B3)
B3
cot
p
−(B1
2 −B2B3)
2
ϱ
. (14) (13) (14) Case 2: When B1
2 −B2B3 > 0 and B3 ̸= 0
Fq(ϱ) = −B1
B3
+
p
(B1
2 −B2B3)
B3
tanh
p
(B1
2 −B2B3)
2
ϱ
,
(15)
Fq(ϱ) = −B1
B3
−
p
(B1
2 −B2B3)
B3
coth
p
(B1
2 −B2B3)
2
ϱ
. (16) 3
3 ̸
Fq(ϱ) = −B1
B3
+
p
(B1
2 −B2B3)
B3
tanh
p
(B1
2 −B2B3)
2
ϱ
,
(15)
Fq(ϱ) = −B1
B3
−
p
(B1
2 −B2B3)
B3
coth
p
(B1
2 −B2B3)
2
ϱ
. (16)
Case 3: When B1
2 + B2B3 > 0 and B3 ̸= 0 and B3 ̸= −B2
Fq(ϱ) = B1
B3
+
p
(B1
2 + B2
2)
B3
tanh
p
(B1
2 + B2
2)
2
ϱ
,
(17)
Fq(ϱ) = B1
B3
+
p
(B1
2 + B2
2)
B3
coth
p
(B1
2 + B2
2)
2
ϱ
. (18)
Case 4: When B1
2 + B2B3 < 0, B3 ̸= 0 and B3 ̸= −B2
Fq(ϱ) = B1
B3
+
p
−(B1
2 + B2
2)
B3
tan
p
−(B1
2 + B2
2)
2
ϱ
,
(19)
Fq(ϱ) = B1
B3
+
p
−(B1
2 + B2
2)
B3
cot
p
−(B1
2 + B2
2)
2
ϱ
. (20) Fq(ϱ) = −B1
B3
+
p
(B1
2 −B2B3)
B3
tanh
p
(B1
2 −B2B3)
2
ϱ
,
(15)
Fq(ϱ) = −B1
B3
−
p
(B1
2 −B2B3)
B3
coth
p
(B1
2 −B2B3)
2
ϱ
. 3.1
New auxiliary approach (20)
Case 5: When B1
2 −B2
2 < 0 and B3 ̸= −B2
Fq(ϱ) = −B1
B3
+
p
−(B1
2 −B2
2)
B3
tan
p
−(B1
2 −B2
2)
2
ϱ
,
(21) Case 4: When B1
2 + B2B3 < 0, B3 ̸= 0 and B3 ̸= −B2
Fq(ϱ) = B1
B3
+
p
−(B1
2 + B2
2)
B3
tan
p
−(B1
2 + B2
2)
2
ϱ
,
(19)
Fq(ϱ) = B1
B3
+
p
−(B1
2 + B2
2)
B3
cot
p
−(B1
2 + B2
2)
2
ϱ
. (20)
Case 5: When B1
2 −B2
2 < 0 and B3 ̸= −B2 Fq(ϱ) = B1
B3
+
p
−(B1
2 + B2
2)
B3
tan
p
−(B1
2 + B2
2)
2
ϱ
,
(19)
Fq(ϱ) = B1
B3
+
p
−(B1
2 + B2
2)
B3
cot
p
−(B1
2 + B2
2)
2
ϱ
. (20)
en B1
2 −B2
2 < 0 and B3 ̸= −B2 Fq(ϱ) = B1
B3
+
p
−(B1
2 + B2
2)
B3
tan
p
−(B1
2 + B2
2)
2
ϱ
,
(19)
Fq(ϱ) = B1 +
p
−(B1
2 + B2
2) cot
p
−(B1
2 + B2
2)ϱ
. (20) (19) (20) (21) (22) Case 6: When B1
2 −B2
2 > 0 and B3 ̸= −B2
Fq(ϱ) = −B1
B3
+
p
(B1
2 −B2
2)
B3
tanh
p
(B1
2 −B2
2)
2
ϱ
,
(23)
Fq(ϱ) = −B1
B3
+
p
(B1
2 −B2
2)
B3
coth
p
(B1
2 −B2
2)
2
ϱ
. (24) Fq(ϱ) = −B1
B3
+
p
(B1
2 −B2
2)
B3
tanh
p
(B1
2 −B2
2)
2
ϱ
,
(23)
Fq(ϱ) = −B1
B3
+
p
(B1
2 −B2
2)
B3
coth
p
(B1
2 −B2
2)
2
ϱ
. (24)
B B
0 B ̸
0
d B
0 (23) (24) Case 7: When B2B3 > 0, B3 ̸= 0 and B1 = 0
Fq(ϱ) =
r
−B2
B3
tanh
√−B2B3
2
ϱ
,
(25) Fq(ϱ) =
r
−B2
B3
tanh
√−B2B3
2
ϱ
,
(25) (25) 5 Fq(ϱ) =
r
−B2
B3
coth
√−B2B3
2
ϱ
. Fq(ϱ) = tan
B2ϱ + c
2
. 3.1
New auxiliary approach (26)
Case 8: When B1 = 0 and B2 = −B3
p Fq(ϱ) =
r
−B2
B3
coth
√−B2B3
2
ϱ
. (26)
Case 8: When B1 = 0 and B2 = −B3
Fq(ϱ) = −(1 + e2B2ϱ) ±
p
2(1 + e2B2ϱ)
e2B2ϱ −1
. (27) (26) Case 8: When B1 = 0 and B2 = −B3 Fq(ϱ) = −(1 + e2B2ϱ) ±
p
2(1 + e2B2ϱ)
e2B2ϱ −1
. (27) Case 9: When B1
2 = B2B3 Case 9: When B1
2 = B2B3 Fq(ϱ) = −B2(B1ϱ + 2)
B1
2ϱ
. Case 10: When B1 = k, B2 = 2k and B3 = 0
Fq(ϱ) = eϱ −1. Case 11: When B1 = k, B3 = 2k and B2 = 0
Fq(ϱ) =
eϱ
1 −eϱ. (28) Case 10: When B1 = k, B2 = 2k and B3 = 0 (29) (30) Case 12: When 2B1 = B2 + B3 Case 12: When 2B1 = B2 + B3 Case 12: When 2B1 = B2 + B3 Fq(ϱ) = 1 + B2e
1
2 (B2−B3)ϱ
1 + B3e
1
2 (B2−B3)ϱ. (31) Case 13: When −2B1 = B2 + B3 Case 13: When −2B1 = B2 + B3 (32) Case 14: When B2 = 0 Case 14: When B2 = 0 (33) Case 15: When B2 = B1 = B3 ̸= 0 Case 15: When B2 = B1 = B3 ̸= 0 (34) Case 16: When B2 = B3, B1 = 0 Case 16: When B2 = B3, B1 = 0 Fq(ϱ) = tan
B2ϱ + c
2
. (35) Case 17: When B3 = 0 Case 17: When B3 = 0 (36) 6 3.2
Multiplier approach Supposing the Eq.(8) and applying the following steps below:
1) D fii
h
l diff
i l Supposing the Eq.(8) and applying the following steps below: 1). Definig the total differential as: 1). Definig the total differential as: Di =
∂
∂χi + Ui
∂
∂U + Uij
∂
∂Uj
+ ...,
i = 1, 2, 3...m,
(37) (37) 2). Defining the Euler operator as below: 2). Defining the Euler operator as below: δ
δU = ∂
∂U −Di
∂
∂Ui
+ Dij
∂
∂Uij
−Dijk
∂
∂Uijk
+ ... ,
(38) (38) 3). Let us define n-tuple f = (f1, f2, f3, ..., fm), i = 1, 2, ...m, Difi = 0,
(39) (39) eq.(39) is said to be the conservation laws and it is fulfils the all results of Eq. (8). 4). The purpose of Λ(χ, τ, U) of the Eq. (8): eq.(39) is said to be the conservation laws and it is fulfils the all results of Eq. (8). 4). The purpose of Λ(χ, τ, U) of the Eq. (8): Difi = Λ(χ, τ, U)H,
(40) (40) for some function U(µ1, µ2, ..., µm). for some function U(µ1, µ2, ..., µm). 5). We will obtain the determining equations for Λ(χ, τ, U) after calculating the derivative
of Λ(χ, τ, U) in Eq.(40):
δ δ
δU (Λ(χ, τ, U)H) = 0. (41) (41) Eq. (41) depends for some function U(µ1, µ2, ..., µm). Finally, when we calculate the
Λ(X, t, U) with use of Eq. (41), the conservation laws can be acquired by Eq. (40). 4
Lie group analysis of Eq. (6) Here, we are supposing the Lie approach for assumed Eq.(6). Now, suppose the one-
parameter Lie group of infinitesimal transformations on (τ, χ, U) given by ¯τ =
τ + ε ζ1(τ, χ, U) + O(ε2),
¯χ =
χ + ε ζ2(τ, χ, U) + O(ε2),
¯U =
u + ε η(τ, χ, U) + O(ε2), and ε ≪1 is a Small parameter. The associated Lie algebra of infinitesimal symmetries
is generated by vector fields and ε ≪1 is a Small parameter. The associated Lie algebra of infinitesimal symmetries
is generated by vector fields X =ζ1(τ, χ, U)∂τ + ζ2(τ, χ, U)∂χ + η(τ, χ, U)∂U. (42) (42) Eq. (42) creates a symmetry of Eq. (6), and X satisfy the Lie group conditions Pr(4)X
∂2U
∂τ 2 = δ2
o
∂2U
∂χ2 + po
∂U
∂χ
∂2U
∂χ2 + qo
∂U
∂χ
2∂2U
∂χ2 + r∂4U
∂χ4
|Eq.(6)=0 = 0. 7 The Pr(4)X for X can be written as: for X can be written as: Pr(4)X =X + ηχ ∂
∂Uχ
+ ηττ
∂
∂Uττ
+ ηχχ
∂
∂Uχχ
+ ηχχχχ
∂
∂Uχχχχ
,
(43) (43) furthermore, we have
ηχ = Dχ(η) −UχDχ(ζ1) −UτDχ(ζ2),
ηχχ = Dχ(ηχ) −UχχDχ(ζ1) −UτχDχ(ζ2),
ητ = Dτ(η) −UχDt(ζ1) −UτDτ(ζ2),
ηττ = Dτ(ητ) −UττDτ(ζ1) −UχτDt(ζ2),
ηχχχ = Dχ(ηχχ) −UχχχDχ(ζ1) −UτχχDχ(ζ2). (44) (44) Let (x1, x2) = (χ, τ), where Di can be written as: Let (x1, x2) = (χ, τ), where Di can be written as: Di =
∂
∂χi + Ui
∂
∂U + Uij
∂
∂Uj
+ ...,
i = 1, 2. Substituting the values of ηi which gives us the following vectors: Substituting the values of ηi which gives us the following vectors: X1 = ∂
∂χ,
X2 = ∂
∂τ ,
X3 = ∂
∂U ,
X4 = τ ∂
∂U . (45) (45) We see that [Xi, Xj] = 0,
where i, j = 1, 2, 3. 5
Optimal system In this section, we observe that from the obtained vector field Eq. (45), the X = {X1, X2}
forms an abelian algebra. So we can use the (42) and get: £1 = < X1 >,
£2 = < X1 + k1X2 > . (46) (46) 5.2
Application of new auxiliary method Here, our aim is to construct the wave patterns for Eq.(6) from Eq.(50) with use of
proposed technique. Using the balancing method and we obtain N = 1. Using the value
of N = 1 in (11) and we have H(ϱ) = Co + C1Fq(ϱ). (51) (51) We have to put Eq.(51) into Eq.(50) and we get the system of equation after comparing
the coefficients of Fq(ϱ). With the use of Maple, we have solved the obtained system of
equations and get the following results. C0 = V1,
C1 = −12rB3
po
,
k1 = ±
q
rB2
1 −4rB2B3 + δ2
o. (52) (52) Using Eq.(52) into Eq.(51) and which gives us the following set of solutions. U(χ, τ) = V1 −12rB3
po
Fq(ϱ),
where
ϱ = χ ±
q
rB2
1 −4rB2B3 + δ2
o τ
(53) U(χ, τ) = V1 −12rB3
po
Fq(ϱ),
where
ϱ = χ ±
q
rB2
1 −4rB2B3 + δ2
o τ
(53) (53) where V1 is orbitraryconstant. 2 where V1 is orbitraryconstant. where V1 is orbitraryconstant. where V1 is orbitraryconstant. y
Case:1 When B1
2 −B2U3 < 0 and B3 ̸= 0 Case:1 When B1
2 −B2U3 < 0 and B3 ̸= 0
U1(χ, τ) = V1 −12rB3
po
−B1
B3
+
p
−(B1
2 −B2B3)
B3
tan
p
−(B1
2 −B2B3)
2
ϱ
, (54)
U2(χ, τ) = V1 −12rB3
po
−B1
B3
+
p
−(B1
2 −B2B3)
B3
cot
p
−(B1
2 −B2B3)
2
ϱ
. (55)
Case:2 When B1
2 −B2B3 > 0 and B3 ̸= 0
U3(χ, τ) = V1 −12rB3
po
−B1
B3
+
p
(B1
2 −B2B3)
B3
tanh
p
(B1
2 −B2B3)
2
ϱ
,
(56)
U4(χ, τ) = V1 −12rB3
po
−B1
B3
−
p
(B1
2 −B2B3)
B3
coth
p
(B1
2 −B2B3)
2
ϱ
. (57)
Case:3 When B1
2 + B2B3 > 0 and B3 ̸= 0 and B3 ̸= −B2
U5(χ, τ) = V1 −12rB3
po
B1
B3
+
p
(B1
2 + B2
2)
B3
tanh
p
(B1
2 + B2
2)
2
ϱ
,
(58)
U6(χ, τ) = V1 −12rB3
po
B1
B3
+
p
(B1
2 + B2
2)
B3
coth
p
(B1
2 + B2
2)
2
ϱ
. 5.1
Similarity reduction of Eq. (6) Here, we will find the similarity variables and analytical results for Eq.(6). 5.1.1
£1 =< X1 > 5.1.1
£1 =< X1 > 5.1.1
£1 =< X1 > Using the vector £1, we get the new variable u(τ, χ) = H(ϱ),
where ϱ = χ,
(47) (47) u(τ, χ) = H(ϱ),
where ϱ = χ, putting the (47) into Eq. (6), which gives us U(τ, χ) = m1τ + m2
(48) (48) U(τ, χ) = m1τ + m2 where m1 and m2 are integration constants. where m1 and m2 are integration constants. 8 5.1.2
£2 =< X1 + k1X2 Using the vector £2, we get the new variable Using the vector £2, we get the new variable u(τ, χ) = H(ϱ),
where ϱ = χ + k1τ,
(49) (49) u(τ, χ) = H(ϱ),
where ϱ = χ + k1τ,
(49)
putting the (49) into Eq. (6), which gives us
6(k2
1 −δ2
o)H′ −3po(H′)2 −2qo(H′)3 −3rH′′′ = 0. (50) putting the (49) into Eq. (6), which gives us putting the (49) into Eq. (6), which gives us 6(k2
1 −δ2
o)H′ −3po(H′)2 −2qo(H′)3 −3rH′′′ = 0. (50) (50) 5.2
Application of new auxiliary method (59) 1
2
3
3 ̸
U1(χ, τ) = V1 −12rB3
po
−B1
B3
+
p
−(B1
2 −B2B3)
B3
tan
p
−(B1
2 −B2B3)
2
ϱ
, (54)
U2(χ, τ) = V1 −12rB3
po
−B1
B3
+
p
−(B1
2 −B2B3)
B3
cot
p
−(B1
2 −B2B3)
2
ϱ
. (55) U3(χ, τ) = V1 −12rB3
po
−B1
B3
+
p
(B1
2 −B2B3)
B3
tanh
p
(B1
2 −B2B3)
2
ϱ
,
(56)
U4(χ, τ) = V1 −12rB3
po
−B1
B3
−
p
(B1
2 −B2B3)
B3
coth
p
(B1
2 −B2B3)
2
ϱ
. (57)
2 Case:3 When B1
2 + B2B3 > 0 and B3 ̸= 0 and B3 ̸= −B2 Case:3 When B1
2 + B2B3 > 0 and B3 ̸= 0 and B3 ̸= −B2 U5(χ, τ) = V1 −12rB3
po
B1
B3
+
p
(B1
2 + B2
2)
B3
tanh
p
(B1
2 + B2
2)
2
ϱ
,
(58)
U6(χ, τ) = V1 −12rB3
po
B1
B3
+
p
(B1
2 + B2
2)
B3
coth
p
(B1
2 + B2
2)
2
ϱ
. (59) (58) (59) 9 Case: 4 When B1
2 + B2B3 < 0, B3 ̸= 0 and B3 ̸= −B2
U7(χ, τ) = V1 −12rB3
po
B1
B3
+
p
−(B1
2 + B2
2)
B3
tan
p
−(B1
2 + B2
2)
2
ϱ
,
(60)
U8(χ, τ) = V1 −12rB3
po
B1
B3
+
p
−(B1
2 + B2
2)
B3
cot
p
−(B1
2 + B2
2)
2
ϱ
. (61)
Case: 5 When B1
2 −B2
2 < 0 and B3 ̸= −B2
U9(χ, τ) = V1 −12rB3
po
−B1
B3
+
p
−(B1
2 −B2
2)
B3
tan
p
−(B1
2 −B2
2)
2
ϱ
,
(62)
U10(χ, τ) = V1 −12rB3
po
−B1
B3
+
p
−(B1
2 −B2
2)
B3
cot
p
−(B1
2 −B2
2)
2
ϱ
. (63)
Case: 6 When B1
2 −B2
2 > 0 and B3 ̸= −B2
U11(χ, τ) = V1 −12rB3
po
−B1
B3
+
p
(B1
2 −B2
2)
B3
tanh
p
(B1
2 −B2
2)
2
ϱ
,
(64)
U12(χ, τ) = V1 −12rB3
po
−B1
B3
+
p
(B1
2 −B2
2)
B3
coth
p
(B1
2 −B2
2)
2
ϱ
. 5.2
Application of new auxiliary method (65)
Case: 7 When B2B3 > 0, B3 ̸= 0 and B1 = 0 Case: 4 When B1
2 + B2B3 < 0, B3 ̸= 0 and B3 ̸= −B2 U7(χ, τ) = V1 −12rB3
po
B1
B3
+
p
−(B1
2 + B2
2)
B3
tan
p
−(B1
2 + B2
2)
2
ϱ
,
(60)
U8(χ, τ) = V1 −12rB3
po
B1
B3
+
p
−(B1
2 + B2
2)
B3
cot
p
−(B1
2 + B2
2)
2
ϱ
. (61)
Case: 5 When B1
2 −B2
2 < 0 and B3 ̸= −B2 (60) (61) U9(χ, τ) = V1 −12rB3
po
−B1
B3
+
p
−(B1
2 −B2
2)
B3
tan
p
−(B1
2 −B2
2)
2
ϱ
, (62) Case: 6 When B1
2 −B2
2 > 0 and B3 ̸= −B2
U11(χ, τ) = V1 −12rB3
po
−B1
B3
+
p
(B1
2 −B2
2)
B3
tanh
p
(B1
2 −B2
2)
2
ϱ
,
(64)
U12(χ, τ) = V1 −12rB3
po
−B1
B3
+
p
(B1
2 −B2
2)
B3
coth
p
(B1
2 −B2
2)
2
ϱ
. (65)
Case: 7 When B2B3 > 0, B3 ̸= 0 and B1 = 0 (64) (65) U13(χ, τ) = V1 −12rB3
po
r
−B2
B3
tanh
√−B2B3
2
ϱ
,
(66)
U14(χ, τ) = V1 −12rB3
po
r
−B2
B3
coth
√−B2B3
2
ϱ
. (67)
Case: 8 When B1 = 0 and B2 = −B3 U13(χ, τ) = V1 −12rB3
po
r
−B2
B3
tanh
√−B2B3
2
ϱ
,
(66) (66) U14(χ, τ) = V1 −12rB3
po
r
−B2
B3
coth
√−B2B3
2
ϱ
. (67)
Case: 8 When B1 = 0 and B2 = −B3 (67) U15(χ, τ) = V1 −12rB3
po
−(1 + e2B2ϱ) ±
p
2(1 + e2B2ϱ)
e2B2ϱ −1
. (68) (68) 15(χ, )
1
po
e2B2ϱ −1
(
)
Case: 9 When B1
2 = B2B3
U16(χ τ) = V1 −12rB3
−B2(B1ϱ + 2)
(69) Case: 9 When B1
2 = B2B3 Case: 9 When B1
2 = B2B3 Case: 9 When B1
2 = B2B3 Case: 9 When B1
2 = B2B3 U16(χ, τ) = V1 −12rB3
po
−B2(B1ϱ + 2)
B1
2ϱ
. 5.2
Application of new auxiliary method (69)
Case: 10 When B1 = k, B2 = 2k and B3 = 0
U17(χ, τ) = V1 −12rB3
po
eϱ −1
. (70)
Case: 11 When B1 = k, B3 = 2k and B2 = 0
U18(χ, τ) = V1 −12rB3
po
eϱ
1 −eϱ
. (71) U16(χ, τ) = V1 −12rB3
po
−B2(B1ϱ + 2)
B1
2ϱ
. (69) U16(χ, τ) = V1 −12rB3
po
−B2(B1ϱ + 2)
B1
2ϱ
. (69)
Case: 10 When B1 = k, B2 = 2k and B3 = 0 (69) U16(χ, τ) = V1 −
po
B1
2ϱ
. (69)
Case: 10 When B1 = k, B2 = 2k and B3 = 0 U17(χ, τ) = V1 −12rB3
po
eϱ −1
. (70)
Case: 11 When B1 = k, B3 = 2k and B2 = 0
U18(χ, τ) = V1 −12rB3
po
eϱ
1 −eϱ
. (71) (70) (71) 10 Case: 12 When 2B1 = B2 + B3 U19(χ, τ) = V1 −12rB3
po
1 + B2e
1
2 (B2−B3)ϱ
1 + B3e
1
2 (B2−B3)ϱ
. (72) (72) Case: 13 When −2B1 = B2 + B3 Case: 13 When −2B1 = B2 + B3 Case: 13 When −2B1 = B2 + B3 Case: 13 When −2B1 = B2 + B3 U20(χ, τ) = V1 −12rB3
po
B2 + B2e
1
2 (B2−B3)ϱ
B3 + B3e
1
2 (B2−B3)ϱ
. (73) (73) Case: 14 When B2 = 0 Case: 14 When B2 = 0 Case: 14 When B2 = 0 U21(χ, τ) = V1 −12rB3
po
B1eB1ϱ
1 + B3
2 eB1ϱ
. (74)
Case: 15 When B2 = B1 = B3 ̸= 0
U22(χ, τ) = V1 −12rB3
−(B2ϱ + 2)
. (75) U21(χ, τ) = V1 −12rB3
po
B1eB1ϱ
1 + B3
2 eB1ϱ
. (74) U21(χ, τ) = V1 −12rB3
po
B1eB1ϱ
1 + B3
2 eB1ϱ
. U21(χ, τ) = V1 −12rB3
po
B1eB1ϱ
1 + B3
2 eB1ϱ
. (74)
Case: 15 When B2 = B1 = B3 ̸= 0
U22(χ, τ) = V1 −12rB3
po
−(B2ϱ + 2)
B2ϱ
. (75)
Case: 16 When B2 = B3, B1 = 0
U23(χ, τ) = V1 −12rB3
tan
B2ϱ + c
. 5.2
Application of new auxiliary method (76) (74)
2
Case: 15 When B2 = B1 = B3 ̸= 0
U22(χ, τ) = V1 −12rB3
po
−(B2ϱ + 2)
B2ϱ
. (75)
C
16 Wh
B
B
B
0 Case: 15 When B2 = B1 = B3 ̸= 0
U22(χ, τ) = V1 −12rB3
po
−(B2ϱ + 2)
B2ϱ
. (75) U22(χ, τ) = V1 −12rB3
po
−(B2ϱ + 2)
B2ϱ
. (75)
Case: 16 When B2 = B3, B1 = 0 U22(χ, τ) = V1 −12rB3
po
−(B2ϱ + 2)
B2ϱ
. (75) (75) po
B2ϱ
Case: 16 When B2 = B3, B1 = 0
U23(χ, τ) = V1 −12rB3
po
tan
B2ϱ + c
2
. (76) Case: 16 When B2 = B3, B1 = 0
U (χ τ)
V
12rB3
tan
B2ϱ + c
(76) U23(χ, τ) = V1 −12rB3
po
tan
B2ϱ + c
2
. (76) (76) Case: 17 When B3 = 0 Case: 17 When B3 = 0 Case: 17 When B3 = 0 Case: 17 When B3 = 0 U24(χ, τ) = V1 −12rB3
po
eB1ϱ −B2
2B1
. (77) (77) Where ϱ = χ ±
p
rB2
1 −4rB2B3 + δ2
o τ is given according to. (a) 3D (b) 2D (a) 3D
(b) 2D
Figure 1: Graphics of u1(χ, τ) for the choice of parameters δo = 1, r = 2, po = 3, B1 = 2,
B2 = 1,B3 = 2, τ = 1. (a) 3D Figure 1: Graphics of u1(χ, τ) for the choice of parameters δo = 1, r = 2, po = 3, B1 = 2,
B2 = 1,B3 = 2, τ = 1. 11 11 (a) 3D (a) 3D
(b) 2D
Figure 2: Graphics of u2(χ, τ) for the choice of parameters δo = 2, r = 1, po = 1, B1 = 3,
B2 = 2,B3 = 1, τ = 2. (b) 2D (a) 3D Figure 2: Graphics of u2(χ, τ) for the choice of parameters δo = 2, r = 1, po = 1, B1 = 3,
B2 = 2,B3 = 1, τ = 2. (a) 3D
(b) 2D
Figure 3: Graphics of u3(χ, τ) for the choice of parameters δo = 1.5, r = 1, po = 4,
B1 = 4, B2 = 3,B3 = 5, τ = 2. 6
Graphics and discussion Graphical representation of obtained solutions is discussed here in this section. By using
the new auxiliary method we have constructed the analytical behaviour of considered
model in the form of trigonometric functions, hyperbolic trigonometric functions, expo-
nential, and algebraic type results. The graph of the tangent function is periodic with a
period of Π and has vertical asymptotes at odd multiples of Pi
2 . As χ approaches these
vertical asymptotes, the tangent function approaches positive or negative infinity depend-
ing on the direction of approach. The graph of the cotangent function is also periodic
with a period of Π and has horizontal asymptotes at even multiples of Π. As χ approaches
these horizontal asymptotes, the cotangent function approaches zero. We have plotted
the behaviour of some obtained results. Fig.(1) shows the graphical behavior of u1(χ, τ)
for the choice of parameters δo = 1, r = 2, po = 3, B1 = 2, B2 = 1,B3 = 2, τ = 1. Fig. (2) represent the behaviour of u2(χ, τ) for the choice of parameters δo = 2, r = 1, po = 1,
B1 = 3, B2 = 2,B3 = 1, τ = 2. Fig. (3) shows the Graphics of u3(χ, τ) for the choice
of parameters δo = 1.5, r = 1, po = 4, B1 = 4, B2 = 3,B3 = 5, τ = 2. Fig. (4) shows
the Graphics of u4(χ, τ) for the choice of parameters δo = 5, r = 0.5, po = 3.5, B1 = 1.5,
B2 = 3.5,B3 = 2.5, τ = 3. Fig. (5) represent the behaviour of u19(χ, τ) for the choice of
parameters δo = 6, r = 3, po = 5, B1 = 1, B2 = 3,B3 = 5, τ = 5. 5.2
Application of new auxiliary method (b) 2D (a) 3D (a) 3D (a) 3D Figure 3: Graphics of u3(χ, τ) for the choice of parameters δo = 1.5, r = 1, po = 4,
B1 = 4, B2 = 3,B3 = 5, τ = 2. (a) 3D
(b) 2D
Figure 4: Graphics of u4(χ, τ) for the choice of parameters δo = 5, r = 0.5, po = 3.5,
B1 = 1.5, B2 = 3.5,B3 = 2.5, τ = 3. (b) 2D (a) 3D (a) 3D Figure 4: Graphics of u4(χ, τ) for the choice of parameters δo = 5, r = 0.5, po = 3.5,
B1 = 1.5, B2 = 3.5,B3 = 2.5, τ = 3. 12 (a) 3D
(b) 2D
Figure 5: Graphics of u19(χ, τ) for the choice of parameters δo = 6, r = 3, po = 5, B1 = 1,
B2 = 3,B3 = 5, τ = 5. (a) 3D (b) 2D (a) 3D Figure 5: Graphics of u19(χ, τ) for the choice of parameters δo = 6, r = 3, po = 5, B1 = 1,
B2 = 3,B3 = 5, τ = 5. 7
Conservation laws (83) (83) Case2 : For C1 = 0, C2 = 1, then Λ1 = 1, we get the following fluxes: T τ
2 = Uτ,
T χ
2 = −1
3 U 3
χqo −1
2U 2
χpo −Uχδ2
o −rUχχχ. (84) (84) 7
Conservation laws In this portion, we will construct the conservation laws by multiplier approach for Eq.(6). We obtain the determinant equation for Λ(X, t, u) by Eq. (41). δ
δU
Λ
∂2U
∂τ 2 −δ2
o
∂2U
∂χ2 −po
∂U
∂χ
∂2U
∂χ2 −qo
∂U
∂χ
2∂2U
∂χ2 −r∂4U
∂χ4
= 0. (78) (78) Using Eq.(38), we can write the Euler operator is of the form g Eq.(38), we can write the Euler operator is of the form δ
δU = ∂
∂U −Dτ
∂
∂Uτ
−Dχ
∂
∂Uχ
+ D2
τ
∂
∂Uττ
+ D2
χ
∂
∂Uχχ
+ DχDτ
∂
∂Uτχ
−...,
(79) δ
δU = ∂
∂U −Dτ
∂
∂Uτ
−Dχ
∂
∂Uχ
+ D2
τ
∂
∂Uττ
+ D2
χ
∂
∂Uχχ
+ DχDτ
∂
∂Uτχ
−...,
(79) δ
δU = ∂
∂U −Dτ
∂
∂Uτ
−Dχ
∂
∂Uχ
+ D2
τ
∂
∂Uττ
+ D2
χ
∂
∂Uχχ
+ DχDτ
∂
∂Uτχ
−...,
(79) (79) 13 defining the total derivative operators Dτ and Dχ from Eq. (37). Dτ = ∂
∂τ + Uτ
∂
∂U + Uττ
∂
∂Uτ
+ Uτχ
∂
∂Uχ
...,
Dχ = ∂
∂χ + Uχ
∂
∂U + Uχχ
∂
∂Uχ
+ Uτχ
∂
∂Uτ
...,
(80) (80) computing Eq. (78) and we obtain the following multipliers and conservation laws as: computing Eq. (78) and we obtain the following multipliers and conservation laws as: Λ = C1τ + C2,
(81) (81) by Eqs. (40) and (81), The following conservation laws are found. by Eqs. (40) and (81), The following conservation laws are found. T τ =Uτ(C1τ + C2) −C1U,
T χ =(C1τ + C2)
−1
3 U 3
χqo −1
2U 2
χpo −Uχδ2
o −rUχχχ
. (82) (82) Using Eq. (82) and we get the following two cases of conservation laws. Case1 : For C1 = 1, C2 = 0, then Λ1 = τ, we get the following fluxes: Using Eq. (82) and we get the following two cases of conservation laws. Case1 : For C1 = 1, C2 = 0, then Λ1 = τ, we get the following fluxes: T τ
1 = τUτ −U,
T χ
1 = −1
3 U 3
χτqo −1
2U 2
χτpo −Uχτδ2
o −rUχχχτ. Acknowledgements: This work has been supported by the Polish National Science
Centre, Poland under the Grant OPUS 18 No.2019/35/B/ST8/00980. 8
Conclusion In this research, nonlinear chains of atoms(NCA) are studied. NCA are complex sys-
tems that exhibit rich dynamics and phenomena, making their study crucial in various
scientific fields. The Lie symmetry method provides a powerful mathematical tool for
analyzing and solving differential equations with symmetries, reducing complexity and
obtaining exact or approximate solutions. The introduction of a new auxiliary scheme
enhances existing techniques, offering additional insights, improved accuracy, or simpli-
fied computations. Travelling wave solutions describe wave-like behavior propagating
through systems, while graphical behavior provides visual representations of relation-
ships and patterns in data or mathematical models. The multiplier method allows for
the identification of conservation laws, which are fundamental principles in physics that
state certain quantities remain constant over time. Understanding conservation laws and
utilizing mathematical techniques such as the Lie symmetry method, travelling wave so-
lutions, and graphical analysis contributes to a deeper understanding of nonlinear chains
of atoms and their dynamics. Acknowledgements: This work has been supported by the Polish National Science
Centre, Poland under the Grant OPUS 18 No.2019/35/B/ST8/00980. 14 Credit authorship contribution statement: M. Junaid-U-Rehman: Conceptualiza-
tion, Methodology, Investigation, Software, Writing - original draft. Jan Awrejcewicz:
Visualization, Investigation. Grzegorz Kudra: Supervision, Software, Validation. Declaration of Competing Interest: The Authors declare that they do not have any Credit authorship contribution statement: M. Junaid-U-Rehman: Conceptualiza-
tion, Methodology, Investigation, Software, Writing - original draft. Jan Awrejcewicz:
Visualization, Investigation. Grzegorz Kudra: Supervision, Software, Validation. Declaration of Competing Interest: The Authors declare that they do not have any
conflicts of interest. tion, Methodology, Investigation, Software, Writing - original draft. Jan Awrejcewicz:
Visualization, Investigation. Grzegorz Kudra: Supervision, Software, Validation. Declaration of Competing Interest: The Authors declare that they do not have any
conflicts of interest. Declaration of Competing Interest: The Authors declare that they do not have any
conflicts of interest. Declaration of Competing Interest: The Authors declare that they do not have any
conflicts of interest. Data availability statements: All data that support the findings of this study are
included within the article (and any supplementary files). Data availability statements: All data that support the findings of this study are
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Risk factors for orofacial clefts in India: A case–control study
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Introduction includes cleft palate (CP) and cleft lip (CL) in isolation or in
combination. Children suffering from OFCs in low income
countries have high morbidity throughout their life. They
face a range of functional and cosmetic problems with poten-
tial long-term adverse impact on health, speech, hearing, psy-
chology, learning ability and social skills (Agbenorku, 2013). The treatment often requires a multidisciplinary approach,
from surgical correction of physical defect to long-term psy-
chological counselling (Mossey and Little, 2009). The burden of orofacial clefts (OFC) is a major concern in
India and throughout the world (Mossey et al., 2009). OFC The burden of orofacial clefts (OFC) is a major concern in
India and throughout the world (Mossey et al., 2009). OFC Additional Supporting information may be found in the online version of this
article. Additional Supporting information may be found in the online version of this
article. 1Indian Institute of Public Health- Delhi, Public Health Foundation of India,
New Delhi, India Estimates from a meta-analysis including 11 hospital
based studies reported a prevalence of OFCs ranging from
0.2 to 2.9 per 1000 total births and a pooled prevalence of
1.3 per 1000 total births in India (Allagh et al., 2015). It is
estimated that every year 28,600 infants are born with
cleft lip and cleft palate, that is, 78 infants per day in the
country (Mossey and Little, 2009). 2Indian Institute of Public Health- Hyderabad, Public Health Foundation of
India, Hyderabad, India 3Centre for Applied Research and Education on Neurodevelopmental
Impairments and Disability related Health Initiatives (CARENIDHI), New Delhi,
India 4Kalawati Saran Hospital and Lady Hardinge Medical College, New Delhi, India 7The UK National Institute for Health Research Bristol Nutrition Biomedical
Research Unit, University of Bristol, Bristol, United Kingdom The etiology of OFC is polygenic and multi-factorial. Both genetic and environmental factors have been identified
though the precise cause in many cases is unclear (Mossey
and Little, 2009). The environmental factors include mater-
nal illness, infections, drugs, radiation, alcohol, and contami-
nation of food and water with pesticides, nitrates, and
mercury (Aylsworth et al., 2015). In vitro fertilization and
intracytoplasmic sperm injection, consanguinity, dietary,
and indigenous medicines used for sex selection are also
implicated as risk factors (Mossey and Little, 2009; Neogi
et al., 2015). Risk Factors for Orofacial Clefts in India:
A Case–Control Study Sutapa Bandyopadhyay Neogi
*1, Samiksha Singh2, Dinesh Raj Pallepogula2, Hira Pant2,
Sunanda Reddy Kolli3, Priyanka Bharti1, Vikram Datta4, Srinivas Reddy Gosla5,
Krishnamurthy Bonanthaya6, Andy Ness7, Sanjay Kinra8, Pat Doyle8, and
Venkata Satyanarayana Murthy Gudlavalleti2 p value 5 0.001), and delayed first conception (AOR, 2.55, 95% CI, 1.25–
5.21, p 5 0.01) were found to be strongly associated with higher risk of
OFCs. Supplementation with folic acid during first 3 months of pregnancy was
not found to be protective against OFCs (AOR, 1.24; 95% CI, 0.59–2.58;
p value 5 0.56). Conclusion: Our study confirmed the importance of family
history as a risk factor for OFC. Our study did not show an association with
folic acid supplementation but was underpowered to detect small effects. Our
finding of higher risk among vegetarians requires replication. Background: Orofacial clefts (OFC) are linked with several genetic and
environmental factors. The aim of this study was to explore the association of
potential risk factors with OFCs in India. Methods: This was a hospital-based,
matched case–control (1:4 ratio; matching done for parity) study conducted
in Hyderabad, Bengaluru, and Delhi-National Capital Region. Cases
(nonsyndromic clefts) were recruited from treatment centers, while controls
(live births) were recruited from maternity centers. Information on exposures
was collected during personal interviews. Exposures of interest included folic
acid supplementation during the peri-conceptional period, consanguineous
marriage, exposure to drugs, infections during pregnancy, family history of
OFC, and dietary factors. Results: A total of 785 participants were included in
the study: 157 cases and 628 controls. A family history of cleft lip/palate
(adjusted odds ratio [AOR], 15.48; 95% confidence interval [CI], 4.36–54.96;
p value 5 0.001), exclusive vegetarianism (AOR, 4.47; 95% CI, 1.83–10.98; Birth Defects Research 109:1284–1291, 2017. V
C 2017 Wiley Periodicals, Inc. Key
words:
Orofacial
clefts;
cleft
lip/palate;
risk
factors;
folic
acid
supplementation Introduction 8London School of Hygiene and Tropical Medicine, London, United Kingdom This work was funded by Wellcome Trust Capacity Strengthening Strategic
Award to the Public Health Foundation of India and a consortium of UK uni-
versities (Grant number WT084754/Z/08/Z). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the
manuscript
This work was funded by Wellcome Trust Capacity Strengthening Strategic Award
to the Public Health Foundation of India and a consortium of UK universities
(Grant number WT084754/Z/08/Z). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript. Conflicts of Interest: None. manuscript. Conflicts of Interest: None. *Correspondence to: Sutapa B. Neogi, Indian Institute of Public Health- Delhi,
Public Health Foundation of India, Delhi, India. E-mail: sutapa.bneogi@iiphd.org
Th
i ht li
f
thi
ti l
h
d
15th M
h 2018 ft
i i
l
*Correspondence to: Sutapa B. Neogi, Indian Institute of Public Health- Delhi,
Public Health Foundation of India, Delhi, India. E-mail: sutapa.bneogi@iiphd.org
The copyright line for this article was changed on 26 June 2019 after original
online publication. The copyright line for this article was changed on 15th March, 2018 after original
online publication. Published online 2 August 2017 in Wiley Online Library (wileyonlinelibrary
This is an open access article under the terms of the Creative Commons
Attribution License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited. Folic acid deficiency has been linked to the causation
of many chronic and developmental disorders (Smithells com). Doi: 10.1002/bdr2.1073
Published online 2 August 2017 in Wiley Online Library (wileyonlinelibrary. com). Doi: 10.1002/bdr2.1073 V
C 2017 Wiley Periodicals, Inc. © 2017 The Authors Birth Defects Research Published by Wiley Periodicals, Inc. V
C 2017 Wiley Periodicals, Inc. © 2017 The Authors Birth Defects Research Published by Wiley Periodicals, Inc. BIRTH DEFECTS RESEARCH 109:1284–1291 (2017) 1285 et al., 1976; Botto et al., 1999; Caudill M, 2008; Molloy
et al., 2008, 2009). Folate deficiency is prevalent globally,
especially in low and middle income countries, ranging
between 20 and 30% (Gamble et al., 2005; Pathak et al.,
2007; McLean et al., 2008). Various studies have suggested
a protective effect of peri-conceptional folic acid consump-
tion from cleft lip with or without palate. However, the
evidence is inconsistent. Materials and Methods
STUDY DESIGN AND SETTINGS This was a hospital-based, matched case–control (1:4
ratio; matching for parity) study conducted in Hyderabad,
Bengaluru, and Delhi. At each site, cases were selected
from a treatment center specialized for managing OFCs. These treatment centers were part of Smile Train, a pro-
gram run by a nongovernmental organization having a
nationwide chain of accredited centers that provide free
treatment for OFCs. The cases were recruited from the fol-
lowing treatment centers: GSR Institute of Craniofacial
Surgery, Hyderabad; Bhagvan Mahaveer Jain Hospital, Ban-
galore,
Karnataka;
Kalawati
Saran
Children’s
hospital,
Delhi; Sant Paramand Hospital, Delhi. For controls, the fol-
lowing centers were selected: Koti Maternity Hospital,
Mahbubnagar;
District
Hospital
Mahbubnagar
District,
Telangana; Kalawati Saran Children’s Hospital, Delhi; Srir-
ampura referral Hospital, Bangalore; Bhanshankari District
Hospital, Bangalore; Ulsoor referral hospital, Bangalore;
Siddipur
referral
Hospital,
Bangalore. Controls
were
selected from maternity centers near/adjoining the treat-
ment centers. Women who delivered in these centers dur-
ing the study period were recruited after they gave their
consent. SAMPLE SIZE For each case, four controls were selected. The estimated
sample size calculated using Open Epi software (version
3.1) (Dean et al., 2013) was 750 (150 cases and 600 con-
trols) assuming an alpha error of 10%, a power of 80% to
detect a 50% decrease in risk (odds ratio [OR] 5 0.5)
associated with folic acid supplementation. The prevalence
of folic acid supplementation in the control group was
assumed to be around 20% with 5% nonresponse rate. Introduction This is further compounded by
heterogeneity resulting from different dosages of folic acid
and types (with or without other multivitamins) (De-Regil
et al., 2010). from other hospitals or who were not from the catchment
area, or those not willing to participate in the study. CASES AND CONTROLS
A
b b Cases. Any baby with nonsyndromic clefts, that is, cleft
lip 6 palate or cleft palate, who visited the treatment cen-
ter within 4 months of birth was considered as a case. The treating physician ascertained the outcomes based on
the standard case definitions used to detect OFCs. OFCs in
this study included an opening in a structure around the
mouth and face, including clefts in the lip, the roof of
the mouth (hard palate), or the tissue in the back of the
mouth (soft palate). We interviewed the mothers of the
cases at the time of recruitment. Most of these studies on risk factors and prevention of
OFCs by folic acid supplementation were done in Western
countries (Figueiredo et al., 2015). Comparatively, India
has a predominantly vegetarian population, low maternal
vitamin-B12 status, and higher prevalence of consanguine-
ous marriages, infections, and maternal illnesses. Thus, the
risk profile of OFCs may be different (Agbenorku, 2013). Therefore, the aim of our study was to assess risk factors
of OFCs, including lack of folic acid supplementation, dur-
ing peri-conceptional period. Controls. The controls were live births without any malfor-
mation born in a maternity center close to the same region
as the treatment center of the case. We interviewed mothers
of the controls within 48 hours of delivery. We verified that
both cases and controls were inhabitants of the same geo-
graphical regions from their addresses, and close to the sites
where the study was conducted. Each control selected pur-
posively was individually matched to her control according
to parity of mother, using the groups: primiparous, parity 1,
2, and 3 1. The study aimed to match four controls to each
case. DATA COLLECTION AND MANAGEMENT Data collection took place between January 2015 and July
2016. Structured data collection questionnaires developed
by the research team captured information on basic socio-
demographic features, details of reproductive, medical and
occupational history and other potential risk factors. We
translated the tools into local languages, pretested, piloted,
and modified before data collection. Trained female field investigators interviewed mothers
to collect information on risk factors. Ascertainment of
exposures was based on the history elicited using a struc-
tured questionnaire designed for the study. Data on folic
acid supplementation were collected based on the history
of intake of folic acid tablets during the peri-conceptional
period and dietary history. The study population consisted of all pregnant women
delivering in these hospitals or visiting treatment centers
for their children with cleft lip/ palate who gave their con-
sent to participate. When selecting controls, we excluded
stillbirths or babies who had any apparent structural or
chromosomal malformations other than OFC, if they were
part of any other on-going studies, had any maternal com-
plications that warranted emergency care, babies referred We collected data on paper forms. Data entry was
done by staff trained by the data manager using custom-
ized forms designed and validated in MS Access with built
in consistency, range, and missing data checks by the data
base administrator. RISK FACTORS FOR OROFACIAL CLEFTS 1286 TABLE 1. DATA COLLECTION AND MANAGEMENT Socio-demographic Profile of Cases and Controls
Variables
Cases (n 5 157)
Control (n 5 628)
p-Value
Age of child in days [mean (SD)]
61.9 (39.1)
14.4 (29.1)
0.000
Age of the child more than or equal to 14 days at time of interview [n (%)]
133 (84.7)
114 (18.2)
0.000
Sex of child - male [n (%)]
64 (40.7)
297 (47.3
0.14
Age of mother (in completed years) [mean (SD)]
24.8 (3.8)
24.0 (3.5)
0.01
Age of father (in completed years) [mean (SD)]
29.7 (4.3)
28.8(4.3)
0.02
Mothers less than 30 years of age
138(87.9)
575 (91.5)
0.15
Fathers less than 30 years of age
80 (50.9)
368 (58.6)
0.08
Education status of mother – illiterate or just literate [n (%)]
18 (11.4)
78 (12.4)
0.74
Education status of father- illiterate or just literate [n (%)]
14 (8.8)
90 (14.3)
0.07
Occupation of mother - homemaker [n (%)]
134 (85.4)
542 (86.3)
0.76
Occupation of father- agriculture/ industry [n (%)]
25 (15.9)
77 (12.2)
0.24
Caste backward caste [n (%)]
74 (47.1)
378 (60.1)
0.003
Family income less than 1 lakh rupees per year [n (%)]
110 (98.2)
391 (99.7)
0.09
Age at marriage [mean (SD)]
20.5 (3.3)
20.1 (3.0)
0.2
Drinking water (ground water) [n (%)]
41 (26.1)
130 (20.7)
0.14
Fuel used in cooking- kerosene/ biomass/ coal [n (%)]
18 (11.5)
89 (14.2)
0.08
Place of cooking inside the house [n (%)]
146 (93)
583 (92.8)
0.6
No. Results A total of 785 participants were included in the study:
260 from north India (Delhi) and the rest from south India
(250 from Hyderabad and 275 from Bengaluru). There
were no refusals. Nearly equal proportion of males and
females were present in the study group, and there was
nearly equal distribution (48.4%) of primiparous and mul-
tigravida (51.6%) mothers. Most of the mothers were liter-
ate. Most households (452; 57.6%) belonged to backward
castes, and 63.8% had family income less than one lakh
Indian rupees (1500 USD) per year. A large proportion
(86%) of women were homemakers. Approximately 7% of
fathers worked in agriculture. The mean age at marriage
for mothers was 20.2 years (SD, 3.1), ranging from 10 to
37 years; 16.4% of the overall study population reported
consanguinity
or
marriage
with
relatives,
with
Delhi
(20%) reporting the maximum (Supplementary Table S1,
which is available online). ETHICAL CONSIDERATION We obtained informed consent from eligible mothers after
giving them detailed information about the study in local
language. We sought approvals from the respective hospi-
tals and Institutional Ethics committee of Public Health
Foundation of India. SOCIO-DEMOGRAPHIC CHARACTERISTICS OF CASES AND CONTROLS
The study had 157 matched case–control pairs (1:4). The
cases and controls were similar with respect to most
of the socio-demographic variables. In both the categories,
the majority of mothers did not have employment outside
the home and most fathers worked in office jobs, as man-
ual laborers or were in business. The majority among
cases and controls belonged to backward castes, and most
cases and controls had an annual family income less than
one lakh Indian rupees (Table 1). EXPOSURES confounders (irrespective of p value) were included in the
adjusted conditional logistic regression model. The risk factors included in the study were lack of folic
acid
supplementation
during
peri-conceptional
period
along with other documented risk factors, which were con-
ception induced by medications or treatment; exposure to
radiation (x-rays); infections; medical illness during preg-
nancy (such as epilepsy, hypertension, diabetes mellitus,
fever, rash, arthritis, common cold, urinary tract infection,
and vomiting); intake of any medicinal drugs; food habits
(vegetarian/nonvegetarian); consumption of coffee, alco-
hol, tobacco, toddy (local alcoholic drink) or any drugs;
exposure to smoking, indoor pollution, pesticides during
the peri-conceptional period; consanguineous marriage;
family history of any malformations; increased maternal
and paternal age (Jha, 2001; Agbenorku, 2013; Neogi
et al., 2015). Information was collected by interviewing
the women as well as by reviewing medical records wher-
ever feasible. The risk factors included in the study were lack of folic
acid
supplementation
during
peri-conceptional
period
along with other documented risk factors, which were con-
ception induced by medications or treatment; exposure to
radiation (x-rays); infections; medical illness during preg-
nancy (such as epilepsy, hypertension, diabetes mellitus,
fever, rash, arthritis, common cold, urinary tract infection,
and vomiting); intake of any medicinal drugs; food habits
(vegetarian/nonvegetarian); consumption of coffee, alco-
hol, tobacco, toddy (local alcoholic drink) or any drugs;
exposure to smoking, indoor pollution, pesticides during
the peri-conceptional period; consanguineous marriage;
family history of any malformations; increased maternal
and paternal age (Jha, 2001; Agbenorku, 2013; Neogi
et al., 2015). Information was collected by interviewing
the women as well as by reviewing medical records wher-
ever feasible. DATA COLLECTION AND MANAGEMENT of living children [mean (SD)]
1.7 (0.07)
1.6 (0.03)
0.07
H/o of consanguineous marriage
28 (17.8)
101 (16.1)
0.6
H/o of abortion
47 (30.0)
131 (20.8)
0.01
H/o neonatal death
5 (3.2)
18 (2.9)
0.83
H/o stillbirths
0
6 (1.0%)
Current multiple pregnancy
5 (3.2)
6 (0.9)
0.03
H/o multiple pregnancy in previous pregnancies
3 (1.9)
1(0.2)
0.00
H/o previous child with cleft lip
3 (1.9)
0
Any family history of cleft lip or palate
27 (17.2)
10 (1.6)
0.000
Any exposure to pesticide/ herbicides
56 (35.7)
235 (37.4)
0.6
More than 1 year for conception
106 (67.5)
324 (51.6)
0.000
H/o any treatment taken for infertility
24 (15.3)
32 (5.1)
0.00
Medical illnesses in the first 3 months of pregnancy
– Fever
23 (14.6)
85 (13.5)
0.71
– Rash
11 (7.0)
17 (2.7)
0.01
– Hypertension
6 (3.8)
15 (2.3)
0.32
– Epilepsy
2 (1.3)
3 (0.5)
0.263
– Arthritis/ chikungunya
1 (0.6)
8 (1.3)
0.50
– Urinary tract Infection
25 (15.9)
65 (10.3)
0.05
– Diabetes
1 (0.6)
2 (0.3)
0.56
H/o intake of medicine in first 3 months for any illness
76 (48.4)
276 (43.9)
0.3
H/o consumption folic acid
– 3 months before pregnancy
9 (5.7)
4 (0.6)
0.00
– First 3 months of pregnancy
48 (30.6)
136 (21.6)
0.02 BIRTH DEFECTS RESEARCH 109:1284–1291 (2017) 1287 TABLE 1. DATA COLLECTION AND MANAGEMENT Continued
Variables
Cases (n 5 157)
Control (n 5 628)
p-Value
H/o consumption of iron
– 3 months before pregnancy
7 (4.4)
5 (0.8)
0.001
– 3 months of pregnancy
70 (44.6)
181 (28.8)
H/o consumption of multivitamin during peri-conceptional period
47 (29.9)
134 (21.3)
0.02
H/o X ray in first 3 months of pregnancy
5 (3.2)
5 (0.8)
0.03
Food habit-vegetarianism
46 (29.3)
72 (11.5)
0.00
Exposure to passive smoking in peri-conceptional period
47 (29.9)
110 (17.5)
0.60
Exposure to active smoking
1 (0.6)
1 (0.2)
0.30
H/o chewing tobacco in peri-conceptional period
1 (0.6)
4 (0.6)
0.31
H/o consumption of any form of alcohol in periconceptional period
5 (3.2)
38 (6.0)
0.10
BIRTH DEFECTS RESEARCH 109:1284 1291 (2017)
1287 Cases (n 5 157)
Control (n 5 628)
p-Value Cases (n 5 157) p
– 3 months before pregnancy
7 (4.4)
5 (0.8)
0.001
– 3 months of pregnancy
70 (44.6)
181 (28.8)
H/o consumption of multivitamin during peri-conceptional period
47 (29.9)
134 (21.3)
0.02
H/o X ray in first 3 months of pregnancy
5 (3.2)
5 (0.8)
0.03
Food habit-vegetarianism
46 (29.3)
72 (11.5)
0.00
Exposure to passive smoking in peri-conceptional period
47 (29.9)
110 (17.5)
0.60
Exposure to active smoking
1 (0.6)
1 (0.2)
0.30
H/o chewing tobacco in peri-conceptional period
1 (0.6)
4 (0.6)
0.31
H/o consumption of any form of alcohol in periconceptional period
5 (3.2)
38 (6.0)
0.10 Factorsa that differed significantly between cases and controls (p < 0.05) (and having at least five respondents in each cate-
ological plausibility as potential confounders (irrespective of the p value). after conception did not seem to have a protective effect
in the final adjusted analysis (AOR, 1.2; 95% CI, 0.6–2.6). Age of child emerged as a strong factor (AOR, 34.3; 95%
CI, 16.7–70.4), reflecting the fact that cases were older at
the time of interview than the controls. (21.6%) during first 3 months of pregnancy. Almost one-
third of the mothers of cases were vegetarians compared
to 11% of mothers of controls. Among the exposures examined, history of previous
abortion, family history of OFC, more than 1 year taken
for conception, history of any treatment taken for infertil-
ity, history of rash, urinary tract infection in first 3 months
of pregnancy, history of consumption of folic acid or iron
and folic acid in first 3 months of pregnancy, multivitamin
consumption, vegetarianism, backward caste, and age of
the child at the time of interview were found to be asso-
ciated (p < 0.05 in unadjusted analysis) with OFCs
(Table 2). STATISTICAL ANALYSIS STATA 11.0 was used for analysis. The socioeconomic char-
acteristics of cases and controls were compared using the
unpaired t test for continuous variables and the Chi-
square test for categorical variables. Associations between
exposures and outcomes were assessed for case–control
pairs using unadjusted and adjusted conditional logistic
regression. For the adjusted analysis, exposures that had
different distributions in cases and controls (p < 0.05)
(and with at least five respondents in each category), and
those
having
a
biological
plausibility
as
potential Association between exposures and OFC. A total of 13 women out
of 785 reported consuming folic acid during 3 months
preceding conception. However, more mothers of cases
reported taking folic acid (30.6%) compared with controls RISK FACTORS FOR OROFACIAL CLEFTS 1288 Factorsa
Unadjusted model
Final model
OR
P value
AOR
p-Value
H/o consumption folic acid in first 3 months of pregnancy
1.61 (1.07-2.41)
0.02
1.24(0.59-2.58)
0.56
H/o consumption of iron in first 3 months of pregnancy
4.80 (1.46-15.73)
0.01
3.29 (0.45- 23.84)
0.24
Food habit -vegetarianism
3.73 (2.32-5.99)
0.000
4.47 (1.83- 10.98)
0.001
Age of the child more than or equal to 14 days at time of interview
31.06(17.08- 56.52)
0.000
34.29 (16.70- 70.42)
0.000
Maternal age more than or equal to 30 years
0.87 (0.47-1.64)
0.68
0.86 (0.29-2.47)
0.77
Backward caste
2.01 (1.34-3.03)
0.001
1.92 (1.01- 3.68)
0.04
H/o of abortion
1.61 (1.06-2.43)
0.02
1.62 (0.75-3.52)
0.22
Current multiple pregnancy
3.33 (1.02-10.92)
0.05
0.6 3(0.07- 6.07)
0.69
Any family history of cleft lip or palate
12.93 (5.86-28.54)
0.000
15.48 (4.36- 54.96)
0.000
More than 1 year for conception
2.10 (1.42-3.12)
0.0001
2.55 (1.25- 5.21)
0.01
H/o any treatment taken for infertility
3.67 (2.05-6.58)
0.000
1.15 (0.42-3.19)
0.78
Medical illnesses in the first 3 months of pregnancy
– Rash
2.58 (1.21-5.52)
0.01
3.37 (0.84-13.49)
0.09
– Urinary Tract Infection
1.58 (0.94-2.65)
0.08
1.22 (0.43-3.47)
0.71
H/o X ray in first 3 months of pregnancy
4.93 (1.32-18.35)
0.02
10.66 (1.03- 110.10)
0.05
aFactors included are those that differed significantly between cases and controls (p < 0.05) (and having at least five respondents in each cate-
gory), and those having a biological plausibility as potential confounders (irrespective of the p value). Discussion Vegetarian populations may be more
exposed to pesticide residues (except for organochlorine
compounds) than the general population (Van Audenhaege
et al., 2009). Nonvegetarians may also be exposed to con-
centrated forms of pesticides because animals eat plant
products, and fat soluble contaminants or those that are
resistant to degradation accumulate in the fat tissues that
ultimately reach humans (Dorea, 2004). It is also important to note that in India supplementation
usually starts only after the first antenatal visit, which usu-
ally takes place after the missed period or approximately 3
to 4 weeks after conception. The fetal lip usually closes by 5
to 6 weeks after conception and the palate by 10 weeks. This gives a narrow or no window of opportunity for peri-
conceptional folic acid supplementation to be administered
and to be effective. The reported intake of supplementation
in the pre-conceptional period was low (5.7% among moth-
ers of cases and 0.6% among controls). Reported intake dur-
ing the first 3 months of pregnancy was higher, 30.6%
among cases and 21.6% among controls, but we have no
details on when they initiated supplementation. One of the limitations of our study was that the data
were primarily based on recall. There is generally no doc-
umentation or medical records available on the frequency
and duration of folic acid or multivitamin supplementation
in our settings. Multivitamin preparations are available in
diverse formulations, these may or may not contain folic
acid, and people tend to interpret supplements as anything
that is given for strength and immunity. These were, there-
fore, excluded from the final analysis. On the other hand,
folic acid preparations are uniform and it is relatively eas-
ier to elicit information from mothers because of its con-
sistency in color and size. Moreover, when Indian women
visit a doctor or a facility in the first trimester of preg-
nancy, any medicine prescribed during the first visit is less
likely to be forgotten. Nonetheless, there could be a prob-
lem with recall bias in both cases and controls giving rise
to nondifferential misclassification of folic acid status. Moreover, in our study, cases were interviewed when they
came for surgical intervention while controls were inter-
viewed 48 hr after the delivery. This could have intro-
duced a selection bias because we selected cases whose
parents sought care. Discussion Previous studies on the association of multivitamins
with OFCs have reported mixed findings, although multivi-
tamin preparations containing folic acid have suggested a
protective effect (Czeizel, 1993; Bailey and Berry, 2005). The independent role of folic acid is still unclear (Shaw
et al., 1995; Itikala et al., 2001). Lack of preventive effect
by folic acid supplementation was reported in some stud-
ies with a large sample size of over 3000 (Hayes et al.,
1996; Czeizelet al., 1999, 2004). Other studies suggested
that folic acid was effective only at higher doses (6–8 mg)
(Czeizel et al., 1999). One study suggested efficacy of mul-
tivitamin and folic acid supplementation in unilateral cleft
with a 83% risk reduction, while no effect was observed
for bilateral cleft (Tolarova and Harris, 1995). A Cochrane
review conducted in 2010 suggests that there is no evi-
dence of any effect of folic acid supplementation on pre-
vention of cleft palate and/or cleft lip (De-Regil et al.,
2010). Yet another study suggests that fortification with
folic acid may create an imbalance between folic acid and
vitamin-B12, further aggravating the risk of having a child
with OFCs (Godbole et al., 2009). Cases and controls should be chosen from the same base
population to reduce selection bias in case–control studies. Evidence shows that for hospital-based case–control stud-
ies, community controls are the best choice if high response
rates can be attained (Neupane et al., 2010). However, con-
sidering the budget constraints for any public health
research in resource-scarce settings, researchers may con-
sider selecting hospital controls for a case–control study if
potential confounders are carefully considered, measured,
and adjusted for (Rahman et al., 2012). In our study, al-
though we attempted to select controls from the same geo-
graphical regions from their addresses, they might not have
represented the population that gave rise to cases. There is,
therefore, a possibility of some degree of selection bias in
our study. Of interest, vegetarianism as compared to nonvegetarian
diet emerged to be strongly associated with OFC in our
study. Women having vegetarian diets have low folate and
vitamin B12 levels (Kirchheimer et al., 2001; Refsum et al.,
2001) and a diet deficient in folate and vitamin B12 was
shown to be related with OFC (Krapels et al., 2004). Most
vegetarians in India adopt traditional cooking methods with
overexposure of food to heat. Discussion In this case–control study of OFCs, we found that a family
history of cleft lip/palate, vegetarianism, and delayed first
conception beyond 1 year were associated with a higher
risk of OFCs. We did not find any evidence to support a
protective association with folic acid supplementation dur-
ing first 3 months of pregnancy. A model of multivariable analysis was developed using
conditional logistic regression (Table 2). In the final model,
a family history of cleft lip/palate, exclusive vegetarianism
and delayed first conception beyond one year were associ-
ated with OFCs. Cases had 15 times higher likelihood of
having a family history than controls (adjusted odds ratio
[AOR], 15.5; 95% confidence interval [CI], 4.4–54.9). Like-
wise, cases were around 4.5 times more likely than con-
trols to be exclusive vegetarians (AOR, 4.5; 95% CI, 1.8–
10.9). Cases were more likely to be born after a delayed
conception compared with controls (AOR, 2.55; 95% CI,
1.25–5.21). Supplementation of folic acid 3 months before
conception had to be excluded from the adjusted analysis
owing to small cell sizes. Supplementation for 3 months A family history of OFC is a strong risk factor for
nonsyndromic OFCs as shown in previous studies (Acuna-
Gonzalez et al., 2011; Rahimov et al., 2012; Burg et al.,
2016). In our study, the strength of association was as
high as 15 but with very wide CIs. Our findings are con-
sistent with other studies (effect size varying from 17 to
56) (Sivertsen et al., 2008; Grosen et al., 2010). Delayed
first conception beyond 1 year emerged as a risk factor in
our analysis. Treatment received for infertility was associ-
ated with OFCs in unadjusted analysis but did not show
any association in the adjusted analysis. In the analysis, supplementation with multivitamin or
folic acid preparations in the peri-conceptional period did
not seem to have any protective effect. However, these BIRTH DEFECTS RESEARCH 109:1284–1291 (2017) 1289 findings should be interpreted in the Indian context where
any preparations consumed for “strength and vitality” are
often termed as “multivitamin preparations” by the people. So, self-reported consumption of multivitamins could in-
clude either multivitamins, folic acid, iron preparations, or
indigenous medicines as well. (Wardlaw, 2004). A vegetarian diet may also increase expo-
sure to pesticides through agricultural use although the evi-
dence is equivocal. References Acuna-Gonzalez G, Medina-Solis CE, Maupome G, et al. 2011. Family history and socioeconomic risk factors for non-syndromic
cleft lip and palate: a matched case-control study in a less devel-
oped country. Biomedica 31:381–391. On the other hand, factors related to medical conditions
are less likely to be missed because every subject was
enrolled from the hospitals where these details are recorded
routinely. Gathering information from medical records leads
to less recall bias. Information on diet is highly subject to
bias because it is likely to get altered during the course of
pregnancy. So, we restricted ourselves only to analyzing
whether the person was vegetarian or nonvegetarian. Vege-
tarianism is strongly linked with sociocultural customs and
generally remains unaltered in Indian culture. The possibil-
ity of a vegetarian person getting misclassified as a nonvege-
tarian is much less, although the converse may be true. However, triangulation was done to overcome this issue by
asking mothers about the frequency of intake of nonvegetar-
ian food before and after pregnancy. Given the limitations of
recall, we could not categorize participants into those con-
suming non vegetarian food daily, weekly, or occasionally,
and we combined them in one group. Agbenorku P. 2013. Orofacial clefts: A worldwide review of the
problem. Available at https://www.hindawi.com/journals/isrn/
2013/348465/. Accessed May 8, 2017. Allagh KP, Shamanna BR, Murthy GV, et al. 2015. Birth preva-
lence of neural tube defects and orofacial clefts in India: a sys-
tematic review and meta-analysis. PLoS One 10:e0118961. Aylsworth AS, Allori AC, Pimenta LA, et al. 2015. Issues involved in
the phenotypic classification of orofacial clefts ascertained through
a state birth defects registry for the North Carolina Cleft Outcomes
Study. Birth Defects Res A Clin Mol Teratol 103:899–903. Bailey LB, Berry RJ. 2005. Folic acid supplementation and the
occurrence of congenital heart defects, orofacial clefts, multiple
births, and miscarriage. Am J Clin Nutr 81:1213S–1217S. Bianchi F, Cianciulli D, Pierini A, Seniori Costantini A. 1997. Con-
genital malformations and maternal occupation: a registry based
case-control study. Occup Environ Med 54:223–228. To our knowledge, this is one of only a few case–control
studies evaluating risk factors of OFC in a largely vegetarian
population. Although, we assumed that there would be a 5%
nonresponse rate, in our study there were no refusals which
adds to its strength. It was conducted in two geographically
and culturally different (north India vs. south India) areas
within India. References This increased the external validity of the study. Cases were taken based on physician’s diagnosis only and all
the subjects were recruited from big hospitals where the sys-
tem of recording is better than many hospitals of the country. Our team reviewed medical records and collected information
from them for most of the risk factors studied, minimizing
information bias as far as possible. In addition, having four
controls for every case improved the power and efficiency of
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was underpowered to detect small differences. The finding
of higher risk among vegetarians requires further explora-
tion and research. More studies are needed to establish the
role of vitamin B12 alone or in combination with folic acid
in prevention of OFCs especially in Indian population where
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most of them are exclusive vegetarians and, therefore, prone
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increase risk of OFCs as vitamin B12 plays a role in many
enzymatic
reactions
linked
to
folic
acid
metabolism There were no disaggregated data on the type of OFC;
literature suggests that determinants (both environmental
and genetic etiologies) of OFC vary with the type (Bianchi
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and contributed significantly. to the mothers and families who participated in the study
and contributed significantly. likelihood of people not revealing the actual caste, we com-
bined all backward castes as a single category and com-
pared it against nonbackward castes in the final analysis. Sociodemographic factors, however, are less amenable to
change during the course of pregnancy and, hence, unlikely
to be affected by recall or misclassification bias. Acknowledgments The authors acknowledge the contribution of doctors,
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ORIGINAL RESEARCH
published: 28 February 2022
doi: 10.3389/fpsyg.2022.847278 Parent–Child Relationship Quality
and Internet Use in a Developing
Country: Adolescents’ Perspectives
Thao Thi Phuong Nguyen 1,2*, Tham Thi Nguyen 1,2, Ha Ngoc Do 3, Thao Bich Thi Vu 3,
Khanh Long Vu 3, Hoang Minh Do 3, Nga Thu Thi Nguyen 4, Linh Phuong Doan 1,2,
Giang Thu Vu 5, Hoa Thi Do 6, Son Hoang Nguyen 5, Carl A. Latkin 7, Cyrus S. H. Ho 8,9 and
Roger C. M. Ho 9,10 1 Institute for Global Health Innovations, Duy Tan University, Da Nang, Vietnam, 2 Faculty of Medicine, Duy Tan University,
Da Nang, Vietnam, 3 Youth Research Institute, Ho Chi Minh Communist Youth Union, Hanoi, Vietnam, 4 Faculty of Social
Sciences and Humanities, Hanoi Metropolitan University, Hanoi, Vietnam, 5 Center of Excellence in Health Services and
System Research, Nguyen Tat Thanh University, Ho Chi Minh City, Vietnam, 6 Institute of Health Economics and Technology
(iHEAT), Hanoi, Vietnam, 7Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, United States,
8 Department of Psychological Medicine, National University Health System, Singapore, Singapore, 9 Department of
Psychological Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore,
10 Institute for Health Innovation and Technology (iHealthtech), National University of Singapore, Singapore, Singapore Edited by:
Quan-Hoang Vuong,
Phenikaa University, Vietnam Objective: The goal of the study was to explore the relationship between parent–children
relationships related to using the internet among kids and potentially associated factors. Objective: The goal of the study was to explore the relationship between parent–children
relationships related to using the internet among kids and potentially associated factors. Reviewed by:
Quy Khuc,
Vietnam National University,
Hanoi, Vietnam
Minh-Hoang Nguyen,
Phenikaa University, Vietnam Reviewed by:
Quy Khuc,
Vietnam National University,
Hanoi, Vietnam
Minh-Hoang Nguyen,
Phenikaa University, Vietnam Reviewed by:
Quy Khuc,
Vietnam National University,
Hanoi, Vietnam
Minh-Hoang Nguyen,
Phenikaa University, Vietnam Materials and Methods: A sample of 1.216 Vietnamese students between the ages of
12 and 18 agreed to participate in the cross-sectional online survey. Data collected
included socioeconomic characteristics and internet use status of participants, their
perceived changes in relationship and communication between parents and children since
using the internet, and parental control toward the child’s internet use. An Ordered Logistic
Regression was carried out to determine factors associated with parent–children
relationship since using the internet. *Correspondence:
Thao Thi Phuong Nguyen
nguyentphuongthao73@duytan.edu.vn Results: The characteristics of the relationship between children and their parents since
using the Internet were divided into three levels: deterioration (7.0%), stability (78.2%),
and improvement (14.8%). The topics that children most often communicate with their
parents include learning, housework, and future directions. Two-way interactive activities,
such as supporting parents to use the Internet, have a positive impact on the parent–child
relationship. Stubborn parental control, such as establishing rules about contact or allowing
Internet access and setting up global positioning system (GPS) to track negatively affecting
parent–child relationships. Specialty section:
This article was submitted to
Health Psychology,
a section of the journal
Frontiers in Psychology Specialty section:
This article was submitted to
Health Psychology,
a section of the journal
Frontiers in Psychology Specialty section:
This article was submitted to
Health Psychology,
a section of the journal
Frontiers in Psychology
Received: 01 January 2022
Accepted: 02 February 2022
Published: 28 February 2022 Received: 01 January 2022
Accepted: 02 February 2022
Published: 28 February 2022 Keywords: internet addiction, internet use, parent–child relationship, children, Vietnam Edited by:
Quan-Hoang Vuong,
Phenikaa University, Vietnam INTRODUCTION control might lead to several mental health issues in children
such as low self-esteem (Younes et al., 2016; Shi et al., 2017),
depression (Younes et al., 2016; Fayazi and Hasani, 2017),
and antisocial behaviors (Barber et al., 2012), which triggered
excessive internet use in children as an adapting way for
satisfying their psychological needs (Yu et al., 2012). Meanwhile,
children who had a high relationship quality with their parents
were found to be more likely to have emotional stability,
better social competence, and fewer behavioral issues (Schneider
et al., 2001). Several publications demonstrated a negative
association between adolescents’ internet addiction and having
an open parent–child relationship (Zhang et al., 2011;
Zhu et al., 2015).h Using the internet has become one of the most popular leisure-
time activities worldwide. In Vietnam, since the internet was
officially connected in 1997, internet users sharply increased
and reached 68.72 million as of January 2021, equivalent to
70.3% of the population. The average internet use/access time
of Vietnamese was 6.5 h every day. Notably, 70.1% of the
internet users in Vietnam were reported to be aged 13–34
(Digital 2021: Vietnam: Datareportal, 2021). A 2016 study
showed that 72% of Vietnamese aged 15–24 used the internet,
of which 74% were reported having the high-risk danger by
cyberbullying (The UNICEF, 2017). The popularity of the
internet among adolescents poses challenges to parents in
protecting their children from excessive internet use and negative
influences from the cyber world (Subrahmanyam et al., 2000;
Greenfield, 2004). Excessive internet use may exhibit similar
patterns and have similar negative consequences to the life of
youths and adolescents compared to other addictive behaviors
(Shapira et al., 2003; Young, 2004). From the parents’ perspective,
the attachment of youths and adolescents to the internet may
lead to the passivity in the young people’s daily activities,
changes in their psychological states and behaviors, and negative
effects on the quality of the parent–child relationship (van
Den Eijnden et al., 2010; Shek et al., 2018; Restrepo et al., 2020;
Li et al., 2021). The research landscape concerning internet use behaviors
of Vietnamese adolescents has expanded significantly in recent
years, in light of the accelerating popularity of the internet
among this population (Sobowale et al., 2016; Tran et al., 2017;
Zhang et al., 2017; Lan et al., 2020). INTRODUCTION Negative effects of excessive parental Existing literature argued that parents play a vital role in
the management of children’s internet use (Shi et al., 2017). Previous studies found an inverse association between children’s
internet problematic use and parental monitoring and bonding
(Siomos et al., 2012; Ding et al., 2017). Meanwhile, the relationship
between parental control and children’s behaviors appears to
be more complicated. Barber et al. (2005) reckoned that parental
control could be in the forms of behavioral or psychological
control. Behavioral control referred to the use of explicit
management strategies by parents including children’s activities
supervision, establishing rules, and restrictions to regulate the
children’s behavior (Shek, 2005), while psychological control
manifested through implicitly adjusting children’s behavior based
on their emotions and thoughts. Psychological tactics that were
used by parents included guilt infusion, love withdrawal, and
authority assertion (Smetana and Daddis, 2002; Rogers et al.,
2003). The existing scientific findings have consistently shown
that parental psychological control diminished child development
and increased parent–child conflicts, while behavioral control
positively related children’s developmental outcomes and well-
being (Barber et al., 2005; Wang et al., 2007; Bleakley et al., 2016). C
i
hild
’
i
i t
t
t di Citation: Nguyen TTP, Nguyen TT, Do HN,
Vu TBT, Vu KL, Do HM, Nguyen NTT,
Doan LP, Vu GT, Do HT, Nguyen SH,
Latkin CA, Ho CSH and
Ho RCM (2022) Parent–Child
Relationship Quality and Internet Use
in a Developing Country:
Adolescents’ Perspectives. Front. Psychol. 13:847278. doi: 10.3389/fpsyg.2022.847278 Conclusion: Findings indicated that changes in the quality of the parent–child relationship
were self-assessed by participants regard to kids’ internet use, especially in the COVID-19
epidemic context. Educational campaigns and programs to raise awareness of parents as
to the dangers and negative influences that their children may encounter online, psychology
of children’s behaviors and effects of different responding strategies are recommended. February 2022 | Volume 13 | Article 847278 1 Frontiers in Psychology | www.frontiersin.org Parent-Child Relationship and Internet Use Nguyen et al. INTRODUCTION Furthermore, in the
context of COVID-19 pandemic outbreaks, the Vietnamese
government implemented proactive measures to prevent
spreading, including school closures (La et al., 2020). During
the stay-at-home period, spending more time on virtual platforms
may influence the parent–children relationship (Cuong et al.,
2021). There have been several studies on internet use among
youths and adolescents in Vietnam (Sobowale et al., 2016;
Tran et al., 2017; Zhang et al., 2017; Lan et al., 2020; Cuong
et al., 2021). However, these studies had not offered a
comprehensive assessment of the effect of Vietnamese parental
management on adolescent Internet use, especially amidst the
pandemic. The current study aimed to (1) assess the relationship
between parent–children relationships related to using the
internet among kids; and (2) identify potentially affected factors. Existing literature argued that parents play a vital role in
the management of children’s internet use (Shi et al., 2017). Previous studies found an inverse association between children’s
internet problematic use and parental monitoring and bonding
(Siomos et al., 2012; Ding et al., 2017). Meanwhile, the relationship
between parental control and children’s behaviors appears to
be more complicated. Barber et al. (2005) reckoned that parental
control could be in the forms of behavioral or psychological
control. Behavioral control referred to the use of explicit
management strategies by parents including children’s activities
supervision, establishing rules, and restrictions to regulate the
children’s behavior (Shek, 2005), while psychological control
manifested through implicitly adjusting children’s behavior based
on their emotions and thoughts. Psychological tactics that were
used by parents included guilt infusion, love withdrawal, and
authority assertion (Smetana and Daddis, 2002; Rogers et al.,
2003). The existing scientific findings have consistently shown
that parental psychological control diminished child development
and increased parent–child conflicts, while behavioral control
positively related children’s developmental outcomes and well-
being (Barber et al., 2005; Wang et al., 2007; Bleakley et al., 2016). Concerning children’s excessive internet use, some studies
found that children were less likely to get addicted to the
internet if their parents managed firmly in proximity and
consistent manner (e.g., establishing the rules for using the
internet, finding how to share internet information; Harakeh
et al., 2004; Van der Vorst et al., 2005). On the other hand,
some empirical evidence showed that the psychological control
of parents was positively associated with children’s internet
addiction (Giles and Price, 2008; Xiuqin et al., 2010;
Cheung et al., 2015). Frontiers in Psychology | www.frontiersin.org Socioeconomic Characteristic We asked participants a number of questions about their
information, including gender (male/female), grade (secondary
school/high school), current location (urban/rural), current
living
with
(parents/grandparents/siblings),
academic
performance (excellent/good/fair/moderate/below moderate),
and the number of family members that participants can
confide in. Measurement and Instrument In this study, we conducted an online questionnaire on
SurveyMonkey’s platform1. This approach yields efficiencies,
such as saving time, reducing cost, and reaching a large of
samples. A structured questionnaire including five major
components: (1) General characteristics; (2) Parent–child
relationship since participants used the internet; (3) Children’s
internet use status; (4) Interactive and supportive activities
regarding internet use between parents and children; and (5)
Social support. A pilot survey was conducted by 10 people
for ensuring logicality and adjusting questions with unclear
before collecting data. Participants spend 20 min completing
the informed consent and questionnaires. Based on “EU Kids Online 2017, questions for children
and young people” and “Children Online in the European
Union 2020” to develop five questions to explore about
activities of students with their parents on the Internet (EU
Kids Online 2017, 2020; The EU kids online network, 2020),
including: Children’s Communication With Their Parents Since
Using the Internet of sample size was added to prevent incomplete survey responses,
thus resulting in a total of 1,460 students being recruited. The
total of participants who agreed to involve in and completed
the questionnaire was 1,216 at the end of data collection period,
with the response rate was 83.2%. To assess the parent–children communication since using
the internet, we asked participants to collect information
about children’s attitude with their parents, and the frequency
of talking to their parents about daily life. In particular,
the question “Do you ignore advice from your parents
about using the internet?” (Yes/No) was used to explore
the children’s attitude toward their parents. Furthermore,
nine popular topics about children’s daily life, such as
future orientation, learning, housework, daily living habits,
hobby, privacy at home, relationship with friends, the
selection of clothing, and the problem of pocket money
were developed to assess the parent–child interaction. For
each topic, a Likert-five scale from 1 (Never) to 5 (Always)
was used to report the frequency of parent communication
in daily life. The Cronbach’s alpha of these questions
was 0.753. Relationship Between Children and Their Parents Since
Using the Internet Relationship Between Children and Their Parents Since
Using the Internet A question “How the relationship between you and your parents
has changed since using the internet?” was used to assess the
internet affects family relationships. The response options for
this question were divided into three levels: deteriorated, constant,
and better relationship. 1. Helping parents when they have difficulty using the Internet. 2. Ask for help from parents when the situation is difficult
to handle on the Internet. 3. Seek advice from parents on what to do on the Internet. 4. Talk about what you do on the Internet with parents. 5. Talk to parents about what is bothering them on the Internet. 1. Helping parents when they have difficulty using the Internet.fi 1. Helping parents when they have difficulty using the Internet.fi fi
2. Ask for help from parents when the situation is difficult
to handle on the Internet. 2. Ask for help from parents when the situation is difficult
to handle on the Internet. 3. Seek advice from parents on what to do on the Internet. 4 T lk
b
t
h t
d
th
I t
t
ith
t y
5. Talk to parents about what is bothering them on the Internet. 5. Talk to parents about what is bothering them on the Internet. Covariates 6. For each question, the response options ranged from 1
(Never) to 5 (Always). The Cronbach’s alpha was 0.825. 6. For each question, the response options ranged from 1
(Never) to 5 (Always). The Cronbach’s alpha was 0.825. Study Design, Sampling Method, and Data
Collection From June to July 2020, an online cross-sectional study was
conducted in Vietnam. The eligibility criteria for participating
in this survey were: (1) studied from grade 6 to grade 12;
(2) currently lived in Vietnam, and (3) agreed to join this
study by providing online informed consent. We recruited
participants via convenience sampling from four cities/provinces
undergoing rapid urbanization, including Can Tho, Ba Ria-Vung
Tau, Hung Yen, and Thanh Hoa. Participants who suffered
from serious illnesses or could not answer questions were
excluded from the recruitment process.h Concerning children’s excessive internet use, some studies
found that children were less likely to get addicted to the
internet if their parents managed firmly in proximity and
consistent manner (e.g., establishing the rules for using the
internet, finding how to share internet information; Harakeh
et al., 2004; Van der Vorst et al., 2005). On the other hand,
some empirical evidence showed that the psychological control
of parents was positively associated with children’s internet
addiction (Giles and Price, 2008; Xiuqin et al., 2010;
Cheung et al., 2015). Negative effects of excessive parental The formula to estimate a population proportion was used
to calculate the sample size with specified relative precision. With confidence level (%) α = 0.05, expected population
proportion with better relationship with parent p = 34.0%
(according to a previous study in Egypt; Moawad and Ebrahem,
2016), and relative precision ε = 0.15, the sample size estimated
to participate by 332 participants per study sites. The total
sample size in four research sites in Vietnam was 1,328. After
calculating the total sample size of four research sites, 10% February 2022 | Volume 13 | Article 847278 Frontiers in Psychology | www.frontiersin.org 2 Parent-Child Relationship and Internet Use Nguyen et al. Children’s Communication With Their Parents Since
Using the Internet 1surveymonkey.com The Parental Management and Care With Internet Use
in Children Based on the survey “EU Kids Online 2017, Questions for
children and young people” (EU Kids Online 2017, 2020), a
series of questions about parental care and management were
developed, including: The Status of Using the Internet Among Participants
Participants were asked to report about time using the internet
per day. Additionally, we based on “EU Kids Online 2017”
to build four questions about the frequency of using the internet
at four places: Family, school, friend’s house, and public areas. For each question, a Likert-seven scale from 1 (Never) to 7
(Almost at the time) was used to collect information from
participants (EU Kids Online 2017, 2020). The total score of
four questions was summed to assess the frequency of using
the internet among students, with the higher score indicated
that the frequency of using the internet among participants
was higher. The Cronbach’s alpha of these questions
was 0.64. Parental Management With Internet Use in Children. We
developed 10 questions to collect information, including: de e oped
0 quest o s to co ect
o
at o ,
c ud
g:
1. Rules about how long I am allowed to go online
2. Rules about when I am allowed to go online
3. Knows about what I look at on the internet and the social
media
4. Rules about who I can contact on social networks
5. Checks my messages on the social media
6. Checks my information on the Internet
7. Technology to track where I am [such as global positioning
system (GPS)]
8. Check out content what I look at on the internet
9. Check out adding my friends and friends list
10. Limits content of what I can see on the internet 1. Rules about how long I am allowed to go online g
g
2. Rules about when I am allowed to go online 3. Knows about what I look at on the internet and the social
media 4. Rules about who I can contact on social networks 5. Checks my messages on the social media 7. Technology to track where I am [such as global positioning
system (GPS)] 8. Check out content what I look at on the int 8. Check out content what I look at on the internet
9. Check out adding my friends and friends list 9. Check out adding my friends and friends list 10. Statistical Analysis y
Both descriptive and analytical statistics were used to address
the main aims of the study by STATA version 16. When screening
data, we used the Listwise Deletion method to clean data before
analyzing it. Listwise Deletion means that any individual in a
data set was excluded from the model if they were missing
data on any variable in the analysis. Continuous variables were
presented as mean and SD, while categorical variables were
presented as frequencies with percentages. In this study, to
compare differences in the parent–children relationship among
participants, the Kruskal-Wallis test for continuous variables and
the χ2 test were used for categorical variables. Multivariate Logistic
Regression was conducted to determine potential predictors of
the parent–child relationship among Vietnamese students
(Deteriorates = 1; and Constant/Better = 0). To identify potential
predictors related to the relationship between parents and children,
firstly, we selected according to univariate analysis, and variables
with value of p ≤ 0.25 were included in the multivariate logistic
regression model (Hosmer et al., 2000). Subsequently, these
potential covariates could be used in multivariate logistics
regression model, including socio-economic characteristics,
parent–children communication, parental management and care,
and social support. Furthermore, we used Hosmer-Lemeshow
test and some criteria, such as R-square, AIC, and BIC to assess Relationship Between Parents and
Children Since Using Internet Multidimensional Scale of Perceived Social Support
Multidimensional Scale of Perceived Social Support (MSPSS)
was a brief, and easy to administer self-report questionnaire
which consisted of 12 items. This scale was used to the extent
to which an individual perceives social support from three
sources: significant others (four items), family (four items),
and friends (four items; Wongpakaran et al., 2011). For each
item, a seven-point scale from 1 (very strongly disagree) to
7 (very strongly agree) was used (Zimet et al., 2020). The
total score of the four items shows that the higher the score,
the higher the degree of support. The Cronbach’s alpha was
excellent at 0.905. Table 1 described the characteristics of the relationship
between children and their parents since they started using
the internet. Participants reported having either deteriorated
(7.0%), constant (78.2%), and better (14.8%) relationship with
their parents. Participants who reported deteriorating
relationships with parents were mainly female (62.4%), high
school students (70.6%), living in a nuclear family (92.9%),
having an academic performance at fair levels (38.1%). Meanwhile, secondary school students (57.8%), having
excellent/good academic performance (68.1%) showed a better
relationship with parents. In addition, most people who had
a bad relationship with their parents ignored advice about
internet use from their parents (91.8%) and had limited
internet access (78.8%). People having deteriorated relationship
with parents spent significantly more time on the internet
compared to those reported having a constant or better
relationship. In particular, the internet use frequency of
students having deteriorated relationships with their parents
was significantly higher than others. The differences were
statistically significant with p < 0.05. The Parental Management and Care With Internet Use
in Children Limits content of what I can see on the internet 10. Limits content of what I can see on the internet February 2022 | Volume 13 | Article 847278 Frontiers in Psychology | www.frontiersin.org 3 Parent-Child Relationship and Internet Use Nguyen et al. Parental Care With Internet Use in Children. Eight questions
were used to collect data, including: Parental Care With Internet Use in Children. Eight questions
were used to collect data, including: Parental Care With Internet Use in Children. Eight questions
were used to collect data, including: the goodness of fit for multivariate logistic regression model
and select the best models in this study. These models were
then combined with the stepwise forward strategies to reduce
models with p < 0.2 as the threshold for including variables
(Hosmer et al., 2013). A value of p < 0.05 was considered
statistically significant. 1. Explains why some online content is good or bad 1. Explains why some online content is good or bad 1. Explains why some online content is good or bad 2. Encourages me to explore and learn things on the Internet 3. Talks to me about what I do on the internet 4. Suggests ways to use the internet safely 5. Helps me when something bothers me on the internet Ethical Consideration 6. Talks to me about what to do if something online bothers
or upsets me All procedures performed in studies involving human participants
were in accordance with the ethical standards of the Youth
Research Institute, Vietnam, and with the 1964 Helsinki Declaration
and its later amendments or comparable ethical standards. Online
Informed consent was obtained from all participants. 7. Helps me when something is difficult to do or find on
the internet 7. Helps me when something is difficult to do or find on
the internet 8. Do sharing activities with me on the internet For all of the above questions, a Likert-five scale was used,
with ranging options from 1 (Never) to 5 (Always). Additionally,
the Cronbach’s alpha values of the questions about parental
management and care were good at 0.857, and 0.859, respectively. Frontiers in Psychology | www.frontiersin.org Communication and Interaction Between
Parents and Children Related to Internet
Use Children’s communication with their parents since using the
internet was shown in Table 2. Learning, housework, and
future orientation were the topics that students communicated
most frequently with their parents. “Helping parents when they
have difficulty using the internet” was the most popular activity
that children do with their parents on the Internet. The majority
of participants having a good relationship with parents showed
the highest frequency of communication about all topics, except
for pocket money issues. Similarly, with the mutual support
in internet use, participants having a good parent–child
relationship were higher frequency of interactive and supportive
activities than the other groups. These differences were statistically
significant with p < 0.05. February 2022 | Volume 13 | Article 847278 Frontiers in Psychology | www.frontiersin.org 4 Parent-Child Relationship and Internet Use Nguyen et al. The Management and Care of Parents
With Children’s Internet Use
The parent’s management and care with the children’s internet
management with their internet use, particularly with rules
about how long (2.9 points)/when they were allowed access
internet (2.6 points). For the group having a better parent–child
TABLE 1 | Characteristics of relationship between parents and children since using internet. Communication and Interaction Between
Parents and Children Related to Internet
Use Parent–children relationships since using the internet management with their internet use, particularly with rules
about how long (2.9 points)/when they were allowed access
internet (2.6 points). For the group having a better parent–child
relationship, the highest frequency of parent’s management was
controlling what children look at on the internet and social The Management and Care of Parents
With Children’s Internet Useh Communication and Interaction Between
Parents and Children Related to Internet
Use Characteristics
Parent–children relationships since using the internet
Total
Value of p
Deteriorates
Constant
Better
n
%
n
%
n
%
n
%
Total
85
7.0
951
78.2
180
14.8
1,216
100.0
Provinces
Ba Ria—Vung Tau
35
41.2
330
34.7
47
26.1
412
33.9
<0.01
Can Tho
12
14.1
184
19.4
54
30.0
250
20.6
Hung Yen
20
23.5
206
21.7
25
13.9
251
20.6
Thanh Hoa
18
21.2
231
24.3
54
30.0
303
24.9
Education
Secondary school
25
29.4
458
48.3
104
57.8
587
48.4
<0.01
High school
60
70.6
491
51.7
76
42.2
627
51.6
Gender
Male
32
37.6
446
46.9
95
52.8
573
47.1
0.07
Female
53
62.4
505
53.1
85
47.2
643
52.9
Current living
Nuclear family
79
92.9
766
80.5
142
78.9
987
81.2
0.05
Extended family
5
5.9
157
16.5
30
16.7
192
15.8
Others
1
1.2
28
2.9
8
4.4
37
3.0
Location
Rural
43
50.6
626
65.8
122
67.8
791
65.0
0.01
Urban
42
49.4
325
34.2
58
32.2
425
35.0
Academic performance
Excellent (≥ 9/10)
11
13.1
143
15.1
59
33.0
213
17.6
<0.01
Good (8–< 9/10)
22
26.2
399
42.1
69
38.5
490
40.5
Fair (7–< 8/10)
32
38.1
264
27.8
36
20.1
332
27.4
Moderate and below (<7/10)
19
22.6
142
15.0
15
8.4
176
14.5
Number of family members can confide in
None
3
3.5
31
3.3
0
0.0
34
2.8
0.19
One
61
71.8
675
71.0
131
72.8
867
71.3
Two and above
21
24.7
245
25.8
49
27.2
315
25.9
Ignore advice of parents
No/unknown
7
8.2
350
36.8
101
56.1
458
37.7
<0.01
Yes
78
91.8
601
63.2
79
43.9
758
62.3
Parents limit my Internet access
No/unknown
18
21.2
333
35.0
63
35.0
414
34.0
<0.01
Yes, a little
37
43.5
498
52.4
84
46.7
619
50.9
Yes, a lot
30
35.3
120
12.6
33
18.3
183
15.0
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Value of p
Time using the internet per day (hour)
5.6
3.3
3.9
2.5
4.0
2.6
4.1
2.6
<0.01
The frequency of using internet (range:
4–28)
16.5
4.6
14.3
4.4
13.2
4.7
14.3
4.5
<0.01
Family (range: 1–7)
5.5
0.9
5.3
1.1
5.1
1.4
5.3
1.1
0.03
School (range: 1–7)
3.0
1.9
2.3
1.7
2.1
1.4
2.3
1.7
<0.01
Friend’s house (range: 1–7)
3.8
1.8
3.5
1.8
3.0
1.8
3.5
1.8
<0.01
Public area (range: 1–7)
4.2
2.1
3.1
1.7
3.0
1.7
3.1
1.8
<0.01
Multidimensional Scale of Perceived Social Support (Unit: 1 score)
Social support score in friend domain
3.4
1.0
4.0
0.8
4.1
0.9
3.9
0.8
<0.01
Social support score in family domain
3.4
1.0
3.6
0.7
3.8
0.8
3.6
0.8
<0.01
Social support score in other domains
3.7
0.9
3.8
0.8
3.7
0.9
3.7
0.8
<0.01 TABLE 1 | Characteristics of relationship between parents and children since using internet. The Management and Care of Parents
With Children’s Internet Useh Characteristics
Parent–child relationships since using the internet
Total
Value of p
Deteriorates
Constant
Better
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Topics and frequency of parent–child communication (range: 1–5)
Learning
2.9
1.1
3.5
0.9
3.9
0.9
3.5
1.0
<0.01
Housework
3.2
1.1
3.3
1.1
3.4
1.3
3.3
1.1
0.14
Future orientation
2.9
1.3
3.1
1.2
3.6
1.3
3.2
1.3
<0.01
Relationship with Friend
2.8
1.0
2.8
1.1
3.3
1.2
2.9
1.1
<0.01
Daily living habits
3.0
1.4
2.9
1.2
3.1
1.3
2.9
1.3
0.04
Hobby
2.3
1.2
2.7
1.2
3.3
1.3
2.8
1.3
<0.01
The selection of clothing
2.4
1.1
2.7
1.1
3.0
1.2
2.7
1.1
<0.01
The problem of pocket money
2.8
1.3
2.4
1.2
2.3
1.2
2.4
1.2
0.01
Privacy at home
2.6
1.4
2.2
1.3
2.7
1.4
2.3
1.3
<0.01
Interactive and supportive activities regarding internet use between parents and children (range: 1–5)
Helping parents when they have difficulty using
Internet
3.2
1.3
3.5
1.2
3.9
1.1
3.5
1.2
<0.01
Ask for help from parents when the situation is
difficult to handle on the Internet
1.8
0.9
2.1
1.2
2.6
1.4
2.1
1.2
<0.01
Ask for advice from parents on what to do on the
Internet
1.5
0.6
2.0
1.1
2.6
1.4
2.1
1.4
<0.01
Talk about what you do on the Internet with
parents
1.8
1.0
2.1
1.1
2.7
1.4
2.1
1.1
<0.01
Talk to parents about what’s bothering them on
the Internet
1.5
0.8
2.0
1.1
2.5
1.4
2.0
1.1
<0.01 TABLE 2 | Topic, frequency communication and interactive activities regarding internet use between parents and children. TABLE 3 | The parent’s management and care with internet use in their children. TABLE 3 | The parent’s management and care with internet use in their children. The Management and Care of Parents
With Children’s Internet Useh Characteristics
Parent-children relationships since using the internet
Total
Value of
p
Deteriorates
Constant
Better
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Parent’s management with internet use in children (range: 1–5)
Rules about how long I am allowed to go online
2.9
1.5
2.5
1.3
2.6
1.3
2.6
1.3
0.14
Rules about when I am allowed to go online
2.6
1.3
2.2
1.2
2.5
1.3
2.3
1.2
<0.01
Knows about what I look at on the internet and social media
2.3
1.1
2.3
1.1
2.6
1.4
2.3
1.2
0.01
Rules about who I can contact on social networks
2.5
1.4
2.0
1.2
2.3
1.4
2.1
1.3
<0.01
Checks my messages on the social media
2.0
1.0
1.6
0.9
1.9
1.2
1.7
1.0
<0.01
Checks my information on the Internet
1.9
1.0
1.7
1.0
2.0
1.3
1.7
1.3
<0.01
Technology to track where I am (such as GPS)
2.4
1.3
1.5
0.9
1.8
1.1
1.6
1.0
<0.01
Check out content what I look at on the internet
1.9
1.1
1.6
0.9
1.8
1.2
1.6
1.0
0.01
Check out adding my friends and friends list
1.3
0.7
1.4
0.8
1.8
1.2
1.5
0.9
<0.01
Limits content of what I can see on the internet
1.6
1.0
1.4
0.8
1.7
1.3
1.4
0.9
<0.01
Parent’s care with internet use in children (range: 1–5)
Explains why some online content is good or bad
3.1
3.2
3.2
1.2
3.4
1.4
3.2
1.4
0.02
Encourages me to explore and learn things on the internet
2.4
1.3
2.8
1.3
3.3
1.4
2.8
1.3
<0.01
Talks to me about what I do on the internet
2.9
1.0
2.6
1.1
2.8
1.3
2.6
1.1
<0.01
Suggests ways to use the internet safely
2.2
1.1
2.5
1.3
2.7
1.4
2.5
1.3
0.03
Helps me when something bothers me on the internet
2.3
1.4
2.4
1.3
3.3
1.4
2.5
1.4
<0.01
Talks to me about what to do if something online bothers or upsets me
2.4
1.2
2.3
1.2
2.8
1.4
2.4
1.3
<0.01
Helps me when something is difficult to do or find on the internet
1.8
0.9
2.2
1.2
2.7
1.6
2.3
1.3
<0.01
Does shared activities with me on the internet
1.9
1.3
2
1.1
2.6
1.2
2.1
1.1
<0.01 Regarding parents’ care with internet use in children, the group
having better parent–child relationship gave higher scores about
all items than the others, excepted “talks to me about what
I do on the internet.” In contrast, the group having deteriorates
relationship with parents showed the item of “talks to me Regarding parents’ care with internet use in children, the group
having better parent–child relationship gave higher scores about
all items than the others, excepted “talks to me about what
I do on the internet.” In contrast, the group having deteriorates
relationship with parents showed the item of “talks to me media (2.6 points), and setting the rules of how long/when
they were allowed access internet. The Management and Care of Parents
With Children’s Internet Useh The parent’s management and care with the children’s internet
use were shown in Table 3. People having deteriorated and
better relationships with parents showed high levels of parental February 2022 | Volume 13 | Article 847278 Frontiers in Psychology | www.frontiersin.org 5 Parent-Child Relationship and Internet Use Nguyen et al. media (2.6 points), and setting the rules of how long/when
they were allowed access internet. Less managed activities
included checking access contents and friends on the internet,
and restricting what they can see on the Internet in all three
groups. The differences were statistically significant with p < 0.05. Regarding parents’ care with internet use in children, the group
having better parent–child relationship gave higher scores about
all items than the others, excepted “talks to me about what
I do on the internet.” In contrast, the group having deteriorates
relationship with parents showed the item of “talks to me
TABLE 2 | Topic, frequency communication and interactive activities regarding internet use between parents and children. The Management and Care of Parents
With Children’s Internet Useh Characteristics
Parent–child relationships since using the internet
Total
Value of p
Deteriorates
Constant
Better
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Topics and frequency of parent–child communication (range: 1–5)
Learning
2.9
1.1
3.5
0.9
3.9
0.9
3.5
1.0
<0.01
Housework
3.2
1.1
3.3
1.1
3.4
1.3
3.3
1.1
0.14
Future orientation
2.9
1.3
3.1
1.2
3.6
1.3
3.2
1.3
<0.01
Relationship with Friend
2.8
1.0
2.8
1.1
3.3
1.2
2.9
1.1
<0.01
Daily living habits
3.0
1.4
2.9
1.2
3.1
1.3
2.9
1.3
0.04
Hobby
2.3
1.2
2.7
1.2
3.3
1.3
2.8
1.3
<0.01
The selection of clothing
2.4
1.1
2.7
1.1
3.0
1.2
2.7
1.1
<0.01
The problem of pocket money
2.8
1.3
2.4
1.2
2.3
1.2
2.4
1.2
0.01
Privacy at home
2.6
1.4
2.2
1.3
2.7
1.4
2.3
1.3
<0.01
Interactive and supportive activities regarding internet use between parents and children (range: 1–5)
Helping parents when they have difficulty using
Internet
3.2
1.3
3.5
1.2
3.9
1.1
3.5
1.2
<0.01
Ask for help from parents when the situation is
difficult to handle on the Internet
1.8
0.9
2.1
1.2
2.6
1.4
2.1
1.2
<0.01
Ask for advice from parents on what to do on the
Internet
1.5
0.6
2.0
1.1
2.6
1.4
2.1
1.4
<0.01
Talk about what you do on the Internet with
parents
1.8
1.0
2.1
1.1
2.7
1.4
2.1
1.1
<0.01
Talk to parents about what’s bothering them on
the Internet
1.5
0.8
2.0
1.1
2.5
1.4
2.0
1.1
<0.01
TABLE 3 | The parent’s management and care with internet use in their children. The Management and Care of Parents
With Children’s Internet Useh Characteristics
Parent-children relationships since using the internet
Total
Value of
p
Deteriorates
Constant
Better
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Parent’s management with internet use in children (range: 1–5)
Rules about how long I am allowed to go online
2.9
1.5
2.5
1.3
2.6
1.3
2.6
1.3
0.14
Rules about when I am allowed to go online
2.6
1.3
2.2
1.2
2.5
1.3
2.3
1.2
<0.01
Knows about what I look at on the internet and social media
2.3
1.1
2.3
1.1
2.6
1.4
2.3
1.2
0.01
Rules about who I can contact on social networks
2.5
1.4
2.0
1.2
2.3
1.4
2.1
1.3
<0.01
Checks my messages on the social media
2.0
1.0
1.6
0.9
1.9
1.2
1.7
1.0
<0.01
Checks my information on the Internet
1.9
1.0
1.7
1.0
2.0
1.3
1.7
1.3
<0.01
Technology to track where I am (such as GPS)
2.4
1.3
1.5
0.9
1.8
1.1
1.6
1.0
<0.01
Check out content what I look at on the internet
1.9
1.1
1.6
0.9
1.8
1.2
1.6
1.0
0.01
Check out adding my friends and friends list
1.3
0.7
1.4
0.8
1.8
1.2
1.5
0.9
<0.01
Limits content of what I can see on the internet
1.6
1.0
1.4
0.8
1.7
1.3
1.4
0.9
<0.01
Parent’s care with internet use in children (range: 1–5)
Explains why some online content is good or bad
3.1
3.2
3.2
1.2
3.4
1.4
3.2
1.4
0.02
Encourages me to explore and learn things on the internet
2.4
1.3
2.8
1.3
3.3
1.4
2.8
1.3
<0.01
Talks to me about what I do on the internet
2.9
1.0
2.6
1.1
2.8
1.3
2.6
1.1
<0.01
Suggests ways to use the internet safely
2.2
1.1
2.5
1.3
2.7
1.4
2.5
1.3
0.03
Helps me when something bothers me on the internet
2.3
1.4
2.4
1.3
3.3
1.4
2.5
1.4
<0.01
Talks to me about what to do if something online bothers or upsets me
2.4
1.2
2.3
1.2
2.8
1.4
2.4
1.3
<0.01
Helps me when something is difficult to do or find on the internet
1.8
0.9
2.2
1.2
2.7
1.6
2.3
1.3
<0.01
Does shared activities with me on the internet
1.9
1.3
2
1.1
2.6
1.2
2.1
1.1
<0.01 TABLE 2 | Topic, frequency communication and interactive activities regarding internet use between parents and children. The Management and Care of Parents
With Children’s Internet Useh Less managed activities
included checking access contents and friends on the internet,
and restricting what they can see on the Internet in all three
groups. The differences were statistically significant with p < 0.05. Frontiers in Psychology | www.frontiersin.org February 2022 | Volume 13 | Article 847278 6 Parent-Child Relationship and Internet Use Nguyen et al. about what I do on the internet” reached highest score than
the other groups (2.9 points); the remaining items almost had
the lowest scores compared to the remaining groups. Overall,
people having a better relationship with parents had significantly
higher scores on most parental care items questions, except
the “Talks to me about what I do on the internet” question. These differences were statistically significant with p < 0.05. parents. It also implied that active family communication
significantly affected the healthy interaction of Internet access
and positive personal behavior.hi This study was conducted during the first year of the
COVID-19 epidemic in Vietnam; the government took proactive
measures to control its spread which included school closures
nationwide (La et al., 2020). Children and adolescents were
promoted to take classes as well as socialize more online. During the stay-at-home period, especially in the condition
of parental supervision, spending more time on virtual platforms
can make a young person feel upset, uncomfortable, and the
parent–child relationship may become more stressed (Tran
et al., 2020; Cuong et al., 2021). Therefore, our results found
that a part of participants reported perceiving relationships
with parents deteriorated related to using the internet. This
issue echoed the previous findings found that children’s internet
usage influences the amount of time spent with family (Jackson
et al., 2003). Moreover, our study also discovered that young
people who had a deteriorated parent–child relationship since
using the internet were likely to confide less and ignore parents’
advice. Negative parent–child communication was found to
have a significant association with the likelihood of Internet
addiction (Xu et al., 2014; Venkatesh et al., 2019). Strict parental
rules about internet access may promote compulsive tendencies
in children, which leads to the children’s opposition status
and decreased frequency of parental communication regarding
internet use (van Den Eijnden et al., 2010). Good communication
regarding children’s internet use has been found to be a
promising tool that parents can use to prevent their teenage
children from developing compulsive Internet use (van Den
Eijnden et al., 2010). Identifying Factors Related to the
Relationship Between Parents and
Children Since Using the Internet Factors related to the relationship between children and their
parents since using the internet were shown in Table 4. Individuals in Hung Yen province had a higher likelihood of
having deteriorated relationships with their parents (OR = 9.59;
95%CI: 3.25; 28.33), meanwhile, the opposite was true for
Thanh Hoa province (OR = 0.18; 95%CI: 0.05; 0.66). Females,
high school students, those having ignored advice from using
the internet from parents, and those having higher time using
the internet per day were more likely to have deteriorated
relationships with their parents. In terms of children’s
communication with their parents, a higher frequency of talking
to parents about learning (OR = 0.29; 95%CI: 0.19; 0.46), selecting
of clothing (OR = 0.34; 95%CI: 0.22; 0.51), as well as the higher
score of “helping parents when they have difficulty using the
internet” (OR = 0.64; 95%CI: 0.45; 0.92), and “ask for advice
from parents on what to do on the Internet” (OR = 0.43; 95%CI:
0.23; 0.80) were positively associated with the relationship
between children and parents. Regarding parental management
and care with internet use in children, higher score of some
parent’s activities such as talking to children about what they
do on the internet (OR = 1.71; 95%CI: 1.10; 2.66), helps children
when something bothers them on the internet (OR = 2.23;
95%CI: 1.46; 3.39), and using technology to track where children
are (OR = 6.92; 95%CI: 3.93; 12.16), rules about who children
can contact (OR = 1.82; 95%CI: 1.24; 2.68)/when children are
allowed to go online (OR = 1.93; 95%CI: 1.28; 2.93), and knows
about what I look at on the internet were increased likelihood
of having deteriorated relationships between parent and children. The Management and Care of Parents
With Children’s Internet Useh Some studies have also suggested that
parent–child communication, which can be enhanced by having
appropriate parental care and supports (Svetaz et al., 2014;
Jaggers et al., 2015; Shek et al., 2019) may be a protective
factor against problematic internet use in adolescents (Kim
and Kim, 2003). It is worth noting that, seemingly positive
parental control strategies like “talk to children about what
they do on the internet” or “help their children when something
bothers them on the internet” can be associated with parent–
child conflict, as our study found. This may be due to the
tendency of young people to express their concerns to people
rather than their parents, possibly because of existing lack of
parent–child communication (Văn, 2018). Thus, having
appropriate parental control strategies is essential in regulating
health internet usage in children, avoiding the vicious cycle
of negative control—impaired parent–child communication—
deteriorated parent–child relationship—unmet psychological
needs—more excessive internet use (Wang et al., 2007; Wang
and Fredricks, 2014; Lam, 2020). In Vietnamese society, the notion of “thuong cho roi
cho vot,” which means “spanking (with a rod) out of love Frontiers in Psychology | www.frontiersin.org DISCUSSION This research has deepened the understanding of the relationship
between parent–child relationship and children’s online behavior. Parental controls or supports of children’s internet use not
only affect the behaviors of the children but can also have
positive or negative consequences in the relationship between
family members. Our results confirmed that parental control
behaviors of their children’s internet use might affect the
children’s excessive internet use, academic performance results,
psychological status as well as the openness of communications
and sharing between parents and children. On the other hand,
our research found that participants who received encouragement,
help, and support from their parents when using the Internet
were more likely to establish better relationships with their In Vietnamese society, the notion of “thuong cho roi
cho vot,” which means “spanking (with a rod) out of love
for the child” suggests that strict discipline was considered
a cultural norm for generations (Vu, 2016; Vuong et al.,
2018). Therefore, our findings indicated that Vietnamese
parents tend to set diehard control strategies such as using
GPS tracking on their children’s devices, establishing rules
about contacts, or allowing online. The majority of parents February 2022 | Volume 13 | Article 847278 Frontiers in Psychology | www.frontiersin.org 7 Parent-Child Relationship and Internet Use Nguyen et al. TABLE 4 | Logistic Regression for identifying factors related to the relationship between parents and children since using the internet. Characteristics
The relationship between parents and children since using internet
(Deteriorates = 1 vs. Constant/Better = 0)
OR
95%CI
INDIVIDUAL CHARACTERISTICS
Province (Vung Tau—Ref)
Can Tho
0.45
0.15; 1.35
Hung Yen
9.59***
3.25; 28.33
Thanh Hoa
0.18***
0.05; 0.66
Gender
Female
3.78***
1.46; 9.77
Grade (vs. secondary school)
High school
7.04***
2.34; 21.19
Ignored advice about using the internet from parents (Yes vs. ***p < 0.01; **p < 0.05; *p < 0.1. DISCUSSION No)
10.25***
2.73; 38.54
The frequency of using internet (per score)
1.00
0.90; 1.11
Time using the internet per day (hour)
1.32***
1.11; 1.57
PARENT-CHILDREN COMMUNICATION SINCE USING THE INTERNET
Frequency of talking to parents (per score)
Learning
0.29***
0.19; 0.46
The selection of clothing
0.34***
0.22; 0.51
Future orientation
0.78
0.53; 1.14
The problem of pocket money
2.09***
1.45; 3.00
Children’ activities with their parents on the internet (per score)
Helping parents when they have difficulty using the Internet
0.64**
0.45; 0.92
Ask for advice from parents on what to do on the Internet
0.43***
0.23; 0.80
THE PARENT’S MANAGEMENT AND CARE WITH INTERNET USE IN CHILDREN
Parent’s care with Internet Use in Children (per score)
Talks to me about what I do on the internet
1.71**
1.10; 2.66
Helps me when something bothers me on the internet
2.23***
1.46; 3.39
Helps me when something is difficult to do or find on the internet
0.67*
0.44; 1.03
Parent’s management with Internet Use in Children (per score)
Technology to track where I am (such as GPS)
6.92***
3.93; 12.16
Checks my messages on the social media
0.42***
0.23; 0.76
Rules about who I can contact on social networks
1.82***
1.24; 2.68
Rules about when I am allowed to go online
1.93***
1.28; 2.93
Knows about what I look at on the internet and the social media
1.87**
1.14; 3.06
Check out adding my friends and friends list
0.13***
0.05; 0.29
SOCIAL SUPPORT
Multidimensional Scale of Perceived Social Support
Social support from family (per score)
0.64*
0.38; 1.08
***p<0 01; **p<0 05; *p<0 1 TABLE 4 | Logistic Regression for identifying factors related to the relationship between parents and children since using the internet. TABLE 4 | Logistic Regression for identifying factors related to the relationship between parents and children since using the internet. The relationship between parents and children since using internet
(Deteriorates = 1 vs. Constant/Better = 0) Frontiers in Psychology | www.frontiersin.org SOCIAL SUPPORT Social support from family (per score) ***p < 0.01; **p < 0.05; *p < 0.1. disciplinary strategies, and setting regulative rules to regulate
children’s behavior has to be associated with positive
developmental results in children (Barber et al., 2005;
Bleakley et al., 2016). regard spending more time on the internet as a distraction
from study and a transgression that warrants discipline
(Cappa and Dam, 2014). However, the current results proved
the diehard parental control strategies negatively influence
the parent–child relationship: the higher magnitude and
frequency of such control, the worse the parent–child
relationship. Negative parental control, which mostly manifests
in the form of psychological control through strategies of
guilt induction and love withdrawal aiming to make children’
dependent emotion on their parents (Steinberg et al., 1989;
Siomos et al., 2012), has been found to harm teenagers’
emotional functioning and the sense of self (Wang et al.,
2007), impair the children’ self-esteem and exacerbate
developmental difficulties and maladjustment in youths and
adolescents (Barber and Harmon, 2002). In contrast, positive
parental control in form of behavioral control through
monitoring, taking interest in children’s activities, developing Changes in the quality of the parent–child relationship were
self-assessed by participants showed improvement or deterioration
since youngsters used the internet. The mindsponge mechanism,
which was proposed by Vuong and Napier (2015) and Vuong
(2016), may be used to explain how and why parents “accept
or not accept” their kids’ behaviors when using the internet. Parents’ awareness and mindset play crucial factors in accepting
children’s behaviors of using online platforms including learning,
exchanging, and entertaining via the internet. Additionally, belief
in their kids is determined as a critical priority card in the
mindsponge mechanism because it encouraged parents in
promoting support, sharing information, and communicating
with their children in participating in social networking activities February 2022 | Volume 13 | Article 847278 Frontiers in Psychology | www.frontiersin.org 8 Parent-Child Relationship and Internet Use Nguyen et al. use behaviors, which might result from general family relations
such as quality of parent–child attachment. Participants
suggested that their family relationship quality had deteriorated
since using the internet, which led to decreasing academic
outcomes, loneliness, and depression. Stubborn parental control
factors, such as establishing rules about contacts or allowing
online, setting up GPS tracking can have a negative impact
on the quality of parent–child relationships. SOCIAL SUPPORT Meanwhile,
parent-adolescent communication quality, frequency (talking
about learning, selection of clothing, and future orientation),
and bidirectional interactive activities such as supporting
their parents for internet use, affected positively the relationship
between children and parents. The present research findings
are of importance for future research on parent–children
relationship quality and internet use in developing countries. Simultaneously, intervention targets the negative impacts of
the internet on young Vietnamese people and increases
parent-child communication quality also mentioned in
this works. (Vuong and Napier, 2015). The positive parent–child relationship
might provide a solid emotional foundation that encourages
positive living and prevents internet addiction in children
(Shek, 2010; Floros and Siomos, 2013).hi The research findings are of importance for future scholarly
works on the parent–children relationship related to using the
internet in developing countries, especially in the context of
prolonged COVID-19 pandemic. Our findings provide insights
for developing evidence-based interventions aiming to lessen
the negative impacts of the internet on young Vietnamese
people and increase the quality of parent–child communication. First, the intervention programs should prioritize improving
parental knowledge, awareness, and attitude about the positive
and negative influences of using online platforms (e-learning,
exchange, and entertainment), and enhancing parent–child
communication. Second, there should be psychological training
programs for parents so that they can understand the psychology
of children’s behavior and have appropriate responding strategies. Third, a sense of self-responsibility among youths and adolescents
should be encouraged through citizen empowerment and
emphasizing responsible behavior, so that young people can
protect themselves online. Fourth, future studies can benefit
from delving deeper into the association between parent–child
relationship and children’s internet use by, for instance, exploring
the specific roles of father and mother in family relationship
quality and their children’s internet use. Last, regardless of the
solutions used by parents or educators for reducing the negative
effects of the internet on young people and improving the
parent–child relationship, it is critical to strictly practice in a
disciplined process for an extended period of time until beneficial
results are obtained (Vuong et al., 2022). ETHICS STATEMENT All procedures performed in studies involving human participants
were in accordance with the ethical standards of the Youth
Research Institute, Vietnam, and with the 1964 Helsinki
Declaration and its later amendments or comparable ethical
standards. The patients/participants provided their written
informed consent to participate in this study. This study should be viewed in light of its limitations. First,
the study employed a cross-sectional design, which did not allow
for drawing causal conclusions between parent–children relationship
quality and associated factors. Second, the use of an online survey
platform might lead to lower response rates as well as lower
reliability and validity of the data compared to that of the
traditional interview method. Even so, in the context of COVID-19
pandemic and resource-scarce setting of Vietnam, the authors
believe that online survey is the optimal approach to reach a
large sample size in a short period. Furthermore, in order to
limit selection bias, we conducted a pilot survey and tried to
present the questionnaire in the most convenient way for
participants in the investigation period. Lastly, due to the length
constraint on the online platform, we could not measure factors
such as individual characteristics of children and parents, or
family context; cultural factors inherently influence the internet
use (awareness, habits to experiences, and practices). It is
recommended that future studies consider evaluating these aspects
in order to possibly draw more objective conclusions from the
quality of the two-way relationship between parents and children. AUTHOR CONTRIBUTIONS TTPN, HND, TV, HMD, NN, CL, CH, and RH: conceptualization. TTPN, TTN, LD, and GV: data curation. TTPN and TTN:
formal analysis. TTPN, TTN, LD, GV, and SN: methodology. HTD, CL, CH, and RH: supervision. TTPN, SN, LD, and
TTN: investigation. TTPN, TTN, GV, and HTD: writing—
original draft. TTPN, TTN, HND, TV, KV, HMD, NN, LD,
GV, HTD, SN, CL, CH, and RH: writing—review and editing. All authors contributed to the article and approved the
submitted version. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will
be made available by the authors, without undue reservation. REFERENCES A., and Ebrahem, G. G. S. (2016). The relationship between
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10 1016/j ijintrel 2015 06 003 Publisher’s Note: All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated organizations,
or those of the publisher, the editors and the reviewers. Any product that may
be evaluated in this article, or claim that may be made by its manufacturer, is
not guaranteed or endorsed by the publisher. Vuong, Q. H., and Napier, N. K. (2015). Acculturation and global mindsponge:
an emerging market perspective. Int. J. Intercult. Relat. 49, 354–367. doi:
10.1016/j.ijintrel.2015.06.003 Copyright © 2022 Nguyen, Nguyen, Do, Vu, Vu, Do, Nguyen, Doan, Vu, Do,
Nguyen, Latkin, Ho and Ho. 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, M. T., and Fredricks, J. A. (2014). The reciprocal links between school
engagement, youth problem behaviors, and school dropout during adolescence. Child Dev. 85, 722–737. doi: 10.1111/cdev.12138h Wang, Q., Pomerantz, E. M., and Chen, H. (2007). The role of parents’ control
in early adolescents’ psychological functioning: a longitudinal investigation
in the United States and China. Child Dev. 78, 1592–1610. doi: 10.1111/j. 1467-8624.2007.01085.x February 2022 | Volume 13 | Article 847278 Frontiers in Psychology | www.frontiersin.org 11
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Rheological Studies Dedicated to the Development of a Novel Injectable Polymeric Blend for Viscosupplementation Treatment
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Introduction OARSI treatment guidelines.22 Viscosupplementa
tion consists in intra-articular, single or repeated,
injections of HA or HA-based derivatives (HAs). Adequate viscoelastic properties are also important
to restore the articular homeostasis.23 HA or HAs
increase the elastoviscosity of the intercellular ma
trix creating a mechanical protective socket that
surrounds nociceptive nerve endings. This results in
a decrease in pain and an increase in joint mobility24
as it has already been demonstrated on animals
models.25,26 OARSI treatment guidelines.22 Viscosupplementa
tion consists in intra-articular, single or repeated,
injections of HA or HA-based derivatives (HAs). Adequate viscoelastic properties are also important
to restore the articular homeostasis.23 HA or HAs
increase the elastoviscosity of the intercellular ma
trix creating a mechanical protective socket that
surrounds nociceptive nerve endings. This results in
a decrease in pain and an increase in joint mobility24
as it has already been demonstrated on animals
models.25,26 Osteoarthritis (OA) is a chronic, progressive
musculoskeletal disorder1–3 that mainly affects the
weight-bearing chondro-synovial joints such as
knee, hip4 and the upper extremities.5,6 The disease
affects the ageing population worldwide7,8 leading
to a high socio-economic burden9 with substantial
financial consequences.10,11 In a healthy condition, the lubricants present in
the synovial fluid, such as Hyaluronic acid (HA),
lubricin12 and the surface-active phospholipids
(SAPL)13,14 act as shock absorbers protecting the
joint against compressive damage.15–17 Hyaluronic
acid (HA) is responsible for the viscoelastic proper
ties.18 One of the most important drawbacks of HA
and HAs is the multiple injection therapy due to the
enzymatic degradation by hyaluronidases in the
body. High molecular weight products and cross-
linked hydrogels have been developed, but they are
able to retard the hyaluronidases attack without pre
venting it.27 Furthermore, inflammatory reactions
sometimes accompanied by swelling and erythema
have also been variably reported.28,29 In OA joint diseases, the concentration and mo
lecular weight of HA decrease16,19 and the synovial
fluid becomes more abundant, less viscous and re
duced in elastoviscous properties that may culmi
nate in a painful condition for the patient. Rheological Studies Dedicated to the Development
of a Novel Injectable Polymeric Blend for Viscosupplementation Treatment doi: 10.15255/CABEQ.2014.2148 Original scientific paper
Received: November 15, 2014
Accepted: November 30, 2015 Viscosupplementation is an intra-articular symptomatic treatment of mild osteoar
thritis. The treatment involves the injection of high-molecular-weight hyaluronan (HA),
and especially of cross-linked HA to restore the lubricating and cushioning properties of
the synovial fluid. This work involves the development of a novel viscosupplementation fluid based on
amidated carboxymethylcellulose and obtained by blending the soluble polymer with its
crosslinked derivative. Rheological analyses carried out under both oscillatory and con
tinuous shear provided a rationale to assess the viscosupplement formulation and the
production process. The hydrogel fraction content and the total polymer concentration
can be properly selected in order to ensure an optimal combination of flowability and
viscoelastic properties. Key words:
amidated carboxymethylcellulose, viscosupplementation, polymeric blend, hydrogel,
rheology amidated carboxymethylcellulose, viscosupplementation, polymeric blend, hydrogel,
rheology *Corresponding author: romano.lapasin@di3.units.it 511 511 R. LAPASIN et al., Rheological Studies Dedicated to the Development of a Novel…, Chem. Biochem. Eng. Q., 29 (4) 511–518 (2015) R. LAPASIN et al., Rheological Studies Dedicated to the Develo Introduction A new derivative similar to HA, but with a bet
ter behaviour in terms of hardness and stability to
wards enzymatic degradation30 has been developed
converting 50 % of the carboxylic groups of car
boxymethylcellulose (CMC) into amidic groups to
obtain
the
amidated
carboxymethylcellulose
(CMCA).31 In vitro results showed that the amida
tion significantly ameliorated the biocompatibility
of the material and diminished the cell expression Viscosupplementation20,21 was identified as an
effective treatment of OA with a small number of
adverse effects. Nowadays, it is approved in Eu
rope/USA for the treatment of pain associated with
OA of the knee, but considerable controversy about
its efficacy is still ongoing as evidenced in the 512 R. LAPASIN et al., Rheological Studies Dedicated to the Development of a Novel…, Chem. Biochem. Eng. Q., 29 (4) 511–518 (2015) of inflammatory cytokines. CMCA also positively
influenced osteoblast activity.30 The vegetal origin
of CMCA potentially avoids the risk of allergic re
actions due to certain protein components present in
the HA and Ha-based products injected. molar ratio with the polymer was 0.5:1 to obtain a
crosslinking degree of about 50 %. The obtained
hydrogel was purified by repeated rinsing in puri
fied water and freeze dried. The two freeze-dried
components, polymer and hydrogel, were milled in
order to have a controlled cut off, up to 2 mm, that
were subsequently blended and hydrated in saline
solution, and eventually filled in syringe and steril
ized. This work involves the development of a novel
viscosupplementation fluid obtained by blending
the soluble CMCA polymer (hereinafter indicated
as CMCAp) with its crosslinked hydrogel deriva
tive (CMCAg) by crosslinking the residual 50 %
fraction of CMCAp carboxylic groups. Materials The raw material for CMCA preparation is so
dium carboxymethylcellulose (average molecular
weight: 750 kDa, substitution degree 0.8) (CP
Kelko). It can be functionalized thanks to the pres
ence of two carboxyl groups in the polymeric unit
through a water-based synthetic pathway. The car
boxylic groups are first activated with NHS/EDC
(N-hydroxysuccinimide purity >97 % (Sigma-Al
drich); N-(3-dimethylaminopropyl)-N’-ethylcarbo
diimide hydrochloride purity >99 % (Actim)). Once
activated, they quickly react with methylamine
(40 % water solution, Sigma-Aldrich), molar ratio
of 1:1. The pH of the solution is always kept at
4.5–5 adjusting hydrochloric acid. The amide-CMCA
was then purified in a buffered solution (about pH
8–8.5 in order to increase the solubility of the poly
mer) through a Tangential Flow Filtration instru
ment using a membrane with 100 kDa cut off. The
obtained solution was separated into two aliquots:
the former was freeze-dried, the latter was used for
the crosslinking process. Recovery tests were performed in order to
evaluate the structural recovery after the application
of continuous shear conditions. They were com
posed of three consecutive segments, alternatively
under small amplitude oscillatory conditions (at 1
Hz) and in continuous shear at constant shear rate
(5 s–1, 180 s). The steady values of G’ and G” at
tained in the oscillatory segments before and after
shear flow were compared. All the experimental tests were done in dupli
cate or triplicate. Rheological experiments CMCA-based fluids were investigated to assess
the rheological properties of the viscosupplement
fluid during the different processing stages. Both
continuous and oscillatory shear tests showed that,
at equal polymer concentration, aqueous CMCAp
and CMCAg systems exhibit quite different rheo
logical behaviours, typical of polymer solutions and
gels, respectively, and hence a wide arc of viscous
and viscoelastic responses can be obtained by
blending the two polymers in different proportions. The gel fraction (CMCAg/CMCAp ratio) and the
total polymer concentration of the blend can be ap
propriately selected in order to ensure an optimal
combination of viscoelastic properties and flow be
haviour (injectability) together with an acceptable
structural recovery after shear. Rheological characterization was carried out
with a rotational controlled stress rheometer
(Rheostress RS150, Haake GmbH, Germany),
equipped with a cone/plate device (d = 60 mm, an
gle = 1°) or a parallel plate geometry (d = 35 mm,
gap = 1 mm) with serrated surfaces in order to avoid
wall slippage effects. The apparatus was comput
er-controlled (RheoWin software program) and the
temperature was kept at 25 ± 0.1 °C (Thermostat
TC500, Haake GmbH, Germany). The flow proper
ties were determined applying a logarithmic se
quence of increasing constant stresses in the range
1-1000 Pa and measuring the corresponding shear
rate. Each stress value was kept constant until the
corresponding time variation of the shear rate satis
fied the pre-set constraint for steady state attain
ment, or at most for 90 s. Oscillatory stress sweep tests were performed
at 1 Hz in order to individuate the extension of the
linear viscoelasticity regime. All the frequency
sweep measurements were performed from 100 to
0.01 Hz within the linear range. Data treatment and modeling The experimental viscosity data can be cor
related quite satisfactorily with the Carreau-Yasuda
model:32,33 η
η
η
η
γ
=
+
−
+
∞
∞
0
1[
(
) ]
a
b p
The amide-CMC polymer is crosslinked by a
solution of NHS/EDC and a bi-functional group. In
particular, 1,3-diaminopropane (purity 99.0 %, Sig
ma-Aldrich) was used as crosslinking agent. The where h0 and h∞ represent the zero-shear rate vis
cosity and the infinite-shear rate viscosity, respec
tively. In this study, the high shear Newtonian pla 513 R. LAPASIN et al., Rheological Studies Dedicated to the Development of a Novel…, Chem. Biochem. Eng. Q., 29 (4) 511–518 (2015) LAPASIN et al., Rheological Studies Dedicated to the Developm Fi g . 2 – Mechanical spectra of aqueous CMC and CMCAp
1 % w/w solutions teau is beyond the experimental window and, hence,
h∞ was arbitrarily set equal to 0.89 mPa s, i.e. to the
water viscosity at 25 °C. The product bp rules the
slope of the flow curve in the power law region,
while the critical shear rate gc marking the onset of
shear thinning is strictly related to the characteristic
time a. The mechanical spectra can be described with
the generalized Maxwell model through the follow
ing expressions: ′=
+
′′=
+
=
=
∑
∑
G
G
G
G
i
i
i
i
N
i
i
i
i
N
ω λ
ω λ
ωλ
ω λ
2
2
2
2
1
2
2
1
1
1 where Gi and li are the modulus and the relaxation
time of the ith Maxwell element, respectively. In or
der to reduce the problem related to parameter cor
relation, a scaling criterion was adopted for the re
laxation time sequence (li+1 = 10 li) and not more
than 5 Maxwell elements were taken into consider
ation for data fitting. Fi g . 2 – Mechanical spectra of aqueous CMC and CMCAp
1 % w/w solutions through the variations of two model parameters: the
zero shear rate viscosity h0 and the critical shear
rate gc. Results and discussion The changes induced by amidation on the lin
ear viscoelastic behaviour of solutions were coher
ent with those observed for their flow properties. At
equal polymer concentration, the mechanical spec
trum moved towards lower values of both moduli
and the crossover point shifted to higher frequen
cies because of the faster relaxation modes. The ex
ample reported in Fig. 2 regards the 1 % solutions. These effects can be evaluated more precisely by
comparing the relaxation time spectra obtained
from data correlation with the Maxwell generalized
model, as illustrated in Fig. 3. The effects produced by amidation on the rheo
logical behaviour of CMC solutions appeared to be
more evident at low shear conditions and higher
polymer concentrations. Comparing the flow curves
of CMC and CMCAp solutions at equal concentra
tion, the zero-shear-rate viscosity diminished after
amidation and the Newtonian plateau became more
extended towards higher shear conditions, as it can
be observed in Fig. 1. Similar rheological effects of
amidic moieties on carboxymethylcellulose have
been observed by Barbucci et al.30 More drastic changes were produced by poly
mer crosslinking, particularly in linear viscoelastic
conditions; this can be seen by comparing the re A satisfactory data correlation was provided by
the Carreau-Yasuda equation. Accordingly, the
above underlined effects can be concisely described Fi g. 1 – Steady viscosity curves of aqueous CMC and CMCAp
solutions at different concentrations (expressed in
% w/w)
Fig. 3 – Relaxation time spectra of aqueous CMC and CMCAp
1 % w/w solutions derived from data correlation
with the Maxwell generalized model Fi g. 1 – Steady viscosity curves of aqueous CMC and CMCAp
solutions at different concentrations (expressed in
% w/w) Fig. 3 – Relaxation time spectra of aqueous CMC and CMCAp
1 % w/w solutions derived from data correlation
with the Maxwell generalized model Fi g. 1 – Steady viscosity curves of aqueous CMC and CMCAp
solutions at different concentrations (expressed in
% w/w) Fig. 3 – Relaxation time spectra of aqueous CMC and CMCAp
1 % w/w solutions derived from data correlation
with the Maxwell generalized model R. LAPASIN et al., Rheological Studies Dedicated to the Development of a Novel…, Chem. Biochem. Eng. Q., 29 (4) 511–518 (2015) 514 514
R. LAPASIN et al., Rheological Studies Dedicated to the D
Fi g. Results and discussion 4 – Viscoelastic responses of 2.5 % CMCAp and 2.5 %
CMCAg systems; a) storage and loss moduli vs strain (from
stress sweep test); b) mechanical spectra (from frequency
sweep test) sions). For CMCAg systems, the critical strain
marking the borderline of the linear viscoelastic re
gime became sensibly lower (two orders of magni
tude or more) than for CMCAp solutions. The quite different profiles of the mechanical
spectra of CMCAp and CMCAg systems reflect the
neat changes produced by crosslinking in linear vis
coelastic properties and relaxation modes, thus evi
dencing the structural transition from solution to
physical gel (Fig. 4b). In the experimental window
explored, the behaviour of the CMCAp solution
was characterized by a prevailing viscous compo
nent (G”>G’) and short relaxation times, as it can
be deduced by the crossover frequency. In the case
of the crosslinked polymer, the storage modulus
curve is placed clearly above G” values, and reach
es a plateau condition at low frequencies, where the
divergence from the linear viscoelastic responses of
CMCAp is quite evident. Studying the steady flow properties, we found
that CMCAg systems exhibited much more pro
nounced shear thinning than CMCAp solutions, ow
ing to their different structural conditions and
shear-induced mechanisms. CMCAg systems be
have as dispersions of gel microdomains, which
may undergo significant deformations and spatial
rearrangements with increasing shear rate. Looking at the problems connected to the de
velopment of a novel polymeric fluid for visco
supplementation treatment, the first decisive step
concerns its formulation in relation to the structural
and rheological properties that the fluid must pos
sess and exhibit. In particular, it should be easily
injectable and display appropriate viscoelastic re
sponses ‘in situ’ in the various kinematic condi
tions experienced by articular joints. Furthermore,
an adequate resistance to enzymatic degradation
and mechanical solicitations for prolonged times
should be ensured. Blending CMCAp solutions
and CMCAg microgel dispersions it is possible to
finely adjust the composition ratio and the total
polymer concentration in order to modulate the
structural features and rheological properties of
both systems. Fi g. 4 – Viscoelastic responses of 2.5 % CMCAp and 2.5 %
CMCAg systems; a) storage and loss moduli vs strain (from
stress sweep test); b) mechanical spectra (from frequency
sweep test) sponses of both of CMCAp and CMCAg systems
with the same concentration of soluble and cross
linked polymer (2.5 % for systems represented in
Figs. Results and discussion 4a and 4b, respectively). Stress sweep tests showed that both viscoelas
tic moduli of the CMCAg system were markedly
higher, and in the nonlinear region, their profiles
were different from those of the corresponding CM
CAp solutions (Fig. 4a). For the latter systems, the
transition from linear to nonlinear regime is de
scribed by monotonic decrease of both moduli. This
behaviour is usually observed in ordinary polymeric
solutions and labelled as type I according to the
classification of LAOS responses.34 Conversely, in
the case of CMCAg systems, the storage modulus
decreased monotonically with increasing strain
whereas a weak overshoot appeared in the G’’ pro
file. Such a pattern (Type III) is typical of weakly
structured fluids (physical gels or colloidal disper The following figures illustrate how the rheo
logical properties of CMCAp-CMCAg blends
changed with increasing gel fraction at equal total
polymer concentration. The steady viscosity strong
ly increased at low shear rates, while its change was
almost negligible at high shear rates. As the gel
fraction increased, the shear thinning character of
the flow behaviour became more and more marked,
being ruled by the level and extension of the New
tonian plateau (Fig. 5). The experimental data can be correlated quite
satisfactorily with the Carreau-Yasuda equation. The effects of increasing gel fraction fg on viscous R. LAPASIN et al., Rheological Studies Dedicated to the Development of a Novel…, Chem. Biochem. Eng. Q., 29 (4) 511–518 (2015) 515 ti
f CMCA
CMCA
bl
d
b
Fi g. 5 – Steady viscosity curves of 2.5 % CMCAp-CMCAg
blends at different gel fractions (1 represents the polymer/gel
ratio of 0/10; 0 represents a polymer/gel ratio of 10/0) Fi g . 7 – Mechanical spectra of 2.5 % CMCAp-CMCAg
blends at different gel fractions (0 represents the polymer/gel
ratio of 10/0; 1 represents a polymer/gel ratio of 0/10) Fi g . 7 – Mechanical spectra of 2.5 % CMCAp-CMCAg
blends at different gel fractions (0 represents the polymer/gel
ratio of 10/0; 1 represents a polymer/gel ratio of 0/10) Fi g. 5 – Steady viscosity curves of 2.5 % CMCAp-CMCAg
blends at different gel fractions (1 represents the polymer/gel
ratio of 0/10; 0 represents a polymer/gel ratio of 10/0) properties of CMCAp-CMCAg blends can be rea
sonably described through the only variations of the
zero-shear rate viscosity h0 and the critical shear
rate gc, marking the border of the Newtonian plateau
(Fig. 6). Results and discussion Thus, it can be noted that the most signifi
cant changes occurred in the fg interval between 0.2
and 0.3, where gc drops significantly, and the rate of
h0 increase became slower. At higher fg values, the
increasing concentration of gel microdomains in the
whole polymeric phase resulted only in an exponen
tial h0 increase with no appreciable effect on the
critical shear rate. Fi g . 7 – Mechanical spectra of 2.5 % CMCAp-CMCAg
blends at different gel fractions (0 represents the polymer/gel
ratio of 10/0; 1 represents a polymer/gel ratio of 0/10) served in the shape of the mechanical spectrum,
passing from CMCAp solution to CMCAg system. According to the Winter-Chambon criterion35,36, the
sol-gel transition could be located near fg = 0.3,
since the traces of G’ and G’’ of system 7 were
almost coincident and obey the same scaling law
(G’ G’’ wn, n 0.45) in the experimentally ex
plored frequency window. Similarly, the linear viscoelastic behaviour of
the blend significantly changed with increasing gel
fraction. To clarify, the graphical representation re
ported in Fig. 7 involves only a partial subset of the
mechanical spectra experimentally determined. As
may be observed, the increasing gel fraction fg led
to an increase in the elastic character of the visco
elastic response, which was more pronounced at
lower frequencies where the storage modulus in
creased more markedly than the viscous compo
nent. Consequently, a progressive change was ob The generalized Maxwell model provides a sat
isfactory data correlation and, consequently, the re
sulting relaxation time spectra can be profitably
used for analysing the blending effects on the linear
viscoelastic responses in the place of the mechani
cal spectra (Fig. 8). As the CMCAg content of the
polymeric phase increased, the relative weight of
the slowest relaxation modes became more import Fig. 8 – Relaxation time spectra derived from data correlation
with the generalized Maxwell model for 2.5 % CMCAp-CMCAg
blends (1 represents the polymer/gel ratio of 0/10, 0 represents
a polymer/gel ratio of 10/0) Fi g. 6 – Zero-shear rate viscosity and critical shear rate vs
gel fraction in 2.5 % CMCAp-CMCAg blends
Fig. 8 – Relaxation time spectra derived from data correlation
with the generalized Maxwell model for 2.5 % CMCAp-CMCAg
blends (1 represents the polymer/gel ratio of 0/10, 0 represents
a polymer/gel ratio of 10/0) Fi g. Results and discussion Typical moist-heat sterilization cycles, based
on exposition to temperature of 121 °C for 15’ have
been applied, and the mechanical spectra obtained
for unsterile and sterile blends of equal total poly
mer concentration and gel fraction have been com
pared. As highlighted in Fig. 9, it can be seen that
steam sterilization induced a loss of both elastic and
viscous moduli due to the polymer degradation, and
hence a reduction in the main properties of the de
vice. Consequently, the initial formulation must be
readjusted in terms of total polymer concentration
and gel fraction in order to compensate the negative
effects produced by the manufacturing process on
the rheological behaviour of the blend and to re
store a correct balance among viscoelasticity, stabil
ity and easy injectability. In fact rheological proper
ties of numerous viscosupplements available on the
market cover a wide spectrum of values and some
times they are quite different from those peculiar to
healthy synovial fluids.20,38–40 Among the several steps of the manufactur
ing process, some of them can strongly affect the
elastoviscosity of the polymer blend. It has been
observed that freeze drying, homogenization in
saline solution and filling in syringes can produce
a drastic decrease in loss and storage moduli in
both soluble and crosslinked CMCA. Moreover,
because viscosupplements are classified as phar
maceutical products or medical devices, an essen
tial requirement for CMCA blends is represent
ed by their capability to resist the sterilization
process. This suggests that the main requirements for
such devices are represented by their viscoelastic
properties in order to exhibit a proper response to
the stress conditions within synovial joints, together
with their capability to be easily injected through an
18-20 G needle. It is also acknowledged that the
values of the linear viscoelastic moduli measured at
0.5 and 2.5 Hz can represent a suitable way to eval
uate how synovial fluid and viscosupplements react
to walking and running conditions, respectively.20,41
Accordingly, they can serve to discriminate the be
haviours of synovial fluids in normal and patholog
ical conditions, as well as to assess the effects of
viscosupplementation therapies.39,40,42 The experi
mental values obtained for a series of commercial
viscosupplements are reported in Fig. 10a. The Several techniques based on chemical agents
(EtO, plasma) or physical processes (heating, b or γ
radiation) are widely used for sterilization in the
pharmaceutical and medical devices industries. Results and discussion 6 – Zero-shear rate viscosity and critical shear rate vs
gel fraction in 2.5 % CMCAp-CMCAg blends Fig. 8 – Relaxation time spectra derived from data correlation
with the generalized Maxwell model for 2.5 % CMCAp-CMCAg
blends (1 represents the polymer/gel ratio of 0/10, 0 represents
a polymer/gel ratio of 10/0) Fi g. 6 – Zero-shear rate viscosity and critical shear rate vs
gel fraction in 2.5 % CMCAp-CMCAg blends 516 R. LAPASIN et al., Rheological Studies Dedicated to the Development of a Novel…, Chem. Biochem. Eng. Q., 29 (4) 511–518 (2015) R. LAPASIN et al., Rheological Studies Dedicated Fi g . 9 – Reduction ratio of viscoelastic moduli between unster
ile and sterile 2.5 % CMCAp-CMCAg blends vs gel
fraction at two reference frequencies (0.5 and 2.5 Hz) ant owing to the increasing contribution of gel mi
crodomains towards a structural state close to jam
ming. The analysis of the reversibility of linear visco
elastic responses (G’, G’’) can be particularly useful
in view of the development of a novel viscosupple
ment, which must be injected, and hence undergoes
short but intense shear conditions in the application
phase. Recovery tests performed on CMCAp-CMCAg
blends showed that the viscoelastic behaviour was
completely reversible for CMCAp solution, since
the values of G’ and G” before and after shear flow
were shown to be equal. On the other hand, only a
partial recovery was attained for CMCAg system
along the time segment following the application of
a constant shear rate. The viscoelastic recovery de
creased with increasing gel fraction. Fi g . 9 – Reduction ratio of viscoelastic moduli between unster
ile and sterile 2.5 % CMCAp-CMCAg blends vs gel
fraction at two reference frequencies (0.5 and 2.5 Hz) manufacturing process, in particular that caused by
steam sterilization. Investigations were also carried out on the de
velopment of a pilot production process and evalu
ations were made on the potential effect of each
processing step on the blend’s final properties. Standard production implies a set of consecutive
steps to obtain both components of the polymeric
blend: CMC solubilization, CMC functionalization
in order to obtain CMCA, purification of CMCA,
concentration and freeze-drying of the polymer,
crosslinking of the CMCA, purification of the ob
tained hydrogel, concentration and freeze-drying of
the hydrogel. The final steps cover rehydration in
saline solution, polymer and gel blending, filling in
syringe and sterilization. Conclusions Blends with tailored viscoelastic properties can
be obtained from CMCA in the form of polymer
solutions and crosslinked hydrogels. More specifically, the entire range between
10/0 and 0/10 of polymer/hydrogel ratio has been
investigated, and the 5/5-6/4-7/3 polymer/hydrogel
compositions seem to create a behaviour very close
to the main characteristics of an HA-based product
for viscosupplementation. Fi g. 10 – a: Viscoelastic moduli (at 0.5 and 2.5 Hz) of the
commercial viscosupplements listed below. b: Viscoelastic moduli
(at 0.5 and 2.5 Hz) of the 6/4 CMCAp/CMCAg blends at 2.5 %,
3 % and 3.61 % w/w concentrations compared with those of two
commercial products (Orthovisc and Monovisc). c: Viscosity
values (at 0.1, 10 and 1000 s–1) of the 6/4 CMCAp/CMCAg blends
at 2.5 %, 3 % and 3.61 % w/w concentrations compared with
those of two commercial products (Orthovisc and Monovisc). Commercial viscosupplement This study has demonstrated the importance of
both polymer and hydrogel form in blends, because,
if singularly used, (10/0 and 0/10 of polymer/hydro
gel samples), they are unable to preserve both stor
age and loss moduli. pp
Suplasyn (Mylan N.V., UK), non-heat degraded highly purified sodium
hyaluronate (500 to 730 kDa), measured values, ref. 39.; Hyalubrix (Fidia
farmaceutici S.p.A., Italy), sodium hyaluronate (1300 to 3600 kDa), from
bacteria fermentation, estimated values, ref. 40.; Orthovisc (Anika Ther
apeutics MA, USA), highly purified sodium hyaluronate in physiologic
saline, measured values, ref. 39.; Monovisc (Anika Therapeutics MA,
USA) highly purified, partially crosslinked sodium hyaluronate in a phos
phate buffered saline solution, estimated values, ref. 40.; Noltrex (JSC
“Research center “Bioform”, Russia), synthetic polyacrylamide hydrogel
with silver ions, measured values in our laboratory; Hymovis (Fidia far
maceutici S.p.A., Italy), partially hydrophobized derivative of hyaluronic
acid, estimated values, ref. 40.; Synvisc (Hylan G-F 20) (Genzyme Bio
surgery, USA). blend 80/20 of Hylan A (high molecular weight hyaluron
ic acid) and Hylan B (crosslinked hyaluronic acid), measured values, ref. 39.: Durolane (Bioventus, USA), crosslinked hyaluronic acid, measured
values in our laboratory.; Fermathron (Hyaltech Ltd. UK), sodium hyal
uronate (1300 to 3600 kDa) in a phosphate buffered saline solution, from
bacterial fermentation, measured values in our laboratory. Properties, such as lubrication and shock ab
sorbing, together with an adequate resistance to
processing and steam sterilization, can be assured
by using a concentration of 3.61 % w/w. Results and discussion Among them, the most suitable and promising
method in terms of efficacy and compatibility with
solution or emulsions is represented by steam or
moist heat. Conversely, it is known that heat induc
es degradation of CMC gels and polymer solutions37
and at high temperatures CMC degrades and perma
nently reduces in viscosity. Therefore, a rheological
investigation has been dedicated to evaluate the po
tential degradation of CMCA blends during the R. LAPASIN et al., Rheological Studies Dedicated to the Development of a Novel…, Chem. Biochem. Eng. Q., 29 (4) 511–518 (2015) 517 Fi g. 10 – a: Viscoelastic moduli (at 0.5 and 2.5 Hz) of the
commercial viscosupplements listed below. b: Viscoelastic moduli
(at 0.5 and 2.5 Hz) of the 6/4 CMCAp/CMCAg blends at 2.5 %,
3 % and 3.61 % w/w concentrations compared with those of two
commercial products (Orthovisc and Monovisc). c: Viscosity
values (at 0.1, 10 and 1000 s–1) of the 6/4 CMCAp/CMCAg blends
at 2.5 %, 3 % and 3.61 % w/w concentrations compared with
those of two commercial products (Orthovisc and Monovisc). Commercial viscosupplement
l
(
l
)
h
d
d d h hl
f d
d quite large interval covered by both moduli at both
frequencies is strictly related to the different me
chanical spectra displayed by the systems consid
ered, spanning from those typical of ordinary poly
meric solutions to classical gel behaviours. Similar
differences can also be found among novel systems
proposed as candidates for viscosupplementa
tion.40,42–45 Tests performed on blends with different gel
fractions have shown that an acceptable structural
recovery after continuous shear application (more
than 80–90 %) is attained when the gel fraction is
lower than 50 %. Thus, the 6/4 CMCAp/CMCAg
blend has been provisionally selected for the final
investigation addressed to individuate the most
suitable total polymer concentration, apt to compen
sate the loss in mechanical properties occurring
during processing and sterilization. The viscoelastic
moduli of three 6/4 CMCAp/CMCAg blends with
different total polymer concentrations are compared
with those of two commercial products in Fig. 10b,
while Fig. 10c reports the viscosity values of the
same systems at three different shear rates. From
both figures, it can be inferred that the 3.61 w/w
concentration provides adequate viscoelastic prop
erties, which are combined with a proper injecta
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The proinflammatory cytokines IL-1beta and TNF-alpha induce the expression of Synoviolin, an E3 ubiquitin ligase, in mouse synovial fibroblasts via the Erk1/2-ETS1 pathway.
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Available online http://arthritis-research.com/content/8/6/R172
Research article
Vol 8 No 6
Open Access
The proinflammatory cytokines IL-1β and TNF-α induce the
expression of Synoviolin, an E3 ubiquitin ligase, in mouse synovial
fibroblasts via the Erk1/2-ETS1 pathway
Beixue Gao1, Karen Calhoun1 and Deyu Fang1,2
1Department
2Department
of Otolaryngology-Head and Neck Surgery, University of Missouri School of Medicine, One Hospital Drive, Columbia, MO 65212, USA
of Molecular Microbiology and Immunology, University of Missouri School of Medicine, One Hospital Drive, Columbia, MO 65212, USA
Corresponding author: Deyu Fang, FangD@missouri.edu
Received: 13 Jun 2006 Revisions requested: 13 Jul 2006 Revisions received: 10 Oct 2006 Accepted: 14 Nov 2006 Published: 14 Nov 2006
Arthritis Research & Therapy 2006, 8:R172 (doi:10.1186/ar2081)
This article is online at: http://arthritis-research.com/content/8/6/R172
© 2006 Gao et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
The overgrowth of synovial tissues is critical in the pathogenesis
of rheumatoid arthritis (RA). The expression of Synoviolin (SYN),
an E3 ubiquitin ligase, is upregulated in arthritic synovial
fibroblasts and is involved in the overgrowth of synovial cells
during RA. However, the molecular mechanisms involved in the
elevated SYN expression are not known. Here, we found that
SYN expression is elevated in the synovial fibroblasts from mice
with collagen-induced arthritis (CIA). The proinflammatory
cytokines interleukin (IL)-1β and tumor necrosis factor-α (TNFα) induce SYN expression in mouse synovial fibroblasts.
Cultivation of mouse synovial fibroblasts with IL-1β activates
mitogen-activated protein kinases, including extra-cellular
signal-regulated kinase (Erk), JNK (c-Jun N-terminal kinase), and
Introduction
Rheumatoid arthritis (RA) is a chronic debilitating disease of
the joints characterized by leukocyte infiltration, hyperproliferation of synovial cells, and bone destruction. Hyperproliferative
synovial fibroblasts play a critical role in the pathogenesis of
RA by the following mechanisms: They directly invade bone
and cartilage, produce proinflammatory cytokines such as
tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β [1],
destroy cartilage through the production of metalloproteinase
[2], and produce the receptor of nuclear factor-kappa B (NFκB) ligand, which augments osteoclast activity for bone
destruction [3-5]. Therefore, inhibition of the proliferative and/
p38, while only Erk-specific inhibitor blocks IL-1β-induced SYN
expression. Expression of transcription factor ETS1 further
enhances IL-1β-induced SYN expression. The dominant
negative ETS1 mutant lacking the transcription activation
domain inhibits SYN expression in a dose-dependent manner.
The activation of both Erk1/2 and ETS1 is increased in the CIA
synovial fibroblasts. Inhibition of Erk activation reduces ETS1
phosphorylation and SYN expression. Our data indicate that the
proinflammatory cytokines IL-1β and TNF-α induce the
overgrowth of synovial cells by upregulating SYN expression via
the Erk1/-ETS1 pathway. These molecules or pathways could
therefore be potential targets for the treatment of RA.
or invasive capacities of synovial fibroblasts should have protective effects against joint destruction.
Synoviolin (SYN), which is also called Hrd1 (3-hydroxy-3methylglutaryl reductase degradation), was identified by
Hampton and co-workers [6] as an E3 ubiquitin ligase in yeast.
SYN is a multispanning membrane protein with its C-terminal
RING (really interesting new gene) finger domain located in
the cytoplasm [6,7]. It has been reported that human SYN is
involved in the elimination of two endoplasmic reticulum (ER)associated degradation substrates, T-cell receptor-α and
CD3-δ, via its E3 ubiquitin ligase activity [8]. Ubiquitination is
a process that covalently conjugates ubiquitin to the target
protein for degradation. This process requires a cascade of
AP-1 = activator protein-1; CFA = complete Freund's Adjuvant; CIA = collagen-induced arthritis; DMEM = Dulbecco's modified Eagle's medium;
EBS = ETS binding site; ER = endoplasmic reticulum; Erk = extracellular signal-regulated kinase; ETS1-DN = dominant negative mutant of ETS1;
FBS = fetal bovine serum; GST = glutathione S-transferase; IACUC = institutional animal care and use committee; IL = interleukin; JNK = c-Jun Nterminal kinase; MAPK = mitogen-activated protein kinase; NF-κB = nuclear factor-kappa B; NP-40 = Nonidet P-40; PCR = polymerase chain reaction; RA = rheumatoid arthritis; RT-PCR = reverse transcription-polymerase chain reaction; SYN = Synoviolin; TNF-α = tumor necrosis factor-α; Ubc
= ubiquitin-conjugation enzyme.
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Gao et al.
three enzymes, E1, E2, and E3. SYN predominantly uses the
ubiquitin-conjugation enzyme 7p (Ubc7p) as an E2 but also
cooperates with Ubc6p and Ubc1p in ER-associated degradation [9]. SYN is also required for the mouse embryo development because the gene knockout mice die in utero at
approximately embryonic day 13.5 [10].
By means of immunoscreening with an anti-rheumatoid synovial cell antibody, SYN was identified and cloned as a rheumatoid regulator. Expression of SYN is highly associated with the
development of RA. Mice with overexpressed SYN (SYN
transgenic mice) develop spontaneous arthropathy. On the
other hand, mice with reduced SYN (SYN+/- mice) are resistant to collagen-induced arthritis (CIA). Further in vitro study
revealed that, through its anti-apoptotic activities, SYN triggers the outgrowth of synovial fibroblasts [11,12]. Therefore,
inhibition of the expression of SYN has potential therapeutic
benefit in the prevention or treatment of RA. However, the
molecular mechanisms involved in overexpression of SYN during RA remain unknown.
In this study, we found that the proinflammatory cytokines, particularly IL-1β, upregulate SYN expression at the transcriptional level. The extra-cellular signal-regulated kinase (Erk)ETS1 signal pathway is involved in IL-1β-induced SYN
expression.
Materials and methods
Reagents, antibodies, and plasmids
All the mitogen-activated protein kinase (MAPK) inhibitors
used in this study, including the Erk activation inhibitor,
PD98059 [13], the c-Jun N-terminal kinase (JNK) inhibitor,
SP600125 [14], p38 inhibitor, SB202190 [15], and the NFκB inhibitor, SN50 [16], were purchased from Calbiochem
(San Diego, CA, USA). Antibodies against JNK1, Erk, and p38
were purchased from Promega Corporation (Madison, WI,
USA). Murine IL-1β, IL-6, and TNF-α were obtained from BD
Pharmingen (San Diego, CA, USA). Anti-actin monoclonal
antibody was purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA, USA), and anti-ETS1 was obtained from
EMD Biosciences, Inc. (San Diego, CA, USA). Antibodies
against Erk, c-Jun, and ATF2, as well as their phosphorylated
forms, were purchased from Promega Corporation. Anti-SYN
polyclonal antibody was generated in our laboratory by immunization of mice with glutathione S-transferase (GST)-fusion
protein of the C-terminal 152 amino acids of SYN. ETS1 fulllength cDNA was purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) (ATCC no. 5844553).
The C-terminal DNA binding domain of ETS1 was amplified by
polymerase chain reaction (PCR) and subcloned into pEF4 his
expression vectors digested by KpnI and Not1.
Mice and CIA
DBA1 mice (The Jackson Laboratory, Bar Harbor, ME, USA)
were bred and maintained in accordance with the guidelines
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of the institutional animal care and use committee (IACUC),
and all the experimental procedures were approved by the
IACUC of the University of Missouri (Columbia, MO, USA).
Native bovine collagen II (Worthington Biochemical Corporation, Lakewood, NJ, USA) was emulsified with an equal volume
of complete Freund's Adjuvant (CFA). Disease was induced
by intradermal injection of DBA1 mice with 50 μl of emulsion
containing 100 μg of collagen in CFA. On day 21, the mice
were boosted by intradermal injection with 100 μg of collagen
in incomplete Freund's Adjuvant. Clinical arthritis was
assessed by the following scoring system: grade 0, no swelling; grade 1, mild but definite redness and swelling of the
ankle or wrist or digits; grade 2, moderate redness and swelling of ankle and wrist; grade 3, severe redness and swelling of
entire paw, including digits; and grade 4, maximally inflamed
limb with involvement of multiple joints. Each limb was graded,
giving a maximum possible score of 16 per mouse. Approximately 80% of DBA1 mice developed arthritis 40 days after
the first injection with collagen (supplemental Figure 1a in
Additional file 1), and most of these mice developed severe
arthritis with an average score of 12 (supplemental Figure 1b
in Additional file 1).
Isolation of synovial fibroblasts
Synovial tissues were obtained from DBA1 mice as described
previously [17]. These joint tissues were minced and incubated with 1 mg/ml of collagenase (Worthington Biochemical
Corporation) in serum-free Dulbecco's modified Eagle's
medium (DMEM) for 3 hours at 37°C, filtered through a nylon
mesh, extensively washed, and cultured in DMEM supplemented with 10% fetal calf serum (Fisher Scientific Co., Pittsburgh, PA, USA), 100 U penicillin, 100 μg/ml streptomycin,
and 50 mg/ml L-glutamine in a humidified atmosphere containing 5% CO2. After overnight culture, we removed the nonadherent cells, trypsinized the adherent cells split at a ratio of
1:3, and cultured them in medium. Synoviocytes were used
from passages 3 to 9 in these experiments, during which they
consisted of a homogeneous population of synovial fibroblasts
monitored by flow cytometry with less than 1% of CD11b,
phagocytic, and Fc receptor II-positive cells.
SDS-PAGE and Western blotting
We analyzed the expression of SYN by SDS-PAGE and Western blotting as described previously [18]. Synovial fibroblasts
were collected from culture dishes and lysed with Nonidet P40 (NP-40) lysis buffer (20 mM Tris-HCl with pH 7.5, 150 mM
NaCl, 1% NP-40, and protease inhibitor cocktail was added
freshly) and boiled in 20 μl of Laemmli's buffer (50 mM TrisHCl, pH 6.8, 30% glycerol, 4% SDS, and 1% β-mercaptoethanol). Samples were subjected to 8% or 10% analysis by
SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes
were probed with the indicated primary antibodies (usually 1
μg/ml), followed by horseradishperoxidase-conjugated
Available online http://arthritis-research.com/content/8/6/R172
Figure 1
Increased Synoviolin (SYN) expression in the synovial fibroblasts from collagen-induced arthritis (CIA) mice.
mice (a) Test of the specificity of anti-SYN
antibody. HEK 293 cells were transfected with (lane 1) or without (lane 2) SYN expression plasmids. Lysates from the transfected cells were analyzed by SDS-PAGE and Western blotting using sera from the mice immunized with GST (glutathione S-transferase)-SYN fusion protein (top panel).
The same membrane was reprobed with anti-actin (bottom panel). (b) Analysis of the protein expression of SYN in mouse synovial fibroblasts.
Mouse synovial fibroblasts were isolated from each of three normal or CIA mice. Cells were lysed and subjected to SDS-PAGE and Western blotting using anti-SYN polyclonal antibodies (top panel). The same membrane was reprobed with anti-actin antibody (bottom panel). (c) Quantification
of SYN expression. The densities of each band in (b) were analyzed, and the ratio of SYN/(10-1 actin) was used. Error bars represent three different
CIA or normal mice. Statistic analysis indicates that the expression of SYN is significantly increased (p < 0.001).
secondary antibodies. Membranes were thenwashed and visualized with an enhanced chemiluminescence detectionsystem (Amersham Pharmacia Biotech, now part of GE
Healthcare, Little Chalfont, Buckinghamshire, UK). When necessary, membranes were stripped by incubation in stripping
buffer (62.5 mM Tris-HCl, pH 5.7,100 mM 2-mercaptoethanol,
and 2% SDS) for 30 minutes at 70°C with constant agitation,
washed, and then reprobed with other antibodies asindicated.
Real-time reverse transcription-PCR
Total cellular RNA extraction was performed using the RNA
purification kit from Promega Corporation. The oligonucleotide
primers used for mouse SYN were forward 5-aggcccatgtacctggccatgagg-3 and reverse 5-caggagcgcaggcagctcgtgtg-3.
The QuantiTect SYBR Green PCR kit (Qiagen Inc., Valencia,
CA, USA) was used. Reactions were loaded on 96-well thinwall plates and sealed with optical-quality sealing tape. Each
reaction was run on an iCycler iQ Multi-Color Real-Time PCR
detection system (Bio-Rad, Hercules, CA, USA) under the following conditions: 95°C for 15 minutes (94°C for 30 seconds,
55°C for 30 seconds, and 72°C for 30 seconds) for 40 cycles
and 72°C for 3 minutes. Samples were run in triplicate, and relative copy numbers were determined.
Results
Elevated expression of SYN during CIA in DBA1 mice
Recent studies demonstrated that SYN functions as a rheumatoid regulator for the overgrowth of synovial tissues in
patients with RA [11]. To investigate the molecular mechanisms involved in regulating SYN expression during arthritis
development, we compared SYN expression in the synovial
fibroblasts from CIA mice with that from normal mice. Because
the antibodies against SYN are not commercially available, we
first generated anti-SYN polyclonal antibody by immunizing a
fusion protein of GST with the C-terminus of SYN. We
detected both overexpressed and endogenous SYN in HEK
293 cells by using the sera from GST-SYN-immunized mice
(Figure 1a). We therefore used these polyclonal anti-SYN antibodies in this study.
With Western blotting analysis, we found a significantly higher
SYN expression in the synovial fibroblasts from CIA mice than
that from normal controls (Figure 1b). The mean relative density values (10 × SYN/actin) were 4.8 ± 0.9 for CIA synovial
fibroblasts and 1.1 ± 0.3 for control synovial cells (p < 0.001)
(Figure 1c). These results indicate that SYN expression is
upregulated during CIA.
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Figure 2
The expression
(CIA)
development
of Synoviolin (SYN) during collagen-induced arthritis
(CIA) development. (a) Synovial fibroblasts were isolated from normal
DBA mice (Normal) and DBA mice 20 days after the first collagen
immunization (Immunized) or 10 days after the second immunization
when CIA is developed (CIA). The expression of SYN in those synovial
fibroblasts was analyzed by Western blotting. (b) The densities of each
band in (a) were analyzed, and the ratio of SYN/(10-1 actin) was used.
Error bars represent three different CIA, immunized, or normal mice.
Statistic analysis indicates that the expression of SYN is significantly
increased during CIA development.
Next, we kinetically analyzed the expression of SYN during the
development of CIA. DBA synovial fibroblasts from normal
DBA mice, DBA mice 20 days after the first immunization with
collagen but without any clinical sign of CIA, and DBA mice 10
days after boosted immunization when these mice developed
severe arthritis (score 8 to 12). Surprisingly, the expression of
SYN has been significantly upregulated when the DBA mice
received only the first immunization and did not develop obvious arthritis. Although statistically not significant (p = 0.06),
boosted immunization further enhanced SYN expression (Figure 2). These results suggest that upregulation of SYN expression is involved in the development of CIA in mice.
Induction of SYN expression by IL-1β and TNF-α in
mouse synovial fibroblasts
The production of proinflammatory cytokines by synovial macrophages and fibroblasts is one reason for synovial cell hyperproliferation. We therefore hypothesized that some of these
cytokines may also be responsible for the induction of SYN
expression. To test this hypothesis, we first examined the
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Figure 3
Induction of Synoviolin (SYN) expression
expression by
by proinflammatory
proinflammatory cytokines.
cytokines
(a) Mouse synovial fibroblasts were starved for 24 hours and then cultivated for 48 hours with each proinflammatory cytokine, including 10
ng/ml of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, or IL-6.
The expression of SYN was tested by Western blotting by using antiSYN antibody (top panel). As a control, the protein level of actin was
detected using the same membrane (bottom panel). (b) The total RNA
from the cultured synovial fibroblasts was isolated and reverse-transcribed into cDNA. The levels of SYN cDNA were analyzed by real-time
polymerase chain reaction by using SYN-specific primers. Error bars
represent the results from three independent experiments (mean ±
standard deviation).
effects of proinflammatory cytokines on the expression of SYN
in mouse synovial fibroblasts. Each of 5 × 105 cells was plated
and cultivated in the presence of IL-6, TNF-α, or IL-1β. Fortyeight hours after the addition of each cytokine, the protein levels of SYN were analyzed. As shown in Figure 3a, TNF-α
slightly induced the protein expression of SYN with a mean
density of 3.1 ± 0.6 compared with 1.1 ± 0.2 of controls from
three independent experiments (p < 0.05 to control), whereas
IL-6 did not affect SYN expression, the mean density of which
was 1.3 ± 0.3 (p = 0.28). The cultivation of synovial fibroblasts
with IL-1β significantly upregulated SYN expression, the mean
density of which was 6.2 ± 1.1 (p < 0.001). These results indicate that both IL-1β and TNF-α induce SYN expression in
mouse synovial fibroblasts.
Available online http://arthritis-research.com/content/8/6/R172
To determine whether these cytokines induce SYN expression
at the transcriptional level, total RNA was isolated from these
synovial fibroblasts, reverse-transcribed into cDNA, and examined by reverse transcription (RT)-PCR using SYN-specific
primers. Real-time RT-PCR revealed that IL-1β induced SYN
expression at the transcriptional level, because the mRNA
level of SYN was upregulated by IL-1β. TNF-α had a weaker
effect on SYN transcription, whereas IL-6 had no effect (Figure 3b). These results indicate that IL-1β induces SYN expression at the mRNA level in mouse synovial fibroblasts.
IL-1β and TNF-α induce SYN expression through
activation of Erk
IL-1β induces the growth of synovial fibroblasts by activating
the MAPK pathways [19-24]. To elucidate the molecular
mechanisms of IL-1β-induced SYN expression, we first tested
the activation of all three MAPK members, including Erk, JNK,
and p38. Because culture of synovial fibroblasts with 10%
fetal bovine serum (FBS) highly activates all these MAPKs, we
first starved the mouse synovial fibroblasts for 24 hours with
media that contain 0.5% FBS, then IL-1β was added. Erk activation was analyzed with anti-phosphorylated Erk. The phosphorylations of c-Jun and ATF2 were used as reporters for the
activation of JNK1 and p38, respectively [19]. The activation
of these MAPKs was not detectable after starvation (Figure
4b–d, lane 1). Cultivation of mouse synovial fibroblasts with IL1β activated all three MAPK members, including Erk (Figure
4b, top panel), p38 (Figure 4c, top panel), and JNK1 (Figure
4d, top panel), and IL-1β significantly upregulated SYN
expression (Figure 4a). These results suggest that MAPK
pathways might be involved in IL-1β-induced SYN expression.
To further elucidate which MAPK pathway is involved in regulating IL-1β-induced SYN expression, specific inhibitors of
MAP kinases were used [13-15,25]. Erk inhibitor, PD98059,
specifically inhibited Erk phosphorylation without affecting the
activation of either JNK or p38 (Figure 4b, lane 3). In the presence of this inhibitor, SYN expression was reduced to a level
that is comparable with non-treated controls (Figure 4a, top
panel, lane 3). p38 inhibitor, SB202190, which specifically
inhibited ATF2 phosphorylation without affecting Erk and
JNK1 activation, had a very mild inhibitory effect on SYN
expression (Figure 4c, lane 4). The specific inhibitor of JNK,
SP600125, which specifically inhibited c-Jun phosphorylation,
had no effects on IL-1β-induced SYN expression (Figure 4d,
lane 5). Similarly, the Erk inhibitor also inhibited TNF-αinduced SYN expression (Figure 4e). Both IL-1β and TNF-α
are also strong activators of the NF-κB pathway [24]. We
therefore tested whether NF-κB activation is also involved in
the induction of SYN expression in mouse synovial fibroblasts.
The NF-κB-specific inhibitor, SN50, strongly inhibited both IL1β and TNF-α-induced NF-κB reporter activities (Figure 4g)
but not the SYN protein expression (Figure 4f). These findings
collectively indicate that IL-1β and TNF-α enhance SYN
expression through the activation of Erk, but not the p38, JNK,
or NF-κB pathways.
Transcription factor ETS1 is involved in IL-1β-induced
SYN expression
ETS1 is a transcription factor, the expression of which is
increased in synovial fibroblasts from patients with RA [27].
Recently, the ETS binding site (EBS), termed EBS-1, from
position -76 to -69 of the proximal promoter, was identified as
being responsible for SYN expression [28]. Interestingly, we
found that overexpression of ETS1 further enhanced IL-1βinduced SYN expression in a dose-dependent manner (Figure
5a, lanes 3 and 4; Figure 5b). To further confirm the involvement of ETS1 in SYN expression, we generated a C-terminal
truncate mutation of ETS1, which has been shown to be a
dominant negative mutant of ETS1 (ETS1-DN) [29]. As shown
in Figure 4a (lanes 5 and 6), ETS1-DN dramatically blocked
SYN expression induced by IL-1β in mouse synovial fibroblasts. These results indicate that the transcription factor is
involved in regulating IL-1β-induced SYN expression in mouse
synovial fibroblasts.
Activation of Erk and ETS1 is involved in the overgrowth
of synovial fibroblasts from CIA mice
We next tested whether the activation of Erk is enhanced in
the synovial fibroblasts from CIA mice, in which SYN expression is increased. As expected, both Erk1 activation and Erk2
activation are slightly increased in CIA synovial fibroblasts
compared with those from normal DBA1 mice (Figure 6a).
Interestingly, in treatment with the Erk-specific inhibitor, the
expression of SYN was reduced to comparable levels
between normal and CIA synovial fibroblasts. These results
suggest that Erk activation is involved in upregulating SYN
expression in vivo during CIA development. It has been
reported that the expression of ETS1 protein is increased in
the synovial fibroblasts from patients with RA [27]. We compared the activation and protein expression of ETS1 in the synovial fibroblasts from CIA mice with those from normal DBA1
mice. As shown in Figure 6a, we found that the activation of
ETS1 but not its protein expression was increased in CIA synovial fibroblasts. These results suggest that the activation of
both Erk and ETS1 is involved in regulating the expression of
SYN in mouse synovial fibroblasts during CIA.
Based on previous studies that have suggested that Erk may
activate ETS1 transcription activity [30] together with our findings that Erk and ETS1 are involved in IL-1β-induced SYN
expression, we proposed that IL-1β induces SYN expression
in mouse synovial fibroblasts via the Erk-ETS1 pathway. To
support this hypothesis, we found that inhibition of Erk activation by Erk-specific inhibitor blocked ETS1 phosphorylation
(Figure 6a). Moreover, Erk inhibitor suppressed the cell growth
of mouse synovial fibroblasts, and the synovial fibroblasts from
CIA mice were more sensitive to Erk inhibition (Figure 6b).
Treatment of these cells with the Erk inhibitor did not
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Gao et al.
Figure 4
Interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) induce Synoviolin (SYN) expression via the Erk pathway
pathway. (a) Synovial fibroblasts were partially starved for 24 hours by cultivation of these cells with media that contain 0.5% fetal bovine serum and then cultured with 10 ng/ml of IL-1β. Cells
were also treated with each of the MAPK inhibitors. The concentrations of each inhibitor used were as follows: Erk inhibitor PD98059, 20 μM; JNK
inhibitor SP600125, 10 μM; p38 inhibitor SB202190, 10 μM; and NF-κB inhibitor SN50, 20 μM. The expression of SYN was examined by Western
blotting (top panel). The protein level of actin was reprobed as a control (bottom panel). (b) The activation of Erk was analyzed by anti-p-Erk antibody
(top panel). The same membrane was reprobed by anti-Erk antibody (bottom panel). (c) p38 activation was analyzed by anti-p-ATF2 antibody (top
panel). The same membrane was reprobed by anti-ATF2 antibody (bottom panel). (d) JNK1 activation was analyzed with anti-phospho-Jun antibody
(top panels). The total protein levels of c-Jun were examined using anti-Jun (bottom panels). (e) Mouse synovial fibroblasts were starved for 24 hours
and then cultured with 10 ng/ml of TNF-α without or with Erk inhibitor. The expression of SYN was analyzed by Western blotting (top panel). The
activation of Erk was determined by anti-phosphorylated Erk antibody (middle panel). The protein level of Erk was analyzed by anti-Erk antibody (bottom panel). (f) The effect of NF-κB inhibitor on SYN expression. Mouse synovial fibroblasts were transfected with NF-κB-luc reporter, which contains firefly luciferase gene under control of NF-κB. The control plasmid pRL-TK encoding renillar luciferase was also included to correct transfection
efficiency. Transfected cells were starved and then cultivated in the presence of 10 ng/ml of IL-1β or TNF-α without or with SN50 for 24 hours. The
expression of SYN in these cells was analyzed by Western blotting (top panel), and the same membrane was reprobed with anti-actin (middle panel).
(g) Parallel prepared cell lysates from (f) were used for testing the NF-κB-driven luciferase activity (bottom panel). Error bars represent three different experiments (mean ± standard deviation). Erk, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-kappa B.
significantly arrest the cell cycle of these synovial fibroblasts
from either normal DBA or CIA mice (Figure 6c). These findings suggest that the activation of Erk and ETS1 is involved in
the overgrowth of mouse synovial fibroblasts during CIA.
Based on these findings, we collectively concluded that the
Erk-ETS1 pathway is involved in SYN expression in mouse
synovial fibroblasts induced by IL-1β and TNF-α.
Discussion
Proinflammatory cytokines, particularly IL-1β and TNF-α, can
induce the cell proliferation of synovial fibroblasts both in vitro
and in vivo [31,32]. The molecular mechanisms of proinflam-
Page 6 of 10
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matory cytokine-induced cell growth of synovial tissues have
been extensively investigated, although there still is room for
debate [33]. IL-1β and TNF-α are key activators of the many
transcription factors, including activator protein-1 (AP-1), Egr1 (early growth response-1), and NF-κB, in synovial fibroblasts. Activation of the NF-κB/Rel transcription family and AP1 complexes, composed of members of the Jun and Fos families, contributes to the hyperproliferation of fibroblast-like synoviocytes [34]. We found that IL-1β induces the proliferation
of synovial fibroblasts by upregulating SYN expression. These
findings provide a new mechanism for IL-1β in synovitis during
RA development.
Available online http://arthritis-research.com/content/8/6/R172
Figure 5
SYN transcription, as indicated by the finding that the specific
inhibitors against Erk, but not JNK and p38, block SYN expression. It has been demonstrated that Erk activation is highly
increased in the synovial fibroblasts from patients with RA.
Consistent with this, we found that the activation of Erk is significantly upregulated in mouse synovial fibroblasts from CIA
mice compared with that from normal DBA1. Interestingly, the
CIA synovial fibroblasts are more sensitive to Erk inhibitormediated cell growth inhibition. Therefore, Erk inhibitors could
be potential candidates for RA treatment.
Recently, EBS-1 was identified as a crucial site for the expression of SYN [28]. EBS-1 is a binding site for ETS family
transcription factors, including ETS1/2, GABP (growth-associated binding protein)-α/β, and Sp-1 [36]. Our findings that
ETS1 expression increases SYN transcription and that ETS1DN blocks SYN expression (Figure 4) confirm that the EBS-1
binding site is the promoter region for IL-1β-induced SYN
expression. ETS1 is activated by Erk-mediated phosphorylation [37]. Therefore, inhibition of Erk activation by specific
inhibitors blocks SYN transcription (Figure 3). Overexpression
of ETS1 family transcription factors has been observed in RA
synovial membranes [19,27]. However, we found that the activation of ETS1 but not its protein expression is upregulated in
the synovial fibroblasts from CIA mice. The elevated ETS1
activation is possibly a direct consequence of Erk activation,
because inhibition of Erk activation blocks ETS1
phosphorylation.
expression
ETS1
is involved in interleukin-1β(IL-1β)-induced Synoviolin (SYN)
expression. (a) ETS1 or ETS1-DN expression plasmids were transfected into mouse synovial fibroblasts. Cells were then cultured in
medium containing 10 ng/ml of IL-1β for 48 hours. The expression of
SYN was detected by Western blotting (top panel). The expression of
ETS1 and its mutant was detected with anti-ETS1 antibody (middle
panel). The same membrane was stripped and reblotted with anti-actin
antibody (bottom panel).(b) The expression level of SYN was quantified. Error bars represent three independent experiments (mean ±
standard deviation). ETS1-DN, dominant negative mutant of ETS1.
MAPKs are especially important in synovitis because they control the proliferation of synovial cells in the rheumatoid joint and
induce the production of MMPs and cytokines that participate
in the rheumatoid process [33,35]. The involvement of all three
MAPK family members, JNK, Erk, and p38, in RA has been
indicated by the fact that their activation is increased in rheumatoid synovial cells. They have also been implicated in the
pathogenesis of RA [33]. The binding of IL-1β to its receptor
expressed on the surface of synovial fibroblasts activates all
three MAPK members, as demonstrated in our study and previous reports [33]. Intriguingly, only Erk activation is involved in
As an E3 ubiquitin ligase on the ER membrane, SYN functions
as an ER-associated degradation system in both yeast and
mammals [8,9]. The biological functions of SYN were analyzed
in SYN transgenic mice and heterozygous knockout mice
because the homozygous mice are embryonic-lethal [11].
Interestingly, the expression level of SYN correlates significantly with the onset of arthropathy: Increased SYN expression causes synovium overgrowth and spontaneous
arthropathy, whereas reduced SYN expression (heterozygous
mutant mice) is associated with resistance to CIA [11]. Our
finding that the suppression of SYN inhibits IL-1β-induced
synovial fibroblast proliferation provides a direct rationale for
SYN as a potential target for the treatment of RA. It will be
extremely interesting to investigate the effects of gene therapeutic delivery of dominant-negative SYN or its siRNA (small
interfering RNA) on the development of arthritis in animals
such as DBA mice with CIA.
Conclusion
This study demonstrated that the proinflammatory cytokines
IL-1β and TNF-α induce synovial fibroblast growth by
upregulating the expression of an E3 ubiquitin ligase, SYN. At
the molecular level, activation of MAPK Erk and transcription
factor ETS1 is required for SYN expression. Based on our
findings and the fact that synovial fibroblasts are among the
major resources for IL-1β and TNF-α in the rheumatoid joints
Page 7 of 10
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Arthritis Research & Therapy
Vol 8 No 6
Gao et al.
Figure 6
Erk inhibition suppresses ETS1 activation and hyperproliferation of mouse synovial fibroblasts from collagen-induced arthritis (CIA) mice
mice. (a) Synovial fibroblasts isolated from CIA or normal mice were cultured without or with Erk inhibitor. The expression of Synoviolin (SYN) was determined by
Western blotting by using anti-SYN antibody (top panel). The activation and protein levels of Erk in the parallel prepared cell lysates were detected
by anti-phospho-Erk and anti-Erk, respectively (the second and third panels). Similarly, the activation and protein levels of ETS1 in the parallel prepared cell lysates were detected by anti-phospho-ETS1 and anti-ETS1, respectively (the bottom two panels). (b) Synovial cells were cultured without or with Erk inhibitor in 12-well plates for 24 hours. One microcurie of 3H-tymidine was added to each well of plated cells and further cultured for
16 hours. 3H-tymidine incorporation was analyzed as described previously [18]. Error bars represent three independent experiments (mean ± standard deviation). (c) Cell cycle analysis of Erk inhibitor-treated synovial fibroblasts. Cells treated with or without the Erk-specific inhibitor were collected
and fixed in cold methanol and then stained with propidium iodide (PI) in the presence of RNase. PI-stained cells were washed once with phosphatebuffered saline and then analyzed by flow cytometry. The cell death was significantly increased when CIA synovial fibroblasts were treated with Erk
inhibitor, PD98059 (PD). CPM, counts per minute; Erk, extracellular signal-regulated kinase; FBS, fetal bovine serum.
[31], we proposed a positive feedback model for SYN in RA
development (Additional file 2). According to this model, IL-1β
induces SYN transcription, SYN enhances IL-1β-induced synovial fibroblast proliferation, and the massive growth of synovial fibroblasts produces more IL-1β for the induction of SYN
expression. This positive feedback process may be critical in
RA development. Molecules in this signal pathway can therefore be potential targets against RA.
Page 8 of 10
(page number not for citation purposes)
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
BG conceived and performed most of the experiments, including anti-SYN antibody generation and signal transduction
analysis. KC participated in drafting the manuscript. DF
designed and organized the study, drafted the manuscript, and
Available online http://arthritis-research.com/content/8/6/R172
performed experiments in inducing arthritis and isolating synovial fibroblasts. All authors read and approved the final
manuscript.
Additional files
The following Additional files are available online:
5.
6.
7.
Additional file 1
Collagen-induced arthritis in DBA/1 mice. DBA/1 mice
at the age of 6 weeks were immunized with 100 μg of
collagen in complete Freund's Adjuvant on day 0 and
boosted with 100 μg of collagen in incomplete Freund's
Adjuvant on day 21. Ten DBA/1 mice were used.
Severity of joint inflammation (a) and incidence of
arthritis (b) were scored.
See http://www.biomedcentral.com/content/
supplementary/ar2081-S1.tiff
9.
Additional file 2
11.
A proposed model for interleukin-1β(IL-1β)-induced
Synoviolin (SYN) expression in rheumatoid arthritis. IL-1β
stimulates synovial fibroblasts and activates Erk.
Activated Erk drives ETS1 activation for the transcription
of SYN mRNA. The upregulated SYN increases the
proliferation of synovial cells, which induces arthritis. The
increased synovial fibroblasts produce more IL-1β and
thereby facilitate the development of arthritis. Erk,
extracellular signal-regulated kinase.
See http://www.biomedcentral.com/content/
supplementary/ar2081-S2.tiff
8.
10.
12.
13.
14.
15.
16.
Acknowledgements
We thank Dr. Christopher H. Evans (Center for Molecular Orthopaedics,
Harvard Medical School, Boston, MA, USA) for providing protocols for
mouse synovial fibroblast isolation. We also thank Dr. Krishna Kannan
(Division of Rheumatology, Department of Internal Medicine, University
of Missouri-Columbia) for his critical comments on the manuscript. This
work was partially supported by a research board grant from the University of Missouri and by an investigator research award from the Arthritis
Foundation to DF.
17.
18.
19.
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Registered report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation
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eLife
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cc-by
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Registered report: COT drives resistance
to RAF inhibition through MAP kinase
pathway reactivation Vidhu Sharma1, Lisa Young1, Miguel Cavadas2, Kate Owen3,
Reproducibility Project: Cancer Biology1* 1Applied Biological Materials, Richmond, Canada; 2University of College Dublin,
Dublin, Ireland; 3University of Virginia, Charlottesville, United States 1Applied Biological Materials, Richmond, Canada; 2University of College Dublin,
Dublin, Ireland; 3University of Virginia, Charlottesville, United States Abstract The Reproducibility Project: Cancer Biology seeks to address growing concerns about
reproducibility in scientific research by conducting replications of selected experiments from a
number of high-profile papers in the field of cancer biology. The papers, which were published
between 2010 and 2012, were selected on the basis of citations and Altmetric scores
(Errington et al., 2014). This Registered Report describes the proposed replication plan of key
experiments from “COT drives resistance to RAF inhibition through MAPK pathway reactivation”
by Johannessen and colleagues, published in Nature in 2010 (Johannessen et al., 2010). The key
experiments to be replicated are those reported in Figures 3B, 3D-E, 3I, and 4E-F. In Figures 3B, D-
E, RPMI-7951 and OUMS023 cells were reported to exhibit robust ERK/MEK activity concomitant
with reduced growth sensitivity in the presence of the BRAF inhibitor PLX4720. MAP3K8 (COT/
TPL2) directly regulated MEK/ERK phosphorylation, as the treatment of RPMI-7951 cells with a
MAP3K8 kinase inhibitor resulted in a dose-dependent suppression of MEK/ERK activity (Figure 3I). In contrast, MAP3K8-deficient A375 cells remained sensitive to BRAF inhibition, exhibiting reduced
growth and MEK/ERK activity during inhibitor treatment. To determine if RAF and MEK inhibitors
together can overcome single-agent resistance, MAP3K8-expressing A375 cells treated with
PLX4720 along with MEK inhibitors significantly inhibited both cell viability and ERK activation
compared to treatment with PLX4720 alone, as reported in Figures 4E-F. The Reproducibility
Project: Cancer Biology is collaboration between the Center for Open Science and Science
Exchange and the results of the replications will be published in eLife. DOI: 10.7554/eLife.11414.001 Funding: See page 31
Received: 17 September 2015
Accepted: 07 March 2016
Published: 21 March 2016 REGISTERED REPORT Introduction Activation of the canonical mitogen activated protein kinase (MAPK) pathway occurs in response to
the binding of growth factors, hormones, or neurotransmitters to receptor tyrosine kinase receptors
located at the cell surface (Dhomen and Marais, 2009; Lopez-Bergami et al., 2008). In untrans-
formed cells, receptor ligation induces the sequential activation of the small GTPase RAS, followed
by RAF, MEK and ERK, which relays proliferative signals generated at the cell periphery into the
nucleus to control cellular survival, differentiation and growth (Inamdar et al., 2010; Panka et al.,
2006). Not surprisingly, dysregulation of MAPK signaling is common in many human cancers includ-
ing melanoma. Mutations in the RAF and RAS genes (Davies et al., 2002; Mercer and Pritchard,
2003) that trigger constitutive activation of the MAPK pathway can result in uncontrolled cell prolif-
eration, invasion, metastasis, survival and angiogenesis (Panka et al., 2006; Sharma et al., 2006;
Smalley et al., 2006; Smalley and Herlyn, 2006). Received: 17 September 2015
Accepted: 07 March 2016
Published: 21 March 2016 Reviewing editor: Tony Hunter,
Salk Institute, United States Copyright Sharma et al. This
article is distributed under the
terms of the Creative Commons
Attribution License, which
permits unrestricted use and
redistribution provided that the
original author and source are
credited. BRAF is one of three members of the RAF family, which includes ARAF, BRAF, and CRAF (or
RAF-1) (Dhomen and Marais, 2009). In melanoma, BRAF represents the most commonly mutated Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 1 of 32 Registered report Biochemistry gene in the MAPK signaling cascade where 90% of tumors carry a valine to glutamic acid transition
at codon 600 (V600E) that renders BRAF constitutively active and hyperactivates the MAPK cascade
(Davies et al., 2002; Dhomen and Marais, 2009; Michaloglou et al., 2008). While preclinical and
clinical studies have shown that targeting BRAF (V600E) melanomas with the use of RAF-selective
inhibitors results in initial tumor regression (Fedorenko et al., 2015; Flaherty et al., 2010;
Shtivelman et al., 2014), responses to RAF inhibitors are transient, with acquired resistance trigger-
ing disease progression (Shtivelman et al., 2014). Although progress has been made in the devel-
opment of drugs that target RAF, the clinical outcome regarding long-term usage and the
mechanisms of acquired resistance warrants further evaluation. In their study, Johannessen and col-
leagues sought to identify kinases involved in mediating resistance to the RAF kinase inhibitor
PLX4720 (Johannessen et al., 2010). Using a kinase open reading frame (ORF) collection and a high throughput screening methodol-
ogy, Johannessen and colleagues identified MAP3K8 (the gene encoding cancer osaka thyroid
(COT)/TPL2), as a driver of resistance to BRAF inhibition with PLX4720 (Johannessen et al., 2010). Johannessen and colleagues first examined basal MAP3K8 expression in multiple cell lines harboring
the V600E mutation (Johannessen et al., 2010). As shown in Figure 3B and reported by others,
RPMI-7951 and OUMS-23 cells were found to express high intrinsic levels of MAP3K8 compared to
A375 cells where MAP3K8 was undetectable (Johannessen et al., 2010; Paraiso et al., 2012). RPMI-7951 and OUMS-23 cells also exhibited robust, undiminished ERK and MEK activity concomi-
tant with reduced growth sensitivity in the presence of PLX4720 (Figure 3D–E; Johannessen et al.,
2010). This is supported by additional findings demonstrating that RPMI-7981 cells treated with the
closely related BRAF inhibitor PLX4032/vemurafenib (a successor of PLX4720) also remain refractory
to inhibitor treatment as assessed by annexin V staining (Paraiso et al., 2012) and MTS assay
(Park et al., 2013). Registered report Materials and methods Materials and methods Protocol 1: MAPK pathway analysis in cells expressing elevated
MAP3K8 This experiment assesses the effect the RAF inhibitor, PLX4720, has on the MAPK pathway, in cells
expressing elevated MAP3K8, as analyzed via Western blot. It utilizes RPMI-7951 and OUMS-23
cells, which express a high level of MAP3K8 and A375 cells, which have undetectable levels. This
protocol replicates the experiments reported in Figures 3B and 3E. p
g
protocol replicates the experiments reported in Figures 3B and 3E. Sampling
Experiment to be repeated a total of 4 times for a minimum power of 80%. The original data is qual-
itative, thus to determine an appropriate number of replicates to initially perform, sample sizes
based on a range of potential variance was determined. . See Power Calculations section for details. Experiment has 3 cohorts:
. Cohort 1: A375 cells
. Cohort 2: RPMI-7951 cells
. Cohort 3: OUMS-23 cells
Each cohort has four conditions:
. Vehicle (DMSO)
. 10 mM PLX4720
. 1 mM PLX4720
. 0.1 mM PLX4720
Each condition will be probed with the following antibodies:
. pERK1/2 (T202/Y204)
. pERK1/2
. pMEK1/2 (S217/221)
. MEK1/2
. MAP3K8
. Actin
Materials and reagents
Reagent
Type
Manufacturer
Catalog #
Comments
RPMI medium with L-glutamine
Cell culture
Sigma
R8758
Replaces Corning cat no. 10-040-CV. Communicated by authors. MEM with L-glutamine
Cell culture
Sigma
M4655-500ML
Replaces Corning cat. No. 10-010-CV. Communicated by authors. Fetal bovine serum
(FBS)
Cell culture
Life Technologies
12483-020
Replaces Corning brand. Communicated by authors. Pen/strep/glutamine
Cell culture
Abm
G255
Replaces Corning brand. Communicated by authors. A375 cells
Cell line
ATCC
CRL-1619
Original brand not specified. RPMI-7951 cells
Cell line
ATCC
HTB-66
OUMS-23 cells
Cell line
JCRB
JCRB1022
Original brand not specified. 6-well plates
Labware
Greiner bio-one
657 160
Original brand not specified. Phosphate buffered saline
(PBS)
Buffer
Sigma
D8537-500ML
Original brand not specified. Trypsin
Cell culture
Sigma
T4049
Original brand not specified. Continued on next page Reviewing editor: Tony Hunter,
Salk Institute, United States However, others have reported RPMI-7981 cells as exhibiting modest sensitivity
to PLX4720 (Schayowitz et al., 2012). In the latter case, ERK activity was reduced by 50% after incu-
bation with inhibitor, although these differences in sensitivity may reflect the significantly shorter
time course and experimental design used by Park and colleagues. Finally, in Figure 3I, Johannessen
and colleagues determined that MAP3K8 kinase activity is required to regulate MEK/ERK activation
in RPMI-7951 cells, findings that further confirm MAP3K8 is an essential upstream activator of the
MEK-ERK signaling cascade (George and Salmeron, 2009; Johannessen et al., 2010). The key
experiments outlined in Figures 3B,D,E, and 3I will be replicated in protocols 1, 2, 3, and 4. p
g
p
p
Resistance to targeted agents, such as BRAF inhibitors, is a frequent cause of therapy failure, as
noted above. Importantly, chronic BRAF inhibition can lead to cross-resistance to several BRAF-
selective inhibitors, indicating that resistance is not likely to be overcome by switching to a new RAF
inhibitor (Corcoran et al., 2010; Villanueva et al., 2011). It has been suggested previously that
combination treatment with MEK and BRAF inhibitors may be useful in preventing the emergence of
resistance or in overcoming resistance to single agent therapies targeting either molecule alone
(Corcoran et al., 2010). To examine whether the combined use of RAF and MEK inhibitors bypass
MAP3K8-driven resistance, Johannessen and colleagues ectopically expressed MAP3K8 in A375 mel-
anoma cells before treatment with BRAF inhibitor (PLX4720) alone or in combination with the MEK
inhibitors CI-1040 or AZD6244. As shown in Figures 4E and 4F, both viability and ERK activation was
dramatically reduced in MAP3K8-expressing cells treated with either of the combination therapies,
similar
to
cells
ectopically
expressing
MEK1,
which
remained
sensitive
to
PLX4720
(Johannessen et al., 2010). Similar results were obtained in RPMI-7951 cells expressing high basal
levels of MAP3K8 treated with PLX4032 and a second MEK inhibitor AS703026 (Park et al., 2013). Interestingly, overexpression of constitutively active MEK (MEK1DD) resulted in increased sensitivity
to BRAF inhibition combined with AZD6244, but not CI-1040 (Johannessen et al., 2010). These find-
ings confirm that MAP3K8 is able to reactivate MAPK signaling despite BRAF inhibition and that tar-
geting RAF and MEK in combination may be an effective anti-melanoma treatment strategy. These
experiments will be replicated in Protocols 5 and 6. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 2 of 32 Registered report Registered report Biochemistry Sampling p
g
Experiment to be repeated a total of 4 times for a minimum power of 80%. The original data is qual-
itative, thus to determine an appropriate number of replicates to initially perform, sample sizes
based on a range of potential variance was determined. . See Power Calculations section for details. . See Power Calculations section for details. Experiment has 3 cohorts:
. Cohort 1: A375 cells
. Cohort 2: RPMI-7951 cells
. Cohort 3: OUMS-23 cells
Each cohort has four conditions:
. Vehicle (DMSO)
. 10 mM PLX4720
. 1 mM PLX4720
. 0.1 mM PLX4720
Each condition will be probed with the following antibodies:
. pERK1/2 (T202/Y204)
. pERK1/2
. pMEK1/2 (S217/221)
. MEK1/2
. MAP3K8
. Actin Materials and reagents
Reagent
Type
Manufacturer
Catalog #
Comments
RPMI medium with L-glutamine
Cell culture
Sigma
R8758
Replaces Corning cat no. 10-040-CV. Communicated by authors. MEM with L-glutamine
Cell culture
Sigma
M4655-500ML
Replaces Corning cat. No. 10-010-CV. Communicated by authors. Fetal bovine serum
(FBS)
Cell culture
Life Technologies
12483-020
Replaces Corning brand. Communicated by authors. Pen/strep/glutamine
Cell culture
Abm
G255
Replaces Corning brand. Communicated by authors. A375 cells
Cell line
ATCC
CRL-1619
Original brand not specified. RPMI-7951 cells
Cell line
ATCC
HTB-66
OUMS-23 cells
Cell line
JCRB
JCRB1022
Original brand not specified. 6-well plates
Labware
Greiner bio-one
657 160
Original brand not specified. Phosphate buffered saline
(PBS)
Buffer
Sigma
D8537-500ML
Original brand not specified. Trypsin
Cell culture
Sigma
T4049
Original brand not specified. 3 of 32 Biochemistry Continued
Reagent
Type
Manufacturer
Catalog #
Comments
10 cm plates
Labware
CellStar
664 160
Original brand not specified. PLX4720
Inhibitor
Selleck
Chemicals
S1152
Replaces Symansis brand. DMSO
Chemical
Sigma
D4540
Original brand not specified. NP-40 buffer
Buffer
Life tech
FNN0021
Original brand not specified. Protease inhibitors
Inhibitor
Roche
04693116001
Original catalog # not specified. Phosphatase inhibitor cocktail I
Inhibitor
Sigma
P2850
Replaces CalBioChem brand. Phosphatase inhibitor cocktail II
Inhibitor
Sigma
P5726
Replaces CalBioChem brand. Cell scraper
Labware
Sarstedt
83.1830
Original brand not specified. BCA kit
Reporter assay
Pierce
23227
Original catalog # not specified. Communicated by authors. Dithiothreitol (DTT)
Chemical
Biobasic
DB0058
Original brand not specified. Sample buffer
Buffer
Abm
G031
Replaces Invitrogen brand. Protein molecular weight
ladder
Western materials
Abm
G252, G494
Original brand not specified. 10% Tris/Glycine gel;
10 well, 1.0 mm thick
Western materials
Abm
Internal
Replaces Invitrogen brand. Running buffer
Buffer
Abm
Internal
Original brand not specified. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 p
p
y
g
.
Cells will be sent for mycoplasma testing and STR profiling. .
RPMI-7951 and OUMS-23 cells maintained in MEM medium supplemented with 10% FBS and
1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2. y
g
p
.
RPMI-7951 and OUMS-23 cells maintained in MEM medium supplemented with 10% FBS an
1% penicillin/streptomycin/L glutamine at 37˚C in a humidified atmosphere at 5% CO Sampling Immobilon P
Western materials
Thermofisher
IPVH00010
Original brand not specified. Transfer buffer
Buffer
Abm
Internal
Original brand not specified. Mouse anti-pERK1/2
(T202/Y204) (clone E10)
antibody (clone E10)
Antibodies
Cell Signaling
9106
Use at 1:1000 dilution. Original catalog # not specified. Rabbit anti-pMEK1/2
(S217/221) (clone 41G9)
antibody
Antibodies
Cell Signaling
9154
Use at 1:11000 dilution. Original catalog # not specified. Mouse anti-p44/42 MAPK
(ERK1/2) (clone L34F12)
antibody
Antibodies
Cell Signaling
4696
Use at 1:11000 dilution. Replaces catalog # 4695. Communicated by authors. Rabbit anti-MEK1/2
(clone D1A5) antibody
Antibodies
Cell Signaling
8727
Use at 1:1000 dilution. Original catalog # not specified. Rabbit anti-MAP3K8
(clone M-20) antibody
Antibodies
Santa Cruz
sc-720
Use at 1:500 dilution. Communicated by authors. Mouse anti-ß-Actin
(clone C4) antibody
Antibodies
Santa Cruz
sc-47778
Use at 1:100 – 1:1000 dilution. Original catalog # not specified. Anti-rabbit IgG – HRP
conjugated antibody
Antibodies
Cell Signaling
7074
Use at 1:1000 dilution. Original catalog # not specified. Anti-mouse IgG – HRP
conjugated antibody
Antibodies
Cell Signaling
7076
Use at 1:1000 dilution. Original catalog # not specified. Chemiluminescent
reagent
Western materials
Life Technologies
WP20005
Replaces Pierce brand. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 4 of 32 4 of 32 Registered report Registered report Biochemistry 1. Plate 500,000 A375 cells, 750,000 RPMI-7951, and 750,000 OUMS-23 cells in 6-well plates
and incubate for 24–36 hr to achieve log phase growth. 1. Plate 500,000 A375 cells, 750,000 RPMI-7951, and 750,000 OUMS-23 cells in 6-well plates
and incubate for 24–36 hr to achieve log phase growth. g p
g
2. 24–36 hr after seeding treat cells with 0.1, 1, and 10 mM PLX4720 or DMSO. Incubate for
24 hr. a. Add drug directly to each well using a 1000X stock (in DMSO). i. Final DMSO concentration kept to 0.1%. 2. 24–36 hr after seeding treat cells with 0.1, 1, and 10 mM PLX4720 or DMSO. Incubate for
24 hr. a
Add drug directly to each well using a 1000X stock (in DMSO) a. Add drug directly to each well using a 1000X stock (in DMSO). i. Final DMSO concentration kept to 0.1%. a. Add drug directly to each well using a 1000X stock (in DMSO). i. Final DMSO concentration kept to 0.1%. 3. Wash cells with 1–2 ml ice-cold PBS and lyse in 1% NP-40 lysis buffer supplemented with 2X
protease inhibitors and 1X phosphatase inhibitor cocktails I and II. 3. Sampling Wash cells with 1–2 ml ice-cold PBS and lyse in 1% NP-40 lysis buffer supplemented with 2X
protease inhibitors and 1X phosphatase inhibitor cocktails I and II. a. Add ~ 100–200 ml 1% NP-40 lysis buffer to ensure that protein concentration is between
2–3 mg/ml. a. Add ~ 100–200 ml 1% NP-40 lysis buffer to ensure that protein concentration is between
2–3 mg/ml. mg m
b. b. Scrape each plate with a rubber cell scraper, collect lysates, and clarify by centrifuga-
tion at max speed (table-top microfuge) at 4˚C. b. b. Scrape each plate with a rubber cell scraper, collect lysates, and clarify by centrifuga- b. b. Scrape each plate with a rubber cell scraper, collect lysates, and clarify by centrifuga-
tion at max speed (table-top microfuge) at 4˚C. tion at max speed (table-top microfuge) at 4˚C. 4. Determine protein concentration by BCA assay, normalize, reduce with DTT, and denature
at 88˚C. 5. Separate 35–50 mg of protein per lane on a 10% Tris/Glycine gel with protein ladder follow-
ing replicating lab’s standard protocol. ing replicating lab’s standard protocol g ep cat g ab s sta da d p otoco . a. Samples run per gel:
i. Protein molecular weight marker
ii. Vehicle (DMSO) treated A375 cells
iii. 10 mM PLX4720 treated A375 cells
iv. 1 mM PLX4720 treated A375 cells
v. 0.1 mM PLX4720 treated A375 cells
vi. Vehicle (DMSO) treated RPMI-7951 cells
vii. 10 mM PLX4720 treated RPMI-7951 cells
viii. 1 mM PLX4720 treated RPMI-7951 cells
ix. 0.1 mM PLX4720 treated RPMI-7951 cells
x. Vehicle (DMSO) treated OUMS-23 cells
xi. 10 mM PLX4720 treated OUMS-23 cells
xii. 1 mM PLX4720 treated OUMS-23 cells
xiii. 0.1 mM PLX4720 treated OUMS-23 cells a. Samples run per gel: a. Samples run per gel:
i. Protein molecular weight marker
ii. Vehicle (DMSO) treated A375 cells
iii. 10 mM PLX4720 treated A375 cells
iv. 1 mM PLX4720 treated A375 cells
v. 0.1 mM PLX4720 treated A375 cells
vi. Vehicle (DMSO) treated RPMI-7951 cells
vii. 10 mM PLX4720 treated RPMI-7951 cells
viii. 1 mM PLX4720 treated RPMI-7951 cells
ix. 0.1 mM PLX4720 treated RPMI-7951 cells
x. Vehicle (DMSO) treated OUMS-23 cells
xi. 10 mM PLX4720 treated OUMS-23 cells
xii. 1 mM PLX4720 treated OUMS-23 cells
xiii. 0.1 mM PLX4720 treated OUMS-23 cells i. Protein molecular weight marker ii. Vehicle (DMSO) treated A375 cells iii. 10 mM PLX4720 treated A375 cells iv. 8. Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation,
d h
d
l
h
l
f ll
f 8. Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation,
and then detect signal using chemiluminescence following manufacturer’s instructions.
a. Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary
antibodies and a low fluorescence membrane will be used instead, and images will be
acquired following manufacturer’s instructions. Confirmatory analysis plan . Statistical Analysis of the Replication Data:
Two-way MANOVA of normalized pERK1/2 and pMEK1/2 levels of A375, RPMI-7951,
and OUMS-23 cells with the following planned comparisons using the Bonferron
Two-way MANOVA of normalized pERK1/2 and pMEK1/2 levels of A375, RPMI-7951,
and OUMS-23 cells with the following planned comparisons using the Bonferron
correction: &
Planned contrast of normalized pERK1/2 levels from A375 cells treated with vehicle
compared to cells treated with PLX4720 (all doses). &
Planned contrast of normalized pERK1/2 levels from A375 cells treated with vehicle &
Planned contrast of normalized pERK1/2 levels from A375 cells tr
compared to cells treated with PLX4720 (all doses). p
compared to cells treated with PLX4720 (all doses). &
Planned contrast of normalized pERK1/2 levels from RPMI-7951 cells treated with
vehicle compared to cells treated with PLX4720 (all doses). &
Planned contrast of normalized pERK1/2 levels from RPMI-
vehicle compared to cells treated with PLX4720 (all doses). p
&
Planned contrast of normalized pERK1/2 levels from OUMS-23 cells treated with
vehicle compared to cells treated with PLX4720 (all doses). p
&
Planned contrast of normalized pMEK1/2 levels from A375 cells treated with vehicle
compared to cells treated with PLX4720 (all doses). p
&
Planned contrast of normalized pMEK1/2 levels from RPMI-7951 cells treated with
vehicle compared to cells treated with PLX4720 (all doses). &
Planned contrast of normalized pMEK1/2 levels from OUMS-23 cells treated with
vehicle compared to cells treated with PLX4720 (all doses). p
. Meta-analysis of original and replication attempt effect sizes:
The replication data (mean and 95% confidence interval) will be plotted with the original
reported data value plotted as a single point on the same plot for comparison. Known differences from the original study The replication will not include the other BRAF (V600E) cell lines reported in the original paper. The
original NP40 cell lysis buffer was composed of: 150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA pH
8, 25 mM NaF, and 1% NP-40. The replication will use a commercial formula, which has the following
composition: 250 mM NaCl, 50 mM Tris pH 7.4, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, and 1%
NP-40. The western blots will use Actin, instead of Vinculin, which was reported in Figure 3B. All
known differences are listed in the materials and reagents section above with the originally used
item listed in the comments section. Provisions for quality control y
The cell line used in this experiment will undergo STR profiling to confirm its identity and will be
sent for mycoplasma testing to ensure there is no contamination. All of the raw data, including the
analysis files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made pub-
lically available. Sampling pERK1/2 (T202/Y204) normalized to MEK1/2 (total). b. pMEK1/2 (S217/221) normalized to ERK1/2 (total). c. MAP3K8 normalized to Actin. 10. Repeat steps 1–9 independently three additional times. Biochemistry Biochemistry 9. Analyze bands with image analysis software and normalize to loading controls. a
pERK1/2 (T202/Y204) normalized to MEK1/2 (total) a. pERK1/2 (T202/Y204) normalized to MEK1/2 (total). p
pMEK1/2 (S217/221) normalized to ERK1/2 (tota c. MAP3K8 normalized to Actin. 10. Repeat steps 1–9 independently three additional times. Deliverables: . Data to be collected: . Data to be collected:
Full image western blot films of all immunoblots including ladder. (Compare to Figures
3B and 3E)
Raw data of band analysis and normalized bands for each sample.
Full image western blot films of all immunoblots including ladder. (Compare to Figures
3B and 3E)
Full image western blot films of all immunoblots including ladder. (Compare to Figures
3B and 3E) )
Raw data of band analysis and normalized bands for each sample. Confirmatory analysis plan All differences have the same capabilities as the original and
are not expected to alter the experimental design. Sampling 1 mM PLX4720 treated A375 cells v. 0.1 mM PLX4720 treated A375 cells m
vi. Vehicle (DMSO) treated RPMI-7951 cells vii. 10 mM PLX4720 treated RPMI-7951 cells viii. 1 mM PLX4720 treated RPMI-7951 cells ix. 0.1 mM PLX4720 treated RPMI-7951 cells x. Vehicle (DMSO) treated OUMS-23 cells xi. 10 mM PLX4720 treated OUMS-23 cells xii. 1 mM PLX4720 treated OUMS-23 cells xiii. 0.1 mM PLX4720 treated OUMS-23 cells 6. Wet transfer with supplied wet-transfer cassette apparatus to immobilon P following repli-
cating lab’s standard protocol. 6. Wet transfer with supplied wet-transfer cassette apparatus to immobilon P following repli-
cating lab’s standard protocol. a. Original transfer protocol was for 120min at 30–35 V at 4˚C. cating lab s standard protocol. a. Original transfer protocol was for 120min at 30–35 V at 4˚C. g
a. Original transfer protocol was for 120min at 30–35 V at 4˚C. g
p
7. After transfer, block non-specific binding and immunoblot membrane with the following pri-
mary antibodies for 18 at 4˚C following manufacturer recommendations:
a. mouse anti-pERK1/2 (T202/Y204); use at 1:1000 dilution; 42, 44 kDa
b. mouse anti-ERK1/2; use at 1:1000 dilution; 42, 44 kDa
c. rabbit anti-pMEK1/2 (S217/221); use at 1:1000 dilution; 45 kDa
d. rabbit anti-MEK1/2; use at 1:1000 dilution; 45 kDa
e. rabbit anti- MAP3K8; use at 1:500 dilution; 52, 58 kDa
f. mouse anti-ß-Actin; use at 1:100 - 1:1000 dilution; 43 kDa 7. After transfer, block non-specific binding and immunoblot membrane with the following pri-
mary antibodies for 18 at 4˚C following manufacturer recommendations:
a. mouse anti-pERK1/2 (T202/Y204); use at 1:1000 dilution; 42, 44 kDa
b. mouse anti-ERK1/2; use at 1:1000 dilution; 42, 44 kDa
c. rabbit anti-pMEK1/2 (S217/221); use at 1:1000 dilution; 45 kDa
d. rabbit anti-MEK1/2; use at 1:1000 dilution; 45 kDa
e. rabbit anti- MAP3K8; use at 1:500 dilution; 52, 58 kDa
f. mouse anti-ß-Actin; use at 1:100 - 1:1000 dilution; 43 kDa Protocol 1 Western Blot Antibody
POI
Loading Control
Independent
Gels
Description
Working
Conc. Description
Working
Conc. 1
Mouse anti-pERK1/2
(T202/Y204) (42, 44kDa)
1:1000
Rabbit anti-MEK1/2
(45 kDa)
1:1000
2
Rabbit anti-pMEK1/2
(S217/221) (45 kDa)
1:1000
Mouse anti-ERK1/2
(42, 44 kDa)
1:1000
3
Rabbit anti-MAP3K8
(52, 58 kDa)
1:500
Mouse anti-ß-Actin
(43 kDa)
1:100 – 1:1000 Protocol 1 Western Blot Antibody Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 5 of 32 Registered report 9. Analyze bands with image analysis software and normalize to loading controls. a. Protocol 2: Determine the range of detection of the replicating lab’s
plate reader This is a general protocol that determines the range of detection of the plate reader in order to cal-
culate the required number of A375, RPMI-7951, and OUMS-23 cells to yield 90–95% confluency in
5 days for Protocols 3 and 5. 6 of 32 6 of 32 Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Registered report Biochemistry Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 .
A375 cells maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/strep-
tomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2.
.
RPMI-7951 and OUMS-23 cells maintained in MEM medium supplemented with 10% FBS
and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2.
.
Cells will be sent for mycoplasma testing and STR profiling. Sampling Sampling
This experiment is performed a total of once with three cell lines (A375, RPMI-7951, and OUMS-23
cells) p
g
This experiment is performed a total of once with three cell lines (A375, RPMI-7951, and OUM
cells). Each cell line has 5 conditions to be performed with six technical replicates per experiment: . A375 cells:
1600 cells/well
1400 cells/well
1200 cells/well
1000 cells/well
800 cells/well
. RPMI-7951 cells
3400 cells/well
3200 cells/well
3000 cells/well
2800 cells/well
2600 cells/well
. OUMS-23 cells
3400 cells/well
3200 cells/well
3000 cells/well
2800 cells/well
2600 cells/well Materials and reagents Materials and reagents
Reagent
Type
Manufacturer
Catalog #
Comments
RPMI medium with
L-glutamine
Cell culture
Sigma
R8758
Replaces Corning
cat no. 10-040-CV. MEM with L-glutamine
Cell culture
Sigma
M4655-500ML
Replaces Corning
cat. no. 10-010-CV. FBS
Cell culture
Life Technologies
12483-020
Replaces Corning brand. Pen/strep/glutamine
Cell culture
Abm
G255
Replaces Corning brand. A375 cells
Cell line
ATCC
CRL-1619
Original brand not specified. RPMI-7951 cells
Cell line
ATCC
HTB-66
OUMS-23 cells
Cell line
JCRB
JCRB1022
Original brand not specified. PBS
Buffer
Sigma
D8537-500ML
Original brand not specified. Trypsin
Cell culture
Sigma
T4049
Original brand not specified. 10 cm plates
Labware
CellStar
664 160
Original brand not specified. 96 well clear plates
Labware
Sarstedt
83.3924
Original brand not specified. WST1 viability assay
Reporter assay
Roche
11644807001
Original catalog # not specified. Microplate reader
(420–480 nm)
Instrument
Molecular Devices
abm
Original brand not specified. Registered report Procedure
N . A375 cells maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/strep-
tomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2. . A375 cells maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/strep-
tomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2. . RPMI-7951 and OUMS-23 cells maintained in MEM medium supplemented with 10% FBS
and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2. . Cells will be sent for mycoplasma testing and STR profiling. . RPMI-7951 and OUMS-23 cells maintained in MEM medium supplemented with 10% FBS
and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2. . Cells will be sent for mycoplasma testing and STR profiling. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 7 of 32 7 of 32 Registered report 1. Plate 800 – 1600 A375 cells, 2600 – 3400 RPMI-7951 cells, and 2600 – 3400 OUMS-23 cells in
96 well plates with 100 ml of medium. Incubate for 5 days. 1. Plate 800 – 1600 A375 cells, 2600 – 3400 RPMI-7951 cells, and 2600 – 3400 OUMS-23 cells in
96 well plates with 100 ml of medium. Incubate for 5 days. a. Plate media alone (no cells) in columns 1 and 12. b
Plate cells in remaining wells (columns 2 11) a. Plate media alone (no cells) in columns 1 and 1 a. Plate media alone (no cells) in columns 1 and 12. b. Plate cells in remaining wells (columns 2–11). b. Plate cells in remaining wells (columns 2–11). b. Plate cells in remaining wells (columns 2–11). c. Exclude plating in the first and last row to avoid edge effects and evaporation. 2. 5 days later estimate confluency and determine cell viability with the WST1 viability as c. Exclude plating in the first and last row to avoid edge effects and evaporation. 2. 5 days later estimate confluency and determine cell viability with the WST1 viability assay
according to manufacturer’s instructions. Briefly described: according to manufacturer’s instructions. Briefly described: according to manufacturer’s instructions. Briefly described: a. Add 11 ml/well reagent WST-1 (1:10 dilution). m
g
b. Incubate cells for 20–30 min. b. Incubate cells for 20–30 min. c. Shake thoroughly for 1 min on a shaker. d. Measure the absorbance against a background control as blank using a microplate
reader at 420–480 nm. Provisions for quality control The cell line used in this experiment will undergo STR profiling to confirm its identity and will be
sent for mycoplasma testing to ensure there is no contamination. This All of the raw data, including
the analysis files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made
publically available. Protocol 3: PLX4720 growth inhibitory analysis in cells expressing
elevated MAP3K8 This experiment assesses the effect the RAF inhibitor, PLX4720, has on cellular viability, in cells
expressing elevated MAP3K8. It utilizes RPMI-7951 and OUMS-23 cells, which express a high level
of MAP3K8 and A375 cells, which have undetectable levels. This protocol replicates the experiment
reported in Figure 3D. Deliverables . Data to be collected: . Data to be collected:
Raw data and background subtracted absorbance at 420–480 nm. Confirmatory analysis plan Confirmatory analysis plan Known differences from the original study All known differences are listed in the materials and reagents section above with the originally used
item listed in the comments section. All differences have the same capabilities as the original and
are not expected to alter the experimental design. Procedure
N (If reference wavelength is to be determined, a filter >600 nm is
recommended) e. Exclude rows A-H due to edge effects/evaporation, thus making each seeding six techni-
cal replicates. f. Calculate viability after background subtraction. f. Calculate viability after background subtraction. g. Use starting cell numbers that give ~ 90–95% confluency in 5 days and is in the linear
range of the viability assay. i. Original report used 1500 A375 cells, 3000 RPMI-7951 cells, and 3000 OUMS-23
cells. Deliverables .
See Power Calculations section for details. Sampling p
g
Experiment to be repeated a total of 3 times for a minimum power of 80%. The original data is from
a single biological replicate, thus to determine an appropriate number of replicates to initially per-
form, sample sizes based on a range of potential variance was determined. . See Power Calculations section for details. Experiment has 3 cohorts: Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 8 of 32 Registered report Registered report Biochemistry . Cohort 1: A375 cells
. Cohort 2: RPMI-7951 cel
. Cohort 3: OUMS-23 cells . Cohort 1: A375 cells . Cohort 2: RPMI-7951 cells . Cohort 3: OUMS-23 cells Each cohort has 9 conditions to be performed with six technical replicates per experiment: . DMSO (vehicle)
. 100 mM PLX4720
. 10 mM PLX4720
. 1 mM PLX4720
. 0.1 mM PLX4720
. 0.01 mM PLX4720
. 0.001 mM PLX4720
. 0.0001 mM PLX4720
. 0.00001 mM PLX4720 Materials and reagents Materials and reagents
Reagent
Type
Manufacturer
Catalog #
Comments
RPMI medium with
L-glutamine
Cell culture
Sigma
R8758
Replaces Corning
cat no. 10-040-CV. MEM with L-glutamine
Cell culture
Sigma
M4655-500ML
Replaces Corning
cat. no. 10-010-CV. FBS
Cell culture
Life Technologies
12483-020
Replaces Corning brand. Pen/strep/glutamine
Cell culture
Abm
G255
Replaces Corning brand. A375 cells
Cell line
ATCC
CRL-1619
Original brand not specified. RPMI-7951 cells
Cell line
ATCC
HTB-66
OUMS-23 cells
Cell line
JCRB
JCRB1022
Original brand not specified. PBS
Buffer
Sigma
D8537-500ML
Original brand not specified. Trypsin
Cell culture
Sigma
T4049
Original brand not specified. 10 cm plates
Labware
CellStar
664 160
Original brand not specified. 96 well clear plates
Labware
Sarstedt
83.3924
Original brand not specified. PLX4720
Inhibitor
Selleck Chemicals
S1152
Replaces Symansis brand. DMSO
Chemical
Sigma
D4540
Original brand not specified. WST1 viability assay
Reporter assay
Roche
11644807001
Original catalog # not specified. Microplate reader
(420-480 nm)
Instrument
Molecular Devices
abm
Original brand not specified. Deliverables . Data to be collected:
Raw data and back . Data to be collected: Data to be collected: Data to be collected:
Raw data and background subtracted absorbance at 420–480 nm. Raw data and background subtracted absorbance
Raw data and background subtracted ab
f GI50 values of each biological replicate.
GI50 values of each biological replicate.
Graph of average GI50 values for each condition. (Compare to Figure 3D.) Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Confirmatory analysis plan . Statistical Analysis of the Replication Data: . Statistical Analysis of the Replication Data:
One way ANOVA of GI50 values from A375, RPMI-7951, and OUMS-23 cells with the fol-
lowing planned comparisons using Fisher’s LSD test. y
p
One way ANOVA of GI50 values from A375, RPMI-7951, and OUMS-23 cells with the fol-
lowing planned comparisons using Fisher’s LSD test. . A375 cells compared to RPMI-7951 cells. . A375 cells compared to OUMS-23 cells. . Meta-analysis of original and replication attempt effect sizes:
The replication data (mean and 95% confidence interval) will be plotted with the original
reported data value plotted as a single point on the same plot for comparison. Known differences from the original study The replication will not include the other BRAF (V600E) cell lines reported in the original paper. All
known differences are listed in the materials and reagents section above with the originally used
item listed in the comments section. All differences have the same capabilities as the original and
are not expected to alter the experimental design. Provisions for quality control Procedure Dilute stock of PLX4720 at 1000X final concentration of serial dilution stocks in DMSO
(100 mM to 0.01 mM). b
Dil t
1000X
i l dil ti
t
k 1 100 i
l t
th
di
t
i ld
10X t
k a. Dilute stock of PLX4720 at 1000X final concentration of serial dilution stocks in DMSO
(100 mM to 0.01 mM). m
b. Dilute 1000X serial dilution stocks 1:100 in complete growth medium to yield a 10X stock
(1 mM to 10-4 mM) that is added directly to the 90 ml of cell/medium. b. Dilute 1000X serial dilution stocks 1:100 in complete growth medium to yield a 10X stock
(1 mM to 10-4 mM) that is added directly to the 90 ml of cell/medium. i. Final DMSO concentration kept to 0.1%. Final DMSO concentration kept to 0.1%. 3. Determine cell viability with the WST1 viability assay according to manufacturer’s instructions. Briefly described: y
a. Add 11 ml/well reagent WST-1 (1:10 dilution). a. Add 11 ml/well reagent WST-1 (1:10 dilution). b. Incubate cells for 20–30 min. c. Shake thoroughly for 1 min on a shaker. c. Shake thoroughly for 1 min on a shaker. d. Measure the absorbance against a background control as blank using a microplate
reader at 420–480 nm. (If reference wavelength is to be determined, a filter >600 nm is
recommended) e. Exclude rows A-H due to edge effects/evaporation, thus making each cohort six techni-
cal replicates, except DMSO (vehicle), which has 12. f. Calculate viability as a percentage of control (DMSO (vehicle) cells) after background
subtraction. g. Determine GI50 value by fitting data using a nonlinear regression curve fit with a sigmoid
dose-response curve (four-parameter log-logistic function). 4. Repeat steps 1–3 independently two additional times. Deliverables Procedure . A375 cells maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/strep-
tomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2. y
g
. RPMI-7951 and OUMS-23 cells maintained in MEM medium supplemented with 10% FBS
and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2. . RPMI-7951 and OUMS-23 cells maintained in MEM medium supplemented with 10% FBS
and 1% penicillin/streptomycin/L-glutamine at 37˚C in a humidified atmosphere at 5% CO2. . Cells will be sent for mycoplasma testing and STR profiling. 1. Plate number of A375, RPMI-7951, and OUMS-23 cells as determined in Protocol 2 in a 96
well plate with 90 ml of medium per well. Incubate for 24 hr. a
Plate media alone (no cells) in columns 1 and 12 a. Plate media alone (no cells) in columns 1 and 12. b. Plate cells in remaining wells (columns 2–11). g
c. Exclude plating in the first and last row to avoid edge effects and evaporation. d. One plate is needed for each cell line. c. Exclude plating in the first and last row to avoid edge effects and evaporation. f p
g
g
p
d. One plate is needed for each cell line. d. One plate is needed for each cell line. d. One plate is needed for each cell line. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 9 of 32 Registered report Biochemistry 2. Treat cells with 10 ml of 10X serial dilutions of PLX4720 to yield final dilutions of 100 mM to
10-5 mM (8 dilutions) (columns 3 through 10), or treat with DMSO (vehicle) control (columns 2
and 11). Incubate for 96 hr. a. Dilute stock of PLX4720 at 1000X final concentration of serial dilution stocks in DMSO
(100 mM to 0.01 mM). b. Dilute 1000X serial dilution stocks 1:100 in complete growth medium to yield a 10X stock
(1 mM to 10-4 mM) that is added directly to the 90 ml of cell/medium. i
Final DMSO concentration kept to 0 1% 2. Treat cells with 10 ml of 10X serial dilutions of PLX4720 to yield final dilutions of 100 mM to
10-5 mM (8 dilutions) (columns 3 through 10), or treat with DMSO (vehicle) control (columns 2
and 11). Incubate for 96 hr. 10-5 mM (8 dilutions) (columns 3 through 10), or treat with DMSO (vehicle) control (columns 2
and 11). Incubate for 96 hr. a. Provisions for quality control Continued on next page Protocol 4: MAPK pathway analysis after MAP3K8 inhibition in cells
expressing elevated MAP3K8 This experiment assesses the effect a MAP3K8 kinase inhibitor has on the MAPK pathway, in cells
expressing elevated MAP3K8, as analyzed via Western blot. It utilizes RPMI-7951 cells, which express
a high level of MAP3K8. This protocol replicates the experiment reported in Figure 3I. Provisions for quality control q
y
The cell line used in this experiment will undergo STR profiling to confirm its identity and will be
sent for mycoplasma testing to ensure there is no contamination. The seeding density of each cell
line was determined in Protocol 2. All of the raw data, including the analysis files, will be uploaded
to the project page on the OSF (https://osf.io/lmhjg/) and made publically available. 10 of 32 Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Registered report Registered report Biochemistry Protocol 4: MAPK pathway analysis after MAP3K8 inhibition in cells
expressing elevated MAP3K8
This experiment assesses the effect a MAP3K8 kinase inhibitor has on the MAPK pathway, in cells
expressing elevated MAP3K8, as analyzed via Western blot. It utilizes RPMI-7951 cells, which express
a high level of MAP3K8. This protocol replicates the experiment reported in Figure 3I. Sampling
Experiment to be repeated a total of 8 times for a minimum power of 80%. The original data is qual-
itative, thus to determine an appropriate number of replicates to initially perform, sample sizes
based on a range of potential variance was determined. . See Power Calculations section for details. Experiment has five conditions:
. Vehicle (DMSO) treated RPMI-7951 cells
. 20 mM MAP3K8 inhibitor treated RPMI-7951 cells
. 10 mM MAP3K8 inhibitor treated RPMI-7951 cells
. 5 mM MAP3K8 inhibitor treated RPMI-7951 cells
. 1 mM MAP3K8 inhibitor treated RPMI-7951 cells
Each condition will be probed with the following antibodies:
. pERK1/2 (T202/Y204)
. pERK1/2
. pMEK1/2 (S217/221)
. MEK1/2
. Vinculin
Materials and reagents
Reagent
Type
Manufacturer
Catalog #
Comments
MEM with
L-glutamine
Cell culture
Sigma
M4655-500ML
Replaces Corningcat. no. 10-010-CV
FBS
Cell culture
Life
Technologies
12483-020
Replaces Corning brand. Pen/strep/glutamine
Cell culture
Abm
G255
Replaces Corning brand. RPMI-7951 cells
Cell line
ATCC
HTB-66
PBS
Buffer
Sigma
D8537-500ML
Original brand not specified. Trypsin
Cell culture
Sigma
T4049
Original brand not specified. 10 cm plates
Labware
CellStar
664 160
Original brand not specified. 6 well plates
Labware
Greiner
bio-one
657 160
Original brand not specified. MAP3K8 kinase inhibitor
Inhibitor
EMD
616373
DMSO
Chemical
Sigma
D4540
Original brand not specified. NP-40 buffer
Buffer
Life
Technologies
FNN0021
Original brand not specified. Protease inhibitors
Inhibitor
Roche
04693116001
Original catalog #
not specified. Phosphatase inhibitor
cocktail I
Inhibitor
Sigma
P2850
Replaces CalBioChem brand. Phosphatase inhibitor
cocktail II
Inhibitor
Sigma
P5726
Replaces CalBioChem brand. Cell scraper
Labware
Sasrstedt
83.1830
Original brand not specified. Registered report Sampling p
g
Experiment to be repeated a total of 8 times for a minimum power of 80%. The original data is qual-
itative, thus to determine an appropriate number of replicates to initially perform, sample sizes
based on a range of potential variance was determined. . See Power Calculations section for details. Experiment has five conditions: . Vehicle (DMSO) treated RPMI-7951 cells
. 20 mM MAP3K8 inhibitor treated RPMI-7951 cells
. 10 mM MAP3K8 inhibitor treated RPMI-7951 cells
. 5 mM MAP3K8 inhibitor treated RPMI-7951 cells
. 1 mM MAP3K8 inhibitor treated RPMI-7951 cells Each condition will be probed with the following antibodies: Each condition will be probed with the following antibodies: Materials and reagents Materials and reagents
Reagent
Type
Manufacturer
Catalog #
Comments
MEM with
L-glutamine
Cell culture
Sigma
M4655-500ML
Replaces Corningcat. no. 10-010-CV
FBS
Cell culture
Life
Technologies
12483-020
Replaces Corning brand. Pen/strep/glutamine
Cell culture
Abm
G255
Replaces Corning brand. RPMI-7951 cells
Cell line
ATCC
HTB-66
PBS
Buffer
Sigma
D8537-500ML
Original brand not specified. Trypsin
Cell culture
Sigma
T4049
Original brand not specified. 10 cm plates
Labware
CellStar
664 160
Original brand not specified. 6 well plates
Labware
Greiner
bio-one
657 160
Original brand not specified. MAP3K8 kinase inhibitor
Inhibitor
EMD
616373
DMSO
Chemical
Sigma
D4540
Original brand not specified. NP-40 buffer
Buffer
Life
Technologies
FNN0021
Original brand not specified. Protease inhibitors
Inhibitor
Roche
04693116001
Original catalog #
not specified. Phosphatase inhibitor
cocktail I
Inhibitor
Sigma
P2850
Replaces CalBioChem brand. Phosphatase inhibitor
cocktail II
Inhibitor
Sigma
P5726
Replaces CalBioChem brand. Cell scraper
Labware
Sasrstedt
83.1830
Original brand not specified. 11 of 32 11 of 32 Biochemistry Continued
Reagent
Type
Manufacturer
Catalog #
Comments
BCA kit
Reporter assay
Pierce
23227
Original catalog # not specified. Communicated by authors. DTT
Chemical
Biobasic
DB0058
Original brand not specified. Sample buffer
Buffer
abm
G031
Replaces Invitrogen brand. Protein molecular weight ladder
Western materials
abm
G252, G494
Original brand not specified. 10% Tris/Glycine gel; 10 well, 1.0 mm thick
Western materials
abm
internal
Replaces Invitrogen brand. Running buffer
Buffer
abm
internal
Original brand not specified. Immobilon P
Western materials
Thermofisher
IPVH00010
Original brand not specified. Transfer buffer
Buffer
abm
internal
Original brand not specified. Mouse anti-pERK1/2
(T202/Y204) (clone E10)
antibody
Antibodies
Cell Signaling
9106
Use at 1:1000 dilution. Original catalog # not specified. Rabbit anti-pMEK1/2
(S217/221) (clone 41G9)
antibody
Antibodies
Cell Signaling
9154
Use at 1:11000 dilution. Original catalog # not specified. Sampling Mouse anti-p44/42
MAPK (ERK1/2)
(clone L34F12) antibody
Antibodies
Cell Signaling
4696
Use at 1:11000 dilution. Replaces catalog # 4695. Communicated by authors. Rabbit anti-MEK1/2
(clone D1A5) antibody
Antibodies
Cell Signaling
8727
Use at 1:1000 dilution. Original catalog # not specified. Rabbit anti-Vinculin
antibody
Antibodies
Sigma
V4139
Use at 1:20,000 dilution. Original catalog # not specified. Anti-rabbit IgG – HRP
conjugated
Antibodies
Cell Signaling
7074
Use at 1:1000 dilution. Original catalog # not specified. Anti-mouse IgG – HRP
conjugated
Antibodies
Cell Signaling
7076
Use at 1:1000 dilution. Original catalog # not specified. Chemiluminescent reagent
Western materials
Life
Technologies
WP20005
Replaces Pierce brand. Registered report
Biochemistry Procedure . RPMI-7951 cells maintained in MEM medium supplemented with 10% FBS and 1% penicillin/
streptomycin at 37˚C in a humidified atmosphere at 5% CO2. . Cells will be sent for mycoplasma testing and STR profiling. 1. Plate 750,000 RPMI-7951 cells in 6-well plates and incubate for 24–36 hr to achieve log
phase growth p
g
2. Wash twice with 1X PBS and incubate overnight in serum-free growth medium. g
g
3. Treat cells with 20, 10, 5, and 1 mM MAP3K8 inhibitor or DMSO for 1 hr. g
g
3. Treat cells with 20, 10, 5, and 1 mM MAP3K8 inhibitor or DMSO for 1 hr. a
Add drug directly to each well 3. Treat cells with 20, 10, 5, and 1 mM MAP3K8 inhibitor or DMSO for 1 hr. a. Add drug directly to each well. g
y
b. Make stocks of MAP3K8 inhibitor at 10 mM in DMSO. (500X dilution for 20 mM) c. Dilute stock of MAP3K8 to achieve 1000X final concentration of serial dilution stocks in
DMSO (10 mM to 1000 mM). m
d. Final DMSO concentration kept to 0.2%. .
RPMI-7951 cells maintained in MEM medium supplemented with 10% FBS and 1% penicilli
streptomycin at 37˚C in a humidified atmosphere at 5% CO2. m
d. Final DMSO concentration kept to 0.2%. (Compare to Figure 3I)
Raw data of band analysis and normalized bands for each sample Full image western blot films of all immunoblots including ladder. (Compare to Figure 3I)
Raw data of band analysis and normalized bands for each sample
Full image western blot films of all immunoblots including ladder. (Compare to Figure 3
Raw data of band analysis and normalized bands for each sample.
Raw data of band analysis and normalized bands for each sample. m
d. Final DMSO concentration kept to 0.2%. d. Final DMSO concentration kept to 0.2%. 4. Wash cells with 1–2 ml ice-cold PBS and lyse in 1% NP-40 lysis buffer (150 mM NaCl, 4. Wash cells with 1–2 ml ice-cold PBS and lyse in 1% NP-40 lysis buffer (150 mM NaCl,
50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40) supplemented with 2X
protease inhibitors and 1X phosphatase inhibitor cocktails I and II. y
y
50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40) supplemented with 2X
protease inhibitors and 1X phosphatase inhibitor cocktails I and II. a. Add ~ 100–200 ml 1% NP-40 lysis buffer to ensure that protein concentration is between
2–3 mg/ml. mg m
b. Scrape each plate with a rubber cell scraper, collect lysates, and clarify by centrifugation
at max speed (table-top microfuge) at 4˚C. p
p
g
5. Determine protein concentration by BCA assay, normalize, reduce with DTT, and denature
at 88˚C. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 12 of 32 Registered report Biochemistry 6. Separate 35–50 mg of protein per lane on a 10% Tris/Glycine gel with protein ladder follow-
ing replicating lab’s standard protocol. 6. Separate 35–50 mg of protein per lane on a 10% Tris/Glycine gel with protein ladder follow-
ing replicating lab’s standard protocol. a. Samples run per gel: a. Samples run per gel: i. Protein molecular weight marker ii. Vehicle (DMSO) treated RPMI-7951 cells iii. 20 mM MAP3K8 inhibitor treated RPMI-7951 cells iv. 10 mM MAP3K8 inhibitor treated RPMI-7951 cells v. 5 mM MAP3K8 inhibitor treated RPMI-7951 cells vi. 1 mM MAP3K8 inhibitor treated RPMI-7951 cells Wet transfer with supplied wet-transfer cassette apparatus (120 min at 30–35 V at 4˚C) to g
p
g
p
8. After transfer, block non-specific binding and immunoblot membrane with the following pri-
mary antibodies for 18 hr at 4˚C following manufacturer recommendations:
a. mouse anti-pERK1/2 (T202/Y204); use at 1:1000 dilution; 42, 44 kDa
b. mouse anti-ERK1/2; use at 1:1000 dilution; 42, 44 kDa
c. rabbit anti-pMEK1/2 (S217/221); use at 1:1000 dilution; 45 kDa
d. rabbit anti-MEK1/2; use at 1:1000 dilution; 45 kDa
e. rabbit anti-Vinculin; use at 1:20,000 dilution; 116 kDa 8. After transfer, block non-specific binding and immunoblot membrane with the following pri-
mary antibodies for 18 hr at 4˚C following manufacturer recommendations:
a. mouse anti-pERK1/2 (T202/Y204); use at 1:1000 dilution; 42, 44 kDa
b. m
d. Final DMSO concentration kept to 0.2%. mouse anti-ERK1/2; use at 1:1000 dilution; 42, 44 kDa
c. rabbit anti-pMEK1/2 (S217/221); use at 1:1000 dilution; 45 kDa
d. rabbit anti-MEK1/2; use at 1:1000 dilution; 45 kDa
e. rabbit anti-Vinculin; use at 1:20,000 dilution; 116 kDa Protocol 4 Western blot antibody combinations
POI
Loading Control
Independent
Gels
Description
Working
Conc. Description
Working
Conc. 1
Mouse anti-pERK1/2
(T202/Y204) (42, 44 kDa)
1:1000
Rabbit anti-MEK1/2
(45 kDa)
1:1000
2
Rabbit anti-pMEK1/2
(S217/221) (45 kDa)
1:1000
Mouse anti-ERK1/2
(42, 44 kDa)
1:1000
3
Rabbit anti-Vinculin
(116 kDa)
1:20000 Protocol 4 Western blot antibody combinations 9. Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation,
and then detect signal using chemiluminescence following manufacturer’s instructions. a. Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary
antibodies and a low fluorescence membrane will be used instead, and images will be
acquired following manufacturer’s instructions. 10. Analyze bands with image analysis software, normalize to loading controls, and normalize
each dose of MAP3K8 inhibitor to Vehicle (DMSO). a. pERK1/2 (T202/Y204) normalized to MEK1/2 (total). b. pMEK1/2 (S217/221) normalized to ERK1/2 (total). 9. Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation,
and then detect signal using chemiluminescence following manufacturer’s instructions. g
g
g
a. Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary
antibodies and a low fluorescence membrane will be used instead, and images will be
acquired following manufacturer’s instructions. g
g
g
a. Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary
antibodies and a low fluorescence membrane will be used instead, and images will be
acquired following manufacturer’s instructions. g
10. Analyze bands with image analysis software, normalize to loading controls, and normalize
each dose of MAP3K8 inhibitor to Vehicle (DMSO). g
0. Analyze bands with image analysis software, normalize to loading controls, and normalize
each dose of MAP3K8 inhibitor to Vehicle (DMSO). a. pERK1/2 (T202/Y204) normalized to MEK1/2 (total). a. pERK1/2 (T202/Y204) normalized to MEK1/2 (total). b
pMEK1/2 (S217/221) normalized to ERK1/2 (total) a. pERK1/2 (T202/Y204) normalized to MEK1/2 (total). b. pMEK1/2 (S217/221) normalized to ERK1/2 (total). p
11. Repeat steps 1–10 independently seven additional times. Deliverables . Data to be collected:
Full image western blot films of all immunoblots including ladder. Provisions for quality control Provisions for quality control The cell line used in this experiment will undergo STR profiling to confirm its identity and will be
sent for mycoplasma testing to ensure there is no contamination. All of the raw data, including the
analysis files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made pub-
lically available. Protocol 5: Viability analysis following combinatorial MAPK pathway
inhibition in cells expressing elevated MAP3K8 This experiment assesses the effect the RAF inhibitor, PLX4720, along with the MEK inhibitors, CI-
1040 or AZD6244, has on cellular viability, in cells expressing MAP3K8. It utilizes A375 cells express-
ing MAP3K8, via ectopic expression of MAP3K8. This protocol replicates the experiment reported in
Figure 4E. Known differences from the original study The original NP40 cell lysis buffer was composed of: 150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA
pH 8, 25 mM NaF, and 1% NP-40. The replication will use a commercial formula, which has the fol-
lowing composition: 250 mM NaCl, 50 mM Tris pH 7.4, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4,
and 1% NP-40. All known differences are listed in the materials and reagents section above with the
originally used item listed in the comments section. All differences have the same capabilities as the
original and are not expected to alter the experimental design. Confirmatory analysis plan . Statistical Analysis of the Replication Data: . Statistical Analysis of the Replication Data:
One-way MANOVA of normalized pERK1/2 and pMEK1/2 levels of RPMI-7951 cells
treated with MAP3K8 inhibitor with the following analysis using the Bonferroni
correction: &
One-way ANOVA of pERK1/2 levels of RPMI-7951 cells treated with MAP3K8
inhibitor. . One-sample t-test of pERK1/2 levels of 20 mM treated cells compared to 1 (vehi-
cle treated cells). Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 13 of 32 Registered report Biochemistry &
One-way ANOVA of pMEK1/2 levels of RPMI-7951 cells treated with MAP3K8
inhibitor. . One-sample t-test of pMEK1/2 levels of 20 mM treated cells compared to 1
(vehicle treated cells). (vehicle treated cells).
IC50 values of normalized pERK1/2 and pMEK1/2 levels treated with vehicle or MAP3K8
inhibitor. . Meta-analysis of original and replication attempt effect sizes: . Meta-analysis of original and replication attempt effect sizes:
The replication data (mean and 95% confidence interval) will be plotted with the original
reported data value plotted as a single point on the same plot for comparison. Known differences from the original study Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Sampling p
g
Generation of A375 cells expressing MEK1, MEK1DD, and MAP3K8 to be performed once. Experiment (steps 4–6) to be repeated a total of 4 times for a minimum power of 80%. The origi-
nal data is from a single biological replicate, thus to determine an appropriate number of replicates
to initially perform, sample sizes based on a range of potential variance was determined. . See Power Calculations section for details. . See Power Calculations section for details
Experiment has 3 cohorts:
. Cohort 1: A375 cells expressing MEK1
. Cohort 2: A375 cells expressing MEK1DD
. Cohort 3: A375 cells expressing MAP3K8 . See Power Calculations section for details. . See Power Calculations section for details. Experiment has 3 cohorts: . Cohort 1: A375 cells expressing MEK1
h
ll
DD . Cohort 2: A375 cells expressing MEK1DD . Cohort 3: A375 cells expressing MAP3K8 Each cohort has 6 conditions to be done with six technical repeats per experiment: . Untreated [additional control]
. DMSO (vehicle)
. 10 mM PLX4720
. 1 mM PLX4720
. 1 mM PLX4720 + 1 mM AZD6244
. 1 mM PLX4720 + 1 mM CI-1040 . Untreated [additional control] . Untreated [additional control]
. DMSO (vehicle)
. 10 mM PLX4720
. 1 mM PLX4720
. 1 mM PLX4720 + 1 mM AZD6244
. 1 mM PLX4720 + 1 mM CI-1040 . 1 mM PLX4720 + 1 mM CI-1040 Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 14 of 32 14 of 32 Materials and reagents
Reagent
Type
Manufacturer
Catalog #
Comments
RPMI medium with L-glutamine
Cell culture
Sigma
R8758
Replaces Corning cat no. 10-040-CV. DMEM medium
Cell culture
Corning
10-013
Original brand not specified. FBS
Cell culture
Life Technologies
12483-020
Replaces Corning brand. Pen/strep/glutamine
Cell culture
Abm
G255
Replaces Corning brand. A375 cells
Cell line
ATCC
CRL-1619
Original brand not specified. 293T cells
Cell line
ATCC
CRL-11268
PBS
Buffer
Sigma
D8537-500ML
Original brand not specified. Trypsin
Cell culture
Sigma
T4049
Original brand not specified. 10 cm plates
Labware
CellStar
664 160
Original brand not specified. 6 cm plates
Labware
Biolite
11825275
Original brand not specified. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Sampling Nucleobond Maxiprep Kit
Kit
Macherey-Nagel
740414
Not originallyspecified
pLX-Blast-V5-MEK1
DNA construct
Provided from original authors
pLX-Blast-V5-MEK1DD
DNA construct
Provided from original authors
pLX-Blast-V5-MAP3K8
DNA construct
Provided from original authors
D8.9 (gag,pol)
DNA construct
Provided from original authors
VSV-G
DNA construct
Provided from original authors
FuGene HD transfection reagent
Transfection reagent
Promega
E2311
Replaces FuGene6 Roche brand. OptiMEM medium
Buffer
Life Tech
51985034
Original brand not specified. 6-well plates
Labware
Greiner bio-one
657 160
Original brand not specified. Polybrene
Cell culture
Sigma
H9268
Original brand not specified. Blasticidin
Cell culture
Invivogen
ant-bl-1
Original brand not specified. 96 well clear plates
Labware
Sarstedt
83.3924
Original brand not specified. PLX4720
Inhibitor
Selleck Chemicals
S1152
Replaces Symansis brand. AZD6244
Inhibitor
Selleck Chemicals
S1008
Original catalog # not
specified. CI-1040
Inhibitor
Selleck Chemicals
S1020
Replaces Shanghai
Lechen International
Trading Co. brand. DMSO
Chemical
Sigma
D4540
Original brand
not specified. WST1 viability assay
Reporter assay
Roche
11644807001
Original catalog #
not specified. Microplate reader
(420–480 nm)
Instrument
Molecular Devices
abm
Original brand not specified. Registered report
Biochemistry Biochemistry y
g
p
. 293T cells maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin
at 37˚C in a humidified atmosphere at 5% CO2. . Cells will be sent for mycoplasma testing and STR profiling. 1. Grow and prepare endotoxin-free plasmid constructs according to the manufacturer’s proto-
col for an endotoxin-free Plasmid Maxiprep Kit. a. Viral packaging vectors:
i. D8.9 (gag,pol) 1. Grow and prepare endotoxin-free plasmid constructs according to the manufacturer’s proto-
col for an endotoxin-free Plasmid Maxiprep Kit. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 15 of 32 Registered report Biochemistry ii.
VSV-G b. DNA construct expression vectors:
i. pLX-Blast-V5-MEK1
ii. pLX-Blast-V5-MEK1DD
iii. pLX-Blast-V5-MAP3K8 b. DNA construct expression vectors: b. DNA construct expression vectors: b. DNA construct expression vectors:
i. pLX-Blast-V5-MEK1
ii. pLX-Blast-V5-MEK1DD
iii. pLX-Blast-V5-MAP3K8 p
i. pLX-Blast-V5-MEK1
ii. pLX-Blast-V5-MEK1DD
iii. pLX-Blast-V5-MAP3K8 2. Sequence expression plasmids to confirm identity and run on gel to confirm vector integrity. U
th
f ll
i
i
i
t
fi
th
id
tif
f th
LX CMV
l
id 2. Sequence expression plasmids to confirm identity and run on gel to confirm vector integrity. Use the following sequencing primers to confirm the identify of the pLX_CMV plasmids:
a. pLX_CMV-ORF-fwd primer: 5’-CACCAAAATCAACGGGACTT-3’
b. pLX-ORF-rev primer: 5’-AGGAGGAGAAAATGAAAGCC-3’ 2. Sequence expression plasmids to confirm identity and run on gel to confirm vector integrity. Use the following sequencing primers to confirm the identify of the pLX_CMV plasmids: Use the following sequencing primers to confirm the identify of the pLX_CMV plasmids a. pLX_CMV-ORF-fwd primer: 5’-CACCAAAATCAACGGGACTT-3
b
LX ORF
i
5’ AGGAGGAGAAAATGAAAGCC 3’ 3. Produce MEK1, MEK1DD, and MAP3K8 lentivirus: 3. Produce MEK1, MEK1DD, and MAP3K8 lentivirus:
a. Seed 8 x 105 293T cells per 6 cm dish, incubate. 3. Produce MEK1, MEK1DD, and MAP3K8 lentivirus:
a. Seed 8 x 105 293T cells per 6 cm dish, incubate. b. 24 hr later, transfect each plate of 293T cells by adding the following:
i. 1 mg pLX-Blast-V5-MEK1, pLX-Blast-V5-MEK1DD, or pLX-Blast-V5-MAP3K8
ii. 900 ng D8.9 (gag,pol)
iii. 100 ng VSV-G
iv. 6 ml FuGene transfection reagent
v. 94 ml OptiMem medium (free of FBS and Pen/Strep). Add OptiMem medium to the
aliquots of DNA, then add the FuGene to the OptiMem/DNA mix. vi. Incubate 30 min at RT, then add to cells. H
t i
72 h
t t
f
ti
li
t
d f
t 80˚C f
t l
t 24 h b f a. Seed 8 x 105 293T cells per 6 cm dish, incubate. b. 24 hr later, transfect each plate of 293T cells by adding the following:
DD i. 1 mg pLX-Blast-V5-MEK1, pLX-Blast-V5-MEK1DD, or pLX-Blast-V5-MAP3K8 ii. 900 ng D8.9 (gag,pol) iii. 100 ng VSV-G iv. 6 ml FuGene transfection reagent v. 94 ml OptiMem medium (free of FBS and Pen/Strep). Add OptiMem medium to the
aliquots of DNA, then add the FuGene to the OptiMem/DNA mix. c. Harvest virus 72 hr post-transfection, aliquot, and freeze at -80˚C for at least 24 hr before
using. ii.
VSV-G i
Note: You will need to freeze down enough virus for both Protocols 5 and 6 i. Note: You will need to freeze down enough virus for both Protocols 5 and 6. 4. Titrate lentivirus: a. Seed 100,000 – 125,000 A375 cells per well in 6 well plates in 2 ml medium. Incubate for
24 hr in normal growth conditions. a. Seed 100,000 – 125,000 A375 cells per well in 6 well plates in 2 ml medium. Incubate fo
24 hr in normal growth conditions. i. Seeding density is such that cells will be near confluent 3 days after removing virus
ii. Seed two wells per viral concentration for each virus (total wells = 30). 24 hr in normal growth conditions. i. Seeding density is such that cells will be near confluent 3 days after removing virus. ii. Seed two wells per viral concentration for each virus (total wells = 30). i. Seeding density is such that cells will be near confluent 3 days after removing virus. d
ll
l
f
h
(
l
ll
) b. Add polybrene (4–10 mg/ml final concentration) to plates, swirl to mix, then infect cells
with varying concentrations of virus (1:5, 1:10, 1:12, 1:15, and 1:20) in duplicate (i.e. two
wells per viral concentration). i. Thaw virus overnight at 4˚C, or in a 37˚C water bath, but without letting the virus
get above 4˚C. ii. Add virus to the wells and swirl to mix. ii. Add virus to the wells and swirl to mix. iii. Spin 6-well plates at 2250 RPM for 30 min at 37˚C. iv. Incubate cells overnight with virus, then change medium the following morning. c. Incubate for 24 hr, then remove medium and replace with growth medium with or with-
out 10 mg/ml blasticidin. d. After 5–7 days of selection, count the cells in all wells and divide the counts with blastici-
din by the non-selected control for each viral dilution. e. Use the lowest viral dilution that yields an 85–95% ratio of selected/non-selected cells
(originally observed to be in the 1:8 – 1:15 range). 5. Infect A375 cells with viral supernatant: a. Seed 100,000 – 125,000 A375 cells per well in 6 well plates in 2 ml medium. Incubate for
24 hr in normal growth conditions. i. Seed three wells per virus. (total wells = 9). a. Biochemistry 6. After 48 hr trypsinize the experimental wells (one per virus), count, and plate number of
A375 infected cells as determined in Protocol 2 in a 96 well plate in 90 ml. Incubate for 24 hr. a. Plate media alone (no cells) in columns 1,2,11, and 12. b. Plate cells in remaining wells (columns 3–10). 6. After 48 hr trypsinize the experimental wells (one per virus), count, and plate number of
A375 infected cells as determined in Protocol 2 in a 96 well plate in 90 ml. Incubate for 24 hr. Pl
di
l
(
ll ) i
l
1 2 11
d 12 6. After 48 hr trypsinize the experimental wells (one per virus), count, and plate number of
A375 infected cells as determined in Protocol 2 in a 96 well plate in 90 ml. Incubate for 24 hr. Pl
di
l
(
ll ) i
l
1 2 11
d 12 a. Plate media alone (no cells) in columns 1,2,11, b. Plate cells in remaining wells (columns 3–10). g
c. Exclude plating in the first and last row to avoid edge effects and evaporation. d. One plate is needed for each of the A375 infected cells. 7. Treat cells with 10 ml of 10X dilutions of PLX4720 with or without AZD6244 or CI-1040 to
yield appropriate final concentrations (columns 5 through 8), or treat with DMSO (vehicle)
control (columns 4 and 9). Add 10 ml medium for untreated control (columns 3 and 10). Incu-
bate for 96 hr. a. Dilute stock of PLX4720 at 1000X final concentration in DMSO (10 mM and 1 mM). b. Dilute stock of AZD6244 and CI-1040 at 1000X final concentration in DMSO (1 mM). c. Dilute 1000X dilution stocks 1:100 in complete growth medium to yield a 10X stock of
appropriate treatment. pp
p
i. Final DMSO concentration kept to 0.2%. 8. Determine cell viability with the WST1 viability assay according to manufacturer’s instructions. Briefly described: a. Add 11 ml/well reagent WST-1 (1:10 dilution). b. Incubate cells for 20–30 min. c. Shake thoroughly for 1 min on a shaker. i. Measure the absorbance against a background control as blank using a microplate
reader at 420–480 nm. (If reference wavelength is to be determined, a filter > 600
nm is recommended) d. Biochemistry Exclude rows A-H due to edge effects/evaporation, thus making each cohort six techni-
cal replicates, except DMSO (vehicle) and untreated control, which has 12. p
p
e. Calculate viability as a percentage of control (DMSO(vehicle) cells) after background
subtraction. 9. Repeat steps 5–8 independently three additional times. Deliverables: . Data to be collected: . Data to be collected:
Raw counts and titration percentages (step 2 above).
Raw counts and infection efficiency (step 3 above).
Raw data and background subtracted absorbance at 420–480 nm. g
Relative viability of A375 cells expressing MEK1, MEK1DD, and MAP3K8 as a percentage g
Relative viability of A375 cells expressing MEK1, MEK1DD, and MAP3K8 as a percentage
of DMSO treatment for each cell line. (Compare to Figure 4E.)
Relative viability of A375 cells expressing MEK1, MEK1DD, and MA
of DMSO treatment for each cell line. (Compare to Figure 4E.) y
p
g
,
,
of DMSO treatment for each cell line. (Compare to Figure 4E.) . Sample for additional protocol:
DD
MEK1, MEK1DD, and MAP3K8 lentivirus for further use (Protocol 6). ii.
VSV-G Seed 100,000 – 125,000 A375 cells per well in 6 well plates in 2 ml medium. Incubate for
24 hr in normal growth conditions. i. Seed three wells per virus. (total wells = . Add polybrene (4–10 mg/ml final concentration) to plates, swirl to mix, then infect cells
with dilution determined by titration protocol (step 4 above). with dilution determined by titration protocol (step 4 above). i. Thaw virus overnight at 4˚C, or in a 37˚C water bath, but without letting the virus
get above 4˚C. y
p
p
i. Thaw virus overnight at 4˚C, or in a 37˚C water bath, but without letting the virus
get above 4˚C. g
ii. Add virus to the wells and swirl to mix. ii. Add virus to the wells and swirl to mix. 1. Alternatively, make a 3X virus/polybrene mixture, such that the addition of 1 ml
of 3X virus to 2 ml of cells/medium yields an appropriate dilution of virus and
polybrene. 1. Alternatively, make a 3X virus/polybrene mixture, such that the addition of 1 ml
of 3X virus to 2 ml of cells/medium yields an appropriate dilution of virus and
polybrene. p
y
iii. Spin 6-well plates at 2250 RPM for 30 min at 37˚C. p
y
iii. Spin 6-well plates at 2250 RPM for 30 min at 37˚C. iv. Incubate cells overnight with virus, then change medium the following morning. b
f
h
h
d
d
l
h
h
d
h
h c. Incubate for 24 hr, then remove medium and replace with growth medium with or with-
out 10 mg/ml blasticidin. c. Incubate for 24 hr, then remove medium and replace with growth medium with or with-
out 10 mg/ml blasticidin. i. Two wells without selection and one well with blasticidin for each virus. out 10 mg/ml blasticidin. i. Two wells without selection and one well with blasticidin for each virus. mg
i. Two wells without selection and one well with blasticidin for each virus. d. After 5–7 days of selection, count cells in one well of no-blasticidin and the blasticidin-
treated well to calculate infection efficiency. y
i. Divide the counts with blasticidin by the non-selected control. y
i. Divide the counts with blasticidin by the non-selected control. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 16 of 32 Registered report Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Confirmatory analysis plan . Statistical Analysis of the Replication Data: . Statistical Analysis of the Replication Data:
One-way ANOVA of normalized viability of A375 cells expressing MAP3K8 treated with
vehicle, 1 mM PLX4720, 1 mM PLX4720 + CI-1040, 1 mM PLX4720 + AZD6244, or 10 mM
PLX4720 with the following planned comparisons using the Bonferroni correction:
&
1 mM PLX4720 treatment compared to 1 mM PLX4720 + CI-1040. &
1 mM PLX4720 treatment compared to 1 mM PLX4720 + AZD6244. &
10 mM PLX4720 treatment compared to 1 mM PLX4720 + CI-1040. 10
M PLX4720
d
1
M PLX4720
AZD6244 y
One-way ANOVA of normalized viability of A375 cells expressing MAP3K8 treated with
vehicle, 1 mM PLX4720, 1 mM PLX4720 + CI-1040, 1 mM PLX4720 + AZD6244, or 10 mM
PLX4720 with the following planned comparisons using the Bonferroni correction: g
g
&
1 mM PLX4720 treatment compared to 1 mM PLX4720 + CI-1040. m
p
m
&
1 mM PLX4720 treatment compared to 1 mM PLX4720 + AZD6244. &
1 mM PLX4720 treatment compared to 1 mM PLX4720 + AZD6244. &
10 mM PLX4720 treatment compared to 1 mM PLX4720 + CI-1040. m
p
m
&
10 mM PLX4720 treatment compared to 1 mM PLX4720 + AZD6244.
Two-way ANOVA of normalized viability of A375 cells expressing MEK1 or MEK1DD
treated with vehicle, 1 mM PLX4720, 1 mM PLX4720 + CI-1040, 1 mM PLX4720 +
AZD6244, or 10 mM PLX4720.
The replication data (mean and 95% confidence interval) will be plotted with the original
reported data value plotted as a single point on the same plot for comparison. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 17 of 32 Registered report Biochemistry Known differences from the original study Known differences from the original study
All known differences are listed in the materials and reagents section above with the originally used
item listed in the comments section. All differences have the same capabilities as the original and
are not expected to alter the experimental design. g
y
All known differences are listed in the materials and reagents section above with the originally used
item listed in the comments section. All differences have the same capabilities as the original and
are not expected to alter the experimental design. Confirmatory analysis plan Provisions for quality control
The cell line used in this experiment will undergo STR profiling to confirm its identity and will be
sent for mycoplasma testing to ensure there is no contamination. The seeding density of the A375
cell line was determined in Protocol 2. Infection efficiency will be determined for each replicate. The expression of the kinase of interest will be assessed using antibodies against the V5 tag as
well as MAP3K8 and MEK1 as described in Protocol 6. All of the raw data, including the analysis
files, will be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made publically
available. Protocol 6 MAPK pathway analysis following combinatorial MAPK
pathway inhibition in cells expressing elevated MAP3K8 p
y
p
g
This experiment assesses the effect the RAF inhibitor, PLX4720, along with the MEK inhibitors, CI-
1040 or AZD6244, has on the MAPK pathway, as analyzed via Western blot. It utilizes A375 cells
expressing MAP3K8, via ectopic expression of MAP3K8. This protocol replicates the experiment
reported in Figure 4F. Registered report Sampling p
g
Experiment to be repeated a total of 3 times for a minimum power of 80%. The original data is qual-
itative, thus to determine an appropriate number of replicates to initially perform, sample sizes
based on a range of potential variance was determined. based on a range of potential variance was determined. . See Power Calculations section for details. Experiment has 3 cohorts:
. Cohort 1: A375 cells expressing MEK1
. Cohort 2: A375 cells expressing MEK1DD
. Cohort 3: A375 cells expressing MAP3K8
Each cohort has 4 conditions:
. DMSO (vehicle)
. 1 mM PLX4720
. 1 mM PLX4720 + 1 mM AZD6244
. 1 mM PLX4720 + 1 mM CI-1040
Each condition will be probed with the following antibodies:
. pERK1/2 (T202/Y204)
. ERK1/2
. V5 [additional]
. Vinculin
. MEK1/2 [additional]
. MAP3K8 [additional]
Materials and reagents
Reagent
Type
Manufacturer
Catalog #
Comments
RPMI medium with L-glutamine
Cell culture
Sigma
R8758
Replaces Corning cat no. 10-040-CV. FBS
Cell culture
Life Technologies
12483-020
Replaces Corning brand. Continued on next page
Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414
18 of 32 . See Power Calculations section for details. Experiment has 3 cohorts:
. Cohort 1: A375 cells expressing MEK1
. Cohort 2: A375 cells expressing MEK1DD
. Cohort 3: A375 cells expressing MAP3K8 . See Power Calculations section for details. Experiment has 3 cohorts: . Cohort 1: A375 cells expressing MEK1
D . Cohort 2: A375 cells expressing MEK1DD . Cohort 3: A375 cells expressing MAP3K8 Each cohort has 4 conditions: . DMSO (vehicle)
. 1 mM PLX4720
. 1 mM PLX4720 + 1 mM AZD6244
. 1 mM PLX4720 + 1 mM CI-1040 . DMSO (vehicle)
. 1 mM PLX4720
. 1 mM PLX4720 + 1 mM AZD6244
. 1 mM PLX4720 + 1 mM CI-1040 Each condition will be probed with the following antibodies: 18 of 32 Biochemistry Continued
Reagent
Type
Manufacturer
Catalog #
Comments
Pen/strep/glutamine
Cell culture
Abm
G255
Replaces Corning brand. A375 cells
Cell line
ATCC
CRL-1619
Original brand not specified. PBS
Buffer
Sigma
D8537-500ML
Original brand not specified. Trypsin
Cell culture
Sigma
T4049
Original brand not specified. 10 cm plates
Labware
CellStar
664 160
Original brand not specified. 6 well plates
Labware
Greiner bio-one
657 160
Original brand not specified. Polybrene
Cell culture
Sigma
H9268
Original brand not specified. Blasticidin
Cell culture
Invivogen
ant-bl-1
Original brand not specified. Sampling PLX4720
Inhibitor
Selleck Chemicals
S1152
Replaces Symansis brand. AZD6244
Inhibitor
Selleck Chemicals
S1008
Original catalog #
not specified. CI-1040
Inhibitor
Selleck Chemicals
S1020
Replaces Shanghai Lechen
International Trading Co. brand. DMSO
Chemical
Sigma
D4540
Original brand not specified. NP-40 buffer
Buffer
Life technologies
FNN0021
Original brand not specified. Protease inhibitors
Inhibitor
Roche
04693116001
Original catalog #
not specified. Phosphatase inhibitor
cocktail I
Inhibitor
Sigma
P2850
Replaces CalBioChem brand. Phosphatase inhibitor
cocktail II
Inhibitor
Sigma
P5726
Replaces CalBioChem brand. Cell scraper
Labware
Sasrstedt
83.1830
Original brand not specified. BCA kit
Reporter assay
Pierce
23227
Original catalog # not specified. Communicated by authors. DTT
Chemical
Biobasic
DB0058
Original brand not specified. Sample buffer
Buffer
abm
G031
Replaces Invitrogen brand. Protein molecular
weight ladder
Western materials
abm
G252, G494
Original brand not specified. 10% Tris/Glycine gel;
10 well, 1.0 mm thick
Western materials
abm
internal
Replaces Invitrogen brand. Running buffer
Buffer
abm
internal
Original brand not specified. Immobilon P
Western materials
Thermofisher
IPVH00010
Original brand not specified. Transfer buffer
Buffer
abm
internal
Original brand not specified. Mouse anti-pERK1/2
(T202/Y204)
(clone E10)
antibody
Antibodies
Cell Signaling
9106
Use at 1:1000 dilution. Original catalog # not specified. Mouse anti-p44/42
MAPK (ERK1/2) (clone L34F12)
antibody
Antibodies
Cell Signaling
4696
Use at 1:11000 dilution. Replaces catalog # 4695. Communicated by authors. Mouse anti-V5-HRP
conjugated antibody
Antibodies
Invitrogen
R961-25
Use at 1:5000 dilution. Original catalog # not specified. Rabbit anti-Vinculin
antibody
Antibodies
Sigma
V4139
Use at 1:20,000 dilution. Original catalog # not specified. Rabbit anti-MEK1/2
(clone D1A5) antibody
Antibodies
Cell Signaling
8727
Use at 1:1000 dilution. Original catalog # not specified. Rabbit anti-MAP3K8
(clone M-20) antibody
Antibodies
Santa Cruz
sc-720
Use at 1:500 dilution. Communicated by authors. Continued on next page 19 of 32 Continued
Reagent
Type
Manufacturer
Catalog #
Comments
Anti-rabbit IgG – HRP
conjugated antibody
Antibodies
Cell Signaling
7074
Use at 1:1000 dilution. Original catalog # not specified. Anti-mouse IgG – HRP
conjugated antibody
Antibodies
Cell Signaling
7076
Use at 1:1000 dilution. Original catalog # not specified. Chemiluminescent reagent
Western materials
Life Technologies
WP20005
Replaces Pierce brand. Registered report
Biochemistry Biochemistry . A375 cells maintained in RPMI medium supplemented with 10% FBS and 1% penicillin/strep-
tomycin at 37˚C in a humidified atmosphere at 5% CO2. y
p
. Cells will be sent for mycoplasma testing and STR profiling. . Cells will be sent for mycoplasma testing and STR profiling. 1. Sampling Infect A375 cells with viral supernatant: 1. Infect A375 cells with viral supernatant:
a. Seed 100,000 – 125,000 A375 cells per well in 6 well plates in 2 ml medium. Incubate for
24 hr in normal growth conditions. i. Seed six wells per virus. (total wells = 18). a. Seed 100,000 – 125,000 A375 cells per well in 6 well plates in 2 ml medium. Incubate for
24 hr in normal growth conditions. b. Add polybrene (4–10 mg/ml final concentration) to plates, swirl to mix, then infect cells
following Protocol 5, step 5 using dilution determined by titration protocol (Protocol 5,
step 4). p
c. Incubate for 24 hr, then remove medium and replace with growth medium with or with-
out 10 mg/ml blasticidin. c. Incubate for 24 hr, then remove medium and replace with growth medium with or with-
out 10 mg/ml blasticidin. i. Five wells without selection and one well with blasticidin for each virus. f
f
f mg
i. Five wells without selection and one well with blasticidin for each virus. d. After 5–7 days of selection, count cells in one well of no-blasticidin and the blasticidin-
treated well to calculate infection efficiency. i. Divide the counts with blasticidin by the non-selected control. 2. 72 hr after infection (96 hr after seeding) treat cells with DMSO or 1 mM PLX4720 with or
without 1 mM AZD6244, 1 mM CI-1040, or DMSO. Incubate for 24 hr. a. Add drugs directly to each well using a 1000X stock (in DMSO). i. Final DMSO concentration kept to 0.2%. 2. 72 hr after infection (96 hr after seeding) treat cells with DMSO or 1 mM PLX4720 with or
without 1 mM AZD6244, 1 mM CI-1040, or DMSO. Incubate for 24 hr. a
Add drugs directly to each well using a 1000X stock (in DMSO) 2. 72 hr after infection (96 hr after seeding) treat cells with DMSO or
without 1 mM AZD6244, 1 mM CI-1040, or DMSO. Incubate for 24 h
a. Add drugs directly to each well using a 1000X stock (in DMSO). (
g)
m
without 1 mM AZD6244, 1 mM CI-1040, or DMSO. Incubate for 24 hr. without 1 mM AZD6244, 1 mM CI 1040, or DMSO. Incubate for 24
a. Add drugs directly to each well using a 1000X stock (in DMSO). i. Final DMSO concentration kept to 0.2% 3. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Sampling Wash cells with 1–2 ml ice-cold PBS and lyse in 1% NP-40 lysis buffer (150 mM NaCl, 3. Wash cells with 1–2 ml ice-cold PBS and lyse in 1% NP-40 lysis buffer (150 mM NaCl,
50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40) supplemented with 2X
protease inhibitors and 1X phosphatase inhibitor cocktails I and II. Add
100 200
l 1% NP 40 l
i b ff
h
i
i
i b 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF, and 1% NP-40) supplemented with 2X
protease inhibitors and 1X phosphatase inhibitor cocktails I and II. a. Add ~ 100–200 ml 1% NP-40 lysis buffer to ensure that protein concentration is between
2–3 mg/ml. mg m
b. Scrape each plate with a rubber cell scraper, collect lysates, and clarify by centrifugation
at max speed (table-top microfuge) at 4˚C. 4. Determine protein concentration by BCA assay, normalize, reduce with DTT, and denature
at 88˚C. 5. Separate 35–50 mg of protein per lane on a 10% Tris/Glycine gel with protein ladder follow-
l
l b
d
d
l 5. Separate 35–50 mg of protein per lane on a 10% Tris/Glycine gel with protein ladder follow-
ing replicating lab’s standard protocol. ing replicating lab’s standard protocol. a. Samples run per gel(s):
i. Protein molecular weight marker
ii. Vehicle (DMSO) treated A375 cells expressing MEK1
iii. 1 mM PLX4720 treated A375 cells expressing MEK1
iv. 1 mM PLX4720 and 1 mM AZD6244 treated A375 cells expressing MEK1
v. 1 mM PLX4720 and 1 mM CI-1040 treated A375 cells expressing MEK1
vi. Vehicle (DMSO) treated A375 cells expressing MEK1DD
vii. 1 mM PLX4720 treated A375 cells expressing MEK1DD
viii. 1 mM PLX4720 and 1 mM AZD6244 treated A375 cells expressing MEK1DD
ix. 1 mM PLX4720 and 1 mM CI-1040 treated A375 cells expressing MEK1DD
x. Vehicle (DMSO) treated A375 cells expressing MAP3K8
xi. 1 mM PLX4720 treated A375 cells expressing MAP3K8
xii. 1 mM PLX4720 and 1 mM AZD6244 treated A375 cells expressing MAP3K8
xiii. 1 mM PLX4720 and 1 mM CI-1040 treated A375 cells expressing MAP3K8 a. Samples run per gel(s): i. Protein molecular weight marker ii. Vehicle (DMSO) treated A375 cells expressing MEK1 ii. Vehicle (DMSO) treated A375 cells expressing MEK1
iii. 1 mM PLX4720 treated A375 cells expressing MEK1
iv. Sampling 1 mM PLX4720 and 1 mM AZD6244 treated A375 cells expressing MEK1
v. 1 mM PLX4720 and 1 mM CI-1040 treated A375 cells expressing MEK1
vi. Vehicle (DMSO) treated A375 cells expressing MEK1DD iii. 1 mM PLX4720 treated A375 cells expressing MEK1 iv. 1 mM PLX4720 and 1 mM AZD6244 treated A375 cells expressing MEK1 v. 1 mM PLX4720 and 1 mM CI-1040 treated A375 cells expressing MEK1 vi. Vehicle (DMSO) treated A375 cells expressing MEK1DD
DD vii. 1 mM PLX4720 treated A375 cells expressing MEK1DD viii. 1 mM PLX4720 and 1 mM AZD6244 treated A375 cells expressing MEK1DD x. Vehicle (DMSO) treated A375 cells expressing MAP3K8 xi. 1 mM PLX4720 treated A375 cells expressing MAP3K8 g
xii. 1 mM PLX4720 and 1 mM AZD6244 treated A375 cells expressing MAP3K8 g
xiii. 1 mM PLX4720 and 1 mM CI-1040 treated A375 cells expressing MAP3K8 Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 20 of 32 Registered report 6. Wet transfer with supplied wet-transfer cassette apparatus (120 min at 30–35 V at 4˚C) to
immobilon P following replicating lab’s standard protocol. 7. After transfer, block non-specific binding and immunoblot membrane with the following pri-
mary antibodies for 18 hr at 4˚C following manufacturer recommendations:
a. mouse anti-pERK1/2 (T202/Y204); use at 1:1000 dilution; 42, 44 kDa
b. mouse anti-ERK1/2; use at 1:1000 dilution; 42, 44 kDa
c. mouse anti-V5-HRP; use at 1:5000 dilution; 45 kDa for MEK1, 52, 58 kDa for MAP3K8
d. rabbit anti-Vinculin; use at 1:20,000 dilution; 116 kDa
e. rabbit anti-MAP3K8; use at 1:500 dilution; 52, 58 kDa
f. rabbit anti-MEK1/2; use at 1:1000 dilution; 45 kDa
Biochemistry Biochemistry 6. Wet transfer with supplied wet-transfer cassette apparatus (120 min at 30–35 V at 4˚C) to
immobilon P following replicating lab’s standard protocol. g
p
g
p
7. After transfer, block non-specific binding and immunoblot membrane with the following pri-
mary antibodies for 18 hr at 4˚C following manufacturer recommendations:
a. mouse anti-pERK1/2 (T202/Y204); use at 1:1000 dilution; 42, 44 kDa
b. mouse anti-ERK1/2; use at 1:1000 dilution; 42, 44 kDa
c. mouse anti-V5-HRP; use at 1:5000 dilution; 45 kDa for MEK1, 52, 58 kDa for MAP3K8
d. rabbit anti-Vinculin; use at 1:20,000 dilution; 116 kDa
e. rabbit anti-MAP3K8; use at 1:500 dilution; 52, 58 kDa
f. Sampling rabbit anti-MEK1/2; use at 1:1000 dilution; 45 kDa Protocol 6 Western blot antibody combinations Protocol 6 Western blot antibody combinations
POI
Loading control
Independent
Gels
Description
Working
Conc. Description
Working
Conc. 1
Mouse anti-pERK1/2
(T202/Y204) (42, 44 kDa)
1:1000
Rabbit anti-MEK1/2
(45 kDa)
1:1000
2
Mouse anti-V5-HRP
(45, 52, 58 kDa)
1:5000
Rabbit anti-Vinculin
(116 kDa)
1:20000
3
Rabbit anti-MAP3K8
(52, 58 kDa)
1:500
Mouse anti-ERK1/2
(42, 44 kDa)
1:1000 8. Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation,
and then detect signal using chemiluminescence following manufacturer’s instructions. a. Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary
antibodies and a low fluorescence membrane will be used instead, and images will be
acquired following manufacturer’s instructions. 9. Analyze bands with image analysis software and normalize to loading controls. a. pERK1/2 (T202/Y204) normalized to MEK1/2 (total). b. V5 (tag on exogenous proteins) normalized to Vinculin. 10. Repeat steps 1–9 independently two additional times. 8. Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation
and then detect signal using chemiluminescence following manufacturer’s instructions. 8. Apply appropriate HRP-linked secondary antibodies for 1 hr at RT with constant agitation,
and then detect signal using chemiluminescence following manufacturer’s instructions. a. Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary
antibodies and a low fluorescence membrane will be used instead, and images will be
acquired following manufacturer’s instructions. a. Note: If a Li-COR Odyssey imaging system is available for use, IR Dye-labeled secondary
antibodies and a low fluorescence membrane will be used instead, and images will be
acquired following manufacturer’s instructions. q
g
9. Analyze bands with image analysis software and normalize to loading controls. y
g
y
a. pERK1/2 (T202/Y204) normalized to MEK1/2 (total). p
b. V5 (tag on exogenous proteins) normalized to Vinculin. p
b. V5 (tag on exogenous proteins) normalized to Vinculin. 10. Repeat steps 1–9 independently two additional times. Deliverables: . Data to be collected: . Data to be collected:
Raw counts and infection efficiency (step 1 above).
Full image western blot films of all immunoblots including ladder. (Compare to Figures
2A and 4F.)
Raw data of band analysis and normalized bands for each sample. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Confirmatory analysis plan . Statistical Analysis of the Replication Data: y
One-way ANOVA of normalized pERK1/2 levels of A375 cells expressing MAP3K8
One-way ANOVA of normalized pERK1/2 levels of A375 cells expressing MAP3K8
treated with vehicle, PLX4720, PLX4720 + CI-1040, or PLX4720 + AZD6244 with the fol
l
l
d
h
f
One-way ANOVA of normalized pERK1/2 levels of A375 cells expressing MAP3K8
treated with vehicle, PLX4720, PLX4720 + CI-1040, or PLX4720 + AZD6244 with the fo
lowing planned comparisons using the Bonferroni correction: &
PLX4720 treatment compared to PLX4720 + CI-1040. &
PLX4720 treatment compared to PLX4720 + AZD6244.
Two-way ANOVA of normalized pERK1/2 levels of A375 cells expressing MEK1 or
DD
Two-way ANOVA of normalized pERK1/2 levels of A375 cells expressing MEK1 or
MEK1DD treated with vehicle, PLX4720, PLX4720 + CI-1040, or PLX4720 + AZD6244 y
p
/
p
g
MEK1DD treated with vehicle, PLX4720, PLX4720 + CI-1040, or PLX4720 + AZD6244. . Meta-analysis of original and replication attempt effect sizes: y
g
p
p
The replication data (mean and 95% confidence interval) will be plotted with the original
reported data value plotted as a single point on the same plot for comparison. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 21 of 32 Registered report Biochemistry Known differences from the original study g
y
The original NP40 cell lysis buffer was composed of: 150 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA
pH 8, 25 mM NaF, and 1% NP-40. The replication will use a commercial formula, which has the fol-
lowing composition: 250 mM NaCl, 50 mM Tris pH 7.4, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4,
and 1% NP-40. All known differences are listed in the materials and reagents section above with the
originally used item listed in the comments section. All differences have the same capabilities as the
original and are not expected to alter the experimental design. Provisions for quality control The cell line used in this experiment will undergo STR profiling to confirm its identity and will be
sent for mycoplasma testing to ensure there is no contamination. Infection efficiency will be deter-
mined for each replicate. The expression of the kinase of interest will be assessed using antibodies
against the V5 tag as well as MAP3K8 and MEK1. All of the raw data, including the analysis files, will
be uploaded to the project page on the OSF (https://osf.io/lmhjg/) and made publically available. Power Calculations
For additional details on power calculations, please see analysis scripts and associated files on the
Open Science Framework:
https://osf.io/sptzv/ Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Registered report h
(
,
)
Comparisons are between DMSO and all PLX4720 doses (10, 1, and 0.1 mM) Protocol 1 The original data presented is qualitative (images of Western blots). We used Image Studio Lite v. 4.0.21 (LI-COR) to perform densitometric analysis of the presented bands to quantify the original
effect size where possible. To identify a suitable sample size, power calculations were performed
using different levels of relative variance. Summary of estimated original data reported in Figure 3E: Summary of estimated original data reported in Figure 3E: Cell line
PLX4720 concentration
(mM)
Normalized
pMEK/ERK
Normalized
pERK/MEK
A375
0 (DMSO)
1.000
1.000
10
0.00212
0.0147
1
0.00283
0.0254
0.1
0.0981
0.00283
RPMI-7951
0 (DMSO)
1.000
1.000
10
0.827
1.849
1
0.380
1.460
0.1
0.471
1.022
OUMS-23
0 (DMSO)
1.000
1.000
10
0.0579
0.926
1
0.197
1.042
0.1
1.100
1.068 Sharma et al. eLife 2016;5:e11414. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Protocol 1 DOI: 10.7554/eLife.11414 22 of 32 Biochemistry Antibody
Cell line
F test statistic
Partial h2
Effect size f
A priori power
Total sample size
2% variance
pMEK
A375
F(1,8) = 20781
0.99962
50.9670
99.9%
12
(4 groups)
RPMI-7951
F(1,8) = 2128.4
0.99626
16.31121
80.0%
12
(4 groups)
OUMS-23
F(1,8) = 3001.0
0.99734
19.36831
80.0%
12
(4 groups)
pERK
A375
F(1,8) = 21841
0.99963
52.2504
99.9%
12
(4 groups)
RPMI-7951
F(1,8) = 582.85
0.98646
8.535621
80.0%
12
(4 groups)
OUMS-23
F(1,8) = 0.8080
0.09173
0.317801
80.0%
12
(4 groups)
15% variance
pMEK
A375
F(1,8) = 369.44
0.97880
6.79560
99.9%
12
(4 groups)
RPMI-7951
F(1,8) = 37.839
0.82547
2.174821
80.0%
12
(4 groups)
OUMS-23
F(1,8) = 53.352
0.86960
2.582441
80.0%
12
(4 groups)
pERK
A375
F(1,8) = 388.28
0.97981
6.96672
99.9%
12
(4 groups)
RPMI-7951
F(1,8) = 10.362
0.56431
1.138081
80.0%
12
(4 groups)
OUMS-23
F(1,8) = 0.01436
0.001792
0.042371
80.0%
12
(4 groups)
28% variance
pMEK
A375
F(1,8) = 106.68
0.92984
3.64050
99.9%
12
(4 groups)
RPMI-7951
F(1,8) = 10.859
0.57581
1.165091
80.0%
12
(4 groups)
OUMS-23
F(1,8) = 15.312
0.65682
1.383451
80.0%
12
(4 groups)
pERK
A375
F(1,8) = 111.43
0.93302
3.73217
99.9%
12
(4 groups)
RPMI-7951
F(1,8) = 2.9737
0.27099
0.609691
80.0%
12
(4 groups)
OUMS-23
F(1,8) = 0.04122
0.000515
0.022701
80.0%
12
(4 groups)
40% variance
pMEK
A375
F(1,8) = 51.952
0.86656
2.54835
99.9%
12
(4 groups)
RPMI-7951
F(1,8) = 5.3211
0.39945
0.815561
80.0%
12
(4 groups)
OUMS-23
F(1,8) = 7.5026
0.48396
0.968421
80.0%
12
(4 groups)
pERK
A375
F(1,8) = 54.602
0.87221
2.61252
99.9%
12
(4 groups)
RPMI-7951
F(1,8) = 1.4571
0.15408
0.426781
80.0%
12
(4 groups)
OUMS-23
F(1,8) = 0.00202
0.000252
0.015891
80.0%
12
(4 groups)
1 This is the calculated effect size using the originally reported value with the indicated variance. Unlike the power
calculations to determine sample size, the aim of these sensitivity calculations are not to detect the original effect
Registered report
Biochemistry Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 23 of 32 Registered report Registered report Biochemistry size, but to understand what effect size could be detected with 80% power and the indicated total sample size of
12. The detectable effect size is 1.29189. size, but to understand what effect size could be detected with 80% power and the indicated total sample size of
12. The detectable effect size is 1.29189. Protocol 1 In order to produce quantitative replication data, we will run the experiment three times. Each
time we will quantify band intensity. We will determine the standard deviation of band intensity
across the biological replicates and combine this with the reported value from the original study
to simulate the original effect size. We will use this simulated effect size to determine the num-
ber of replicates necessary to reach a power of at least 80%. We will then perform additional
replicates, if required, to ensure that the experiment has more than 80% power to detect the
original effect. Protocol 3 The original data is from a single biological replicate. To identify a suitable sample size, power calcu-
lations were performed using different levels of relative variance. Summary of estimated original data reported in Figure 3D: Cell line
GI50 (mM)
A375
0.2276
RPMI-7951
10.862
OUMS-23
8.731 Test family
&
2 tailed t test, difference between two independent means, Fisher’s LSD: alpha error = 0.05
Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). Group 1
Group 2
Effect size d
A priori power
Group 1 sample size
Group 2 sample size
2% variance
A375
RPMI-7951
69.2139
99.9%
2
2
A375
OUMS-23
55.3442
99.9%
2
2
15% variance
A375
RPMI-7951
68.3898
99.9%
2
2
A375
OUMS-23
7.37923
93.3%
2
2
28% variance
A375
RPMI-7951
4.94385
99.2%
3
3
A375
OUMS-23
3.95316
94.4%
3
3
40% variance
A375
RPMI-7951
3.46070
88.0%
3
3
A375
OUMS-23
2.76721
90.0%
4
4
In order to produce quantitative replication data, we will run the experiment three times. Each
time we will calculate the GI50 value. We will determine the standard deviation of GI50 values across
the biological replicates and combine this with the reported value from the original study to simulate
the original effect size. We will use this simulated effect size to determine the number of replicates
necessary to reach a power of at least 80%. We will then perform additional replicates, if required,
to ensure that the experiment has more than 80% power to detect the original effect. Test family
&
2 tailed t test, difference between two independent means, Fisher’s LSD: alpha error = 0.05
Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). In order to produce quantitative replication data, we will run the experiment three times. Each
time we will calculate the GI50 value. We will determine the standard deviation of GI50 values across
the biological replicates and combine this with the reported value from the original study to simulate
the original effect size. We will use this simulated effect size to determine the number of replicates
necessary to reach a power of at least 80%. We will then perform additional replicates, if required,
to ensure that the experiment has more than 80% power to detect the original effect. In order to produce quantitative replication data, we will run the experiment three times. Each
time we will calculate the GI50 value. Registered report Protocol 3 We will determine the standard deviation of GI50 values across
the biological replicates and combine this with the reported value from the original study to simulate
the original effect size. We will use this simulated effect size to determine the number of replicates
necessary to reach a power of at least 80%. We will then perform additional replicates, if required,
to ensure that the experiment has more than 80% power to detect the original effect. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 24 of 32 Biochemistry Protocol 4 The original data does not indicate the error associated with multiple biological replicates. To iden-
tify a suitable sample size, power calculations were performed using different levels of relative
variance. Summary of original data reported in Figure 3I: Summary of original data reported in Figure 3I: IC50 values performed with R software, version 3.2.1 (Team, 2015). Summary of original data reported in Figure 3I: We will determine the standard deviation of band intensity
across the biological replicates and combine this with the reported value from the original study to
simulate the original effect size. We will use this simulated effect size to determine the number of
replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if
required, to ensure that the experiment has more than 80% power to detect the original effect. Protocol 5
The original data is from a single biological replicate. To identify a suitable sample size, power calcu-
lations were performed using different levels of relative variance. Summary of original data reported in Figure 4E (provided by authors)
Cell line
Drug(s)
Relative
viability
Technical replicate
stdev
MEK1
DMSO
100
5.09
PLX4720 (10 mM)
5.78
0.67
PLX4720 (1 mM)
11.32
0.97
PLX4720 (1 mM) + CI-1040 (1 mM)
10.13
0.60
PLX4720 (1 mM) + AZD6244 (1 mM)
10.92
0.91
MEK1DD
DMSO
96.96
9.40
PLX4720 (10 mM)
97.56
6.25
PLX4720 (1 mM)
92.10
8.23
PLX4720 (1 mM) + CI-1040 (1 mM)
102.24
8.55
PLX4720 (1 mM) + AZD6244 (1 mM)
42.44
2.74
Continued on next page
Registered report
Biochemistry Test family
&
t-test: Means: Difference from constant (one sample case): Bonferroni’s correction: alpha
error = 0.025
Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). MAP3K8 inhibitor dose
Constant (vehicle)
Antibody
Effect size d
A priori power
Sample size
per group
2% variance
20 mM
0 mM
pMEK
88.8889
99.9%
3
pERK
20.4225
99.9%
3
15% variance
20 mM
0 mM
pMEK
11.8519
99.9%
3
pERK
2.72300
80.2%
4
28% variance
20 mM
0 mM
pMEK
6.34921
95.1%
3
pERK
1.45875
86.6%
8
40% variance
20 mM
0 mM
pMEK
4.44444
99.2%
4
pERK
1.02113
81.1%
12
Registered report
Biochemistry Test family
&
t-test: Means: Difference from constant (one sample case): Bonferroni’s correction: alpha
error = 0.025
ed report
Biochemistry Biochemistry Test family
&
t-test: Means: Difference from constant (one sample case): Bonferroni’s correction: alpha
error = 0.025 Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). In order to produce quantitative replication data, we will run the experiment eight times. Each
time we will quantify band intensity. We will determine the standard deviation of band intensity
across the biological replicates and combine this with the reported value from the original study to
simulate the original effect size. Summary of original data reported in Figure 3I: Cell line
MAP3K8 inhibitor concentration (mM)
Normalized pMEK/ERK
Normalized pERK/MEK
RPMI-7951
0 (DMSO)
1.00
1.00
1
1.35
1.00
5
1.15
0.92
10
0.70
0.75
20
0.36
0.71 IC50 values performed with R software, version 3.2.1 (Team, 2015). IC50 value of normalized pMEK values
IC50 value of normalized pERK values
9.4725
6.4091 Test family
&
F test, ANOVA: Fixed effects, omnibus, one-way, Bonferroni’s correction: alpha error =
0.025
Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h2 performed with R software, version 3.2.1 (Team, 2015). Partial h2 calculated from (Lakens, 2013). Groups
Antibody
F test statistic
Partial h2
Effect size f
A priori power
Total sample size
2% variance
MAP3K8 inhibitor (1, 5, 10, 20 mM)
pMEK
F(3,8) = 1583.7
0.99832
24.3697
99.9%
8
(4 groups)
pERK
F(3,8) = 195.33
0.98653
8.55863
99.9%
8
(4 groups)
15% variance
MAP3K8 inhibitor (1, 5, 10, 20 mM)
pMEK
F(3,8) = 28.155
0.91348
3.24931
96.8%
8
(4 groups)
pERK
F(3,8) = 3.4726
0.56563
1.14114
83.6%
16
(4 groups)
28% variance
MAP3K8 inhibitor (1, 5, 10, 20 mM)
pMEK
F(3,8) = 8.0801
0.75186
1.74070
94.7%
12
(4 groups)
pERK
F(3,8) = 0.9966
0.27205
0.61133
80.8%
40
(4 groups)
40% variance
MAP3K8 inhibitor (1, 5, 10, 20 mM)
pMEK
F(3,8) = 3.9593
0.59754
1.21849
88.7%
16
(4 groups)
pERK
F(3,8) = 0.4883
0.15478
0.42793
80.5%
76
(4 groups) Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 25 of 32 Test family
&
t-test: Means: Difference from constant (one sample case): Bonferroni’s correction: alpha
error = 0.025
Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). MAP3K8 inhibitor dose
Constant (vehicle)
Antibody
Effect size d
A priori power
Sample size
per group
2% variance
20 mM
0 mM
pMEK
88.8889
99.9%
3
pERK
20.4225
99.9%
3
15% variance
20 mM
0 mM
pMEK
11.8519
99.9%
3
pERK
2.72300
80.2%
4
28% variance
20 mM
0 mM
pMEK
6.34921
95.1%
3
pERK
1.45875
86.6%
8
40% variance
20 mM
0 mM
pMEK
4.44444
99.2%
4
pERK
1.02113
81.1%
12
In order to produce quantitative replication data, we will run the experiment eight times. Each
time we will quantify band intensity. Summary of original data reported in Figure 3I: We will use this simulated effect size to determine the number of
replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if
required, to ensure that the experiment has more than 80% power to detect the original effect. Protocol 5 The original data is from a single biological replicate. To identify a suitable sample size, power calcu-
lations were performed using different levels of relative variance. Summary of original data reported in Figure 4E (provided by authors) Summary of original data reported in Figure 4E (provided by authors) Cell line
Drug(s)
Relative
viability
Technical replicate
stdev
MEK1
DMSO
100
5.09
PLX4720 (10 mM)
5.78
0.67
PLX4720 (1 mM)
11.32
0.97
PLX4720 (1 mM) + CI-1040 (1 mM)
10.13
0.60
PLX4720 (1 mM) + AZD6244 (1 mM)
10.92
0.91
MEK1DD
DMSO
96.96
9.40
PLX4720 (10 mM)
97.56
6.25
PLX4720 (1 mM)
92.10
8.23
PLX4720 (1 mM) + CI-1040 (1 mM)
102.24
8.55
PLX4720 (1 mM) + AZD6244 (1 mM)
42.44
2.74 Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 26 of 32 Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Continued
Cell line
Drug(s)
Relative
viability
Technical replicate
stdev
MAP3K8
DMSO
100
10.66
PLX4720 (10 mM)
43.44
3.48
PLX4720 (1 mM)
62.81
5.05
PLX4720 (1 mM) + CI-1040 (1 mM)
13.42
2.42
PLX4720 (1 mM) + AZD6244 (1 mM)
12.52
1.65
Registered report
Biochemistry Biochemistry Cell line
Drug(s)
Relative
viability
Technical replicate
stdev
MAP3K8
DMSO
100
10.66
PLX4720 (10 mM)
43.44
3.48
PLX4720 (1 mM)
62.81
5.05
PLX4720 (1 mM) + CI-1040 (1 mM)
13.42
2.42
PLX4720 (1 mM) + AZD6244 (1 mM)
12.52
1.65 PLX4720 (1 mM) + AZD6244 (1 mM)
12.52
1.65
Test family
&
F test, ANOVA: Fixed effects, omnibus, one-way, alpha error = 0.05. Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h2 performed with R software, version 3.2.1 (Team, 2015). MAP3K8 values
Groups
F test statistic
Partial h2
Effect size f
A priori power
Total sample size
2% variance
All treatments
F(4,25) = 6246.6
0.99900
31.60696
99.9%
10
(5 groups)
15% variance
All treatments
F(4,25) = 111.04
0.94672
4.21509
99.9%
15
(5 groups)
28% variance
All treatments
F(4,25) = 31.868
0.83604
2.25807
99.9%
20
(5 groups)
40% variance
All treatments
F(4,25) = 15.615
0.71416
1.58066
99.9%
25
(5 groups)
Test family
&
2 tailed t test, difference between two independent means, Bonferroni’s correction: alpha
error = 0.0125
Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Protocol 5 Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h2 performed with R software, version 3.2.1 (Team, 2015). MEK1 and MEK1DD values
Groups
F test statistic
Partial h2
Effect size f
A priori power
Total sample size
2% variance
All treatments
F(4,50) = 2716.2
0.99542
14.7409
99.9%
20
(10 groups)
15% variance
All treatments
F(4,50) = 48.287
0.79437
1.96545
99.9%
30
(10 groups)
28% variance
All treatments
F(4,50) = 13.858
0.52576
1.05292
99.9%
40
(10 groups)
40% variance
All treatments
F(4,50) = 6.7904
0.35201
0.73704
98.7%
50
(10 groups)
In order to produce quantitative replication data, we will run the experiment four times. Each
time we will quantify relative viability. We will determine the standard deviation of viability values
across the biological replicates and combine this with the reported value from the original study to
simulate the original effect size. We will use this simulated effect size to determine the number of
replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if
i
d t
th t th
i
t h
th
80%
t
d t
t th
i i
l
ff
t
Registered report
Biochemistry Test family
&
F test, ANOVA: Fixed effects, special, main effects and interactions, alpha error = 0.05. Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h2 performed with R software, version 3.2.1 (Team, 2015). MEK1 and MEK1DD values
Groups
F test statistic
Partial h2
Effect size f
A priori power
Total sample size
2% variance
All treatments
F(4,50) = 2716.2
0.99542
14.7409
99.9%
20
(10 groups)
15% variance
All treatments
F(4,50) = 48.287
0.79437
1.96545
99.9%
30
(10 groups)
28% variance
All treatments
F(4,50) = 13.858
0.52576
1.05292
99.9%
40
(10 groups)
40% variance
All treatments
F(4,50) = 6.7904
0.35201
0.73704
98.7%
50
(10 groups)
In order to produce quantitative replication data, we will run the experiment four times. Each
time we will quantify relative viability. We will determine the standard deviation of viability values
across the biological replicates and combine this with the reported value from the original study to
simulate the original effect size. We will use this simulated effect size to determine the number of
replicates necessary to reach a power of at least 80%. Protocol 5 MAP3K8 values
Group 1
Group 2
Effect
size d
A priori
power
Group 1
sample size
Group 2
sample size
2% variance
1 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
67.45484
99.9%
2
2
1 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
55.51810
99.9%
2
2
10 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
46.68351
99.9%
2
2
10 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
48.35265
99.9%
2
2
15% variance
Continued on next page 27 of 32 Continued
Group 1
Group 2
Effect
size d
A priori
power
Group 1
sample size
Group 2
sample size
1 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
7.24974
99.4%
3
3
1 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
7.40241
99.6%
3
3
10 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
6.22447
97.2%
3
3
10 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
6.44702
97.9%
3
3
28% variance
1 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
3.88379
93.0%
4
4
1 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
3.96558
94.0%
4
4
10 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
3.33454
82.9%
4
4
10 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
3.45376
85.7%
4
4
40% variance
1 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
2.71865
82.6%
5
5
1 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
2.77591
84.3%
5
5
10 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
2.33418
81.5%
6
6
10 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
2.41763
84.5%
6
6
Registered report
Biochemistry Biochemistry Biochemistry Continued
Group 1
Group 2
Effect
size d
A priori
power
Group 1
sample size
Group 2
sample size
1 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
7.24974
99.4%
3
3
1 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
7.40241
99.6%
3
3
10 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
6.22447
97.2%
3
3
10 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
6.44702
97.9%
3
3
28% variance
1 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
3.88379
93.0%
4
4
1 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
3.96558
94.0%
4
4
10 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
3.33454
82.9%
4
4
10 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
3.45376
85.7%
4
4
40% variance
1 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
2.71865
82.6%
5
5
1 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
2.77591
84.3%
5
5
10 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
2.33418
81.5%
6
6
10 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
2.41763
84.5%
6
6
Test family
&
F test, ANOVA: Fixed effects, special, main effects and interactions, alpha error = 0.05. Protocol 5 Summary of estimated original data reported in Figure 4F:
Cell line
Drug(s)
Normalized pERK/ERK
MEK1
DMSO
1.0000
PLX4720 (1 mM)
0.0799
PLX4720 (1 mM) + CI-1040 (1 mM)
0.0021
PLX4720 (1 mM) + AZD6244 (1 mM)
0.0002
MEK1DD
DMSO
1.0000
PLX4720 (1 mM)
1.0203
PLX4720 (1 mM) + CI-1040 (1 mM)
0.3015
PLX4720 (1 mM) + AZD6244 (1 mM)
0.0166
MAP3K8
DMSO
1.0000
PLX4720 (1 mM)
0.7689
PLX4720 (1 mM) + CI-1040 (1 mM)
0.0045
PLX4720 (1 mM) + AZD6244 (1 mM)
0.0063
Test family
. F test, ANOVA: Fixed effects, omnibus, one-way, alpha error = 0.05. Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h2 performed with R software, version 3.2.1 (Team, 2015). MAP3K8 values
Groups
F test statistic
Partial h2
Effect size f
A priori
power
Total
sample size
2% variance
Registered report
Biochemistry Biochemistry Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Protocol 5 We will then perform additional replicates, if
required, to ensure that the experiment has more than 80% power to detect the original effect. Test family
&
F test, ANOVA: Fixed effects, special, main effects and interactions, alpha error = 0.05. Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h2 performed with R software, version 3.2.1 (Team, 2015). MEK1 and MEK1DD values
Groups
F test statistic
Partial h2
Effect size f
A priori power
Total sample size
2% variance
All treatments
F(4,50) = 2716.2
0.99542
14.7409
99.9%
20
(10 groups)
15% variance
All treatments
F(4,50) = 48.287
0.79437
1.96545
99.9%
30
(10 groups)
28% variance
All treatments
F(4,50) = 13.858
0.52576
1.05292
99.9%
40
(10 groups)
40% variance
All treatments
F(4,50) = 6.7904
0.35201
0.73704
98.7%
50
(10 groups) MEK1 and MEK1DD values
Groups
F test statistic
Partial h2
Effect size f
A priori power
Total sample size
2% variance
All treatments
F(4,50) = 2716.2
0.99542
14.7409
99.9%
20
(10 groups)
15% variance
All treatments
F(4,50) = 48.287
0.79437
1.96545
99.9%
30
(10 groups)
28% variance
All treatments
F(4,50) = 13.858
0.52576
1.05292
99.9%
40
(10 groups)
40% variance
All treatments
F(4,50) = 6.7904
0.35201
0.73704
98.7%
50
(10 groups) In order to produce quantitative replication data, we will run the experiment four times. Each
time we will quantify relative viability. We will determine the standard deviation of viability values
across the biological replicates and combine this with the reported value from the original study to
simulate the original effect size. We will use this simulated effect size to determine the number of
replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if
required, to ensure that the experiment has more than 80% power to detect the original effect. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 28 of 32 Protocol 6
The original data presented is qualitative (images of Western blots). We used Image Studio Lite v. 4.0.21 (LI-COR) to perform densitometric analysis of the presented bands to quantify the original
effect size where possible. Protocol 6 We will determine the standard deviation of band intensity
across the biological replicates and combine this with the reported value from the original study to
sim late the original effect si e We
ill
se this sim lated effect si e to determine the n mber of Group 1
Group 2
Effect size d
A priori
power
Group 1
sample size
Group 2
sample size
2% variance
1 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
68.8057
99.9%
2
2
1 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
70.5269
99.9%
2
2
15% variance
1 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
9.17409
87.8%
2
2
1 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
9.40358
89.0%
2
2
28% variance
1 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
4.91469
95.5%
3
3
1 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
5.03763
96.3%
3
3
40% variance
1 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
3.44028
93.7%
4
4
1 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
3.52634
94.7%
4
4 In order to produce quantitative replication data, we will run the experiment three times. Each
time we will quantify band intensity. We will determine the standard deviation of band intensity
across the biological replicates and combine this with the reported value from the original study to
simulate the original effect size. We will use this simulated effect size to determine the number of
replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if
required, to ensure that the experiment has more than 80% power to detect the original effect. Protocol 6 The original data presented is qualitative (images of Western blots). We used Image Studio Lite v. 4.0.21 (LI-COR) to perform densitometric analysis of the presented bands to quantify the original
effect size where possible. p
Summary of estimated original data reported in Figure 4F: Summary of estimated original data reported in Figure 4F: Cell line
Drug(s)
Normalized pERK/ERK
MEK1
DMSO
1.0000
PLX4720 (1 mM)
0.0799
PLX4720 (1 mM) + CI-1040 (1 mM)
0.0021
PLX4720 (1 mM) + AZD6244 (1 mM)
0.0002
MEK1DD
DMSO
1.0000
PLX4720 (1 mM)
1.0203
PLX4720 (1 mM) + CI-1040 (1 mM)
0.3015
PLX4720 (1 mM) + AZD6244 (1 mM)
0.0166
MAP3K8
DMSO
1.0000
PLX4720 (1 mM)
0.7689
PLX4720 (1 mM) + CI-1040 (1 mM)
0.0045
PLX4720 (1 mM) + AZD6244 (1 mM)
0.0063 Test family
. F test, ANOVA: Fixed effects, omnibus, one-way, alpha error = 0.05. Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h2 performed with R software, version 3.2.1 (Team, 2015). MAP3K8 values
Groups
F test statistic
Partial h2
Effect size f
A priori
power
Total
sample size
2% variance
All treatments
F(3,8) = 5024.1
0.99947
43.4257
99.9%
8
(4 groups)
15% variance
All treatments
F(3,8) = 89.317
0.97101
5.78735
99.9%
8
(4 groups)
28% variance
All treatments
F(3,8) = 25.633
0.90577
3.10038
99.9%
12
(4 groups)
40% variance
All treatments
F(3,8) = 12.560
0.82487
2.17026
99.9%
16
(4 groups)
Test family
. 2 tailed t test, difference between two independent means, Bonferroni’s correction: alpha
error = 0.025
Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). MAP3K8 values
Groups
F test statistic
Partial h2
Effect size f
A priori
power
Total
sample size
2% variance
All treatments
F(3,8) = 5024.1
0.99947
43.4257
99.9%
8
(4 groups)
15% variance
All treatments
F(3,8) = 89.317
0.97101
5.78735
99.9%
8
(4 groups)
28% variance
All treatments
F(3,8) = 25.633
0.90577
3.10038
99.9%
12
(4 groups)
40% variance
All treatments
F(3,8) = 12.560
0.82487
2.17026
99.9%
16
(4 groups)
Test family
2
il d
diff
b
i d
d
B
f
i’
i
l h Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). Sharma et al. eLife 2016;5:e11414. Protocol 6 DOI: 10.7554/eLife.11414 29 of 32 Biochemistry Group 1
Group 2
Effect size d
A priori
power
Group 1
sample size
Group 2
sample size
2% variance
1 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
68.8057
99.9%
2
2
1 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
70.5269
99.9%
2
2
15% variance
1 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
9.17409
87.8%
2
2
1 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
9.40358
89.0%
2
2
28% variance
1 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
4.91469
95.5%
3
3
1 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
5.03763
96.3%
3
3
40% variance
1 mM PLX4720
1 mM PLX4720 + 1 mM CI1040
3.44028
93.7%
4
4
1 mM PLX4720
1 mM PLX4720 + 1 mM AZD6244
3.52634
94.7%
4
4
Test family
&
F test, ANOVA: Fixed effects, special, main effects and interactions, alpha error = 0.05. Power Calculations performed with G*Power software, version 3.1.7 (Faul et al., 2007). ANOVA F test statistic and partial h2 performed with R software, version 3.2.1 (Team, 2015). MEK1 and MEK1DD values
Groups
F test statistic
Partial h2
Effect size f
A priori power
Total sample size
2% variance
All treatments
F(3,16) = 1847.2
0.99712
18.6103
99.9%
16
(8 groups)
15% variance
All treatments
F(3,16) = 32.840
0.86029
2.48143
99.9%
16
(8 groups)
28% variance
All treatments
F(3,16) = 9.4247
0.63862
1.32934
99.9%
24
(8 groups)
40% variance
All treatments
F(3,16) = 4.6181
0.46406
0.93053
99.0%
32
(8 groups)
In order to produce quantitative replication data, we will run the experiment three times. Each
time we will quantify band intensity. Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Acknowledgements g
The Reproducibility Project: Cancer Biology core team would like to thank the original authors, in
particular Cory Johannessen, for generously sharing critical information as well as reagents to ensure
the fidelity and quality of this replication attempt. We thank Courtney Soderberg at the Center for 30 of 32 Sharma et al. eLife 2016;5:e11414. DOI: 10.7554/eLife.11414 Registered report Biochemistry Open Science for assistance with statistical analyses. We would also like to thank the following com-
panies for generously donating reagents to the Reproducibility Project: Cancer Biology; American
Type Culture Collection (ATCC), Applied Biological Materials, BioLegend, Charles River Laborato-
ries, Corning Incorporated, DDC Medical, EMD Millipore, Harlan Laboratories, LI-COR Biosciences,
Mirus Bio, Novus Biologicals, Sigma-Aldrich, and System Biosciences (SBI). Author contributions Author contributions
VS, LY, MC, KO, Drafting or revising the article; RP:CB, Conception and design, Drafting or revising
the article Reproducibility Project: Cancer Biology Elizabeth Iorns: Science Exchange, Palo Alto, United States; William Gunn: Mendeley, London,
United Kingdom; Fraser Tan: Science Exchange, Palo Alto, United States; Joelle Lomax: Science
Exchange, Palo Alto, United States; Nicole Perfito: Science Exchange, Palo Alto, United States;
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The landscape of microRNA interactions annotation: analysis of three rare disorders as case study
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bioRxiv preprint * Presently Astra Zeneca, Data Office, Data Science and AI, UK. Academy House,
136 Hills Road, Cambridge CB2 8PA UK The landscape of microRNA interactions annotation:
analysis of three rare disorders as case study Panni Simona 1, Kalpana Panneerselvam,2 Pablo Porras2*, Margaret
Duesbury2, Livia Perfetto3, Luana Licata4, Henning Hermjakob2, Sandra
Orchard2 Panni Simona 1, Kalpana Panneerselvam,2 Pablo Porras2*, Margaret
Duesbury2, Livia Perfetto3, Luana Licata4, Henning Hermjakob2, Sandra
Orchard2 1. Università della Calabria, Dipartimento di Biologia, Ecologia e Scienze della Terra,
Rende, IT 2. European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-
EBI), Wellcome Genome Campus, Hinxton, Cambridge. CB10 1SD. UK. 3. Department of Biology and Biotechnologies "Charles Darwin", La Sapienza
University of Rome, IT 4. Department of Biology University of Rome Tor Vergata, Rome, IT . CC-BY 4.0 International license
made available under a
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bioRxiv preprint Keywords: microRNA, interaction database, rare disease, biocuration,
RRI Abstract In recent years, a huge amount of data on ncRNA interactions has been
described in scientific papers and databases. Although considerable effort
has been made to annotate the available knowledge in public repositories,
there are still significant discrepancies in how different resources capture
and interpret data on ncRNAs functional and physical associations. In the present paper, we have focused on microRNAs which regulate
genes associated with rare diseases, as a case study to investigate data
availability. The list of protein-coding genes with a known role in specific rare diseases
was retrieved from the Genome England PanelApp, and associated
microRNA-mRNA interactions were annotated in the IntAct database, and
compared with other datasets. The annotation follows recognised
standard criteria approved by the IMEX consortium. RNAcentral identifiers
were used for unambiguous, stable identification of ncRNAs. The
information about the interaction was enhanced by a detailed description
of the cell types and experimental conditions, providing a computer-
interpretable summary of the published data, integrated with the huge
amount of protein interactions already gathered in the database. Furthermore, for each interaction, the binding sites of the microRNA are
precisely mapped on a well-defined mRNA transcript of the target gene. This information is crucial to conceive and design optimal microRNA
mimics or inhibitors, to interfere in vivo with a deregulated process. As
these approaches become more feasible, high-quality, reliable networks
of microRNA interactions are needed to help, for instance, in the selection
of the best target to be inhibited and to predict potential secondary off-
target effects. . CC-BY 4.0 International license
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bioRxiv preprint Introduction In recent years, it has become increasingly clear that complex and
dynamic interactions between ncRNA molecules or ncRNAs and proteins
contribute to virtually any biological process. MicroRNAs are probably the
best characterised ncRNAs, as they can be identified with bioinformatics
approaches, thanks to the conserved hairpin shape of precursor
transcripts, and their possible targets can be predicted [1]. MicroRNAs
regulate gene expression by guiding the RNA induced silencing complex
(RISC) to those mRNAs which display sequences complementary to the
microRNA “seed” i.e. nucleotides 2-7 from the 5’ end [2,3]. Predicted and
verified microRNAs from more than 270 species are annotated in
miRbase [4], with their precursor and mature sequences and other useful
information. The latest release contains 1917 human hairpin precursors
and 2654 human mature sequences, of which 26% are high-confidence
microRNAs [4]. Several microRNA precursors encode two active
microRNAs, named 3p and 5p respectively, which recognise different
targets. Identifying the exact function for each miRNA is challenging and
time consuming, and only a subset of them have been characterised. As
mentioned above, it is possible to predict all possible targets for any
microRNA, based on the sequence complementarity, site conservation
and other features [1,5]. However, these methods provide a list of
hundreds of targets for each miRNAs, many of which are not bona-fide
interactors, and need to be experimentally verified [6,7]. To this aim,
several techniques have been developed, for low and high throughput
interaction detection, which we briefly summarise here. One of the most
frequently used assays is the luciferase reporter assay, which is an
adaptation of the homonymous test used to identify the regulatory regions
on DNA promoters (Figure 1). The method not only permits verification of
the interaction, but also the mapping of the precise binding site on the
mRNA, through mutagenesis of the predicted sequence [8]. QRT-PCR
and western blot allow the quantification of the mRNA and protein levels,
respectively, following microRNA over-expression, and can be used to
discriminate between mRNA degradation and translation inhibition, which
are the two possible results of microRNA interaction (Figure 1). Pull down
and immunoprecipitation approaches, commonly used to demonstrate
protein binding, have been adapted to demonstrate microRNA
interactions. Cross-linking ligation and sequencing of hybrids (CLASH)
allows high-throughput identification of RNA-RNA interactions. Introduction In this
approach, the RNA is cross-linked to the bait protein, and, after the
immunoprecipitation, the ends of co-precipitated, interacting RNAs, are
ligated together and sequenced to identify the couples [9]. Other high- In recent years, it has become increasingly clear that complex and
dynamic interactions between ncRNA molecules or ncRNAs and proteins
contribute to virtually any biological process. MicroRNAs are probably the
best characterised ncRNAs, as they can be identified with bioinformatics
approaches, thanks to the conserved hairpin shape of precursor
transcripts, and their possible targets can be predicted [1]. MicroRNAs
regulate gene expression by guiding the RNA induced silencing complex
(RISC) to those mRNAs which display sequences complementary to the
microRNA “seed” i.e. nucleotides 2-7 from the 5’ end [2,3]. Predicted and
verified microRNAs from more than 270 species are annotated in
miRbase [4], with their precursor and mature sequences and other useful
information. The latest release contains 1917 human hairpin precursors
and 2654 human mature sequences, of which 26% are high-confidence
microRNAs [4]. Several microRNA precursors encode two active
microRNAs, named 3p and 5p respectively, which recognise different
targets. Identifying the exact function for each miRNA is challenging and
time consuming, and only a subset of them have been characterised. As
mentioned above, it is possible to predict all possible targets for any
microRNA, based on the sequence complementarity, site conservation
and other features [1,5]. However, these methods provide a list of
hundreds of targets for each miRNAs, many of which are not bona-fide
interactors, and need to be experimentally verified [6,7]. To this aim,
several techniques have been developed, for low and high throughput
interaction detection, which we briefly summarise here. One of the most
frequently used assays is the luciferase reporter assay, which is an
adaptation of the homonymous test used to identify the regulatory regions
on DNA promoters (Figure 1). The method not only permits verification of
the interaction, but also the mapping of the precise binding site on the
mRNA, through mutagenesis of the predicted sequence [8]. QRT-PCR
and western blot allow the quantification of the mRNA and protein levels,
respectively, following microRNA over-expression, and can be used to
discriminate between mRNA degradation and translation inhibition, which
are the two possible results of microRNA interaction (Figure 1). Pull down
and immunoprecipitation approaches, commonly used to demonstrate
protein binding, have been adapted to demonstrate microRNA
interactions. Introduction Cross-linking ligation and sequencing of hybrids (CLASH)
allows high-throughput identification of RNA-RNA interactions. In this
approach, the RNA is cross-linked to the bait protein, and, after the
immunoprecipitation, the ends of co-precipitated, interacting RNAs, are
ligated together and sequenced to identify the couples [9]. Other high- . CC-BY 4.0 International license
made available under a
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bioRxiv preprint throughput sequencing approaches of immunoprecipitated RNAs, after
cross-linking to proteins of the RISC complex (CLIP-seq, PAR-CLIP etc.),
provide datasets of potential RNA-RNA interactions [10], largely improving
the performance of the binding predictors, although specific couples of
miRNA-mRNA should be further verified [11]. While the number of
publications describing microRNA interactions is constantly increasing,
several databases have begun to collect them, often including high
throughput indirect or weak evidence. It is worth noting that miRTarBase
[12] and RAID [13,14] allow filtering of collected ncRNAs associations, to
reduce false positive rates when selecting “strong evidence” interactions. The inclusion of potentially erroneous targets in network analysis may
result in misleading data interpretation, as extensively discussed in
Huntley et al., 2018. The UCL Functional Gene Annotation group has
focused on the gene ontology annotation of human microRNAs collecting
a set of highly reliable microRNA-mRNA pairs [15,16]. g y
p
[
,
]
Since 2002 the HUPO Proteomic Standard Initiative (HUPO-PSI) has
provided a standardised annotation system for molecular interactions, and
has defined the minimal information requirements and the syntax of terms
used to describe an interaction experiment (MIMIx) [17], approved by
members of the International Molecular Exchange (IMEx) Consortium
[18,19]. Common guidelines help to elude false positives, and increase
the level of detail and contextual information captured when describing
molecules, such as protein binding [20]. The IntAct database, which is a
member of the Consortium, has expanded its activities into the annotation
of ncRNA interactions, initially focusing on S. cerevisiae ncRNAs [21], and
subsequently on mammalian ncRNAs. Introduction In this paper, we present the
collection of microRNA-mRNA interactions annotated in IntAct and we
integrate and compare it with other resources. In particular, we have
focused on the microRNAs that regulate genes associated with rare
diseases, in order to provide information potentially useful for the
development of new drugs. A rare disease is a health condition that affects
a minority of people compared with other diseases that are prevalent in
the population [22]. Although the increased focus on these diseases in the
last few years, very little is known on microRNAs involved in rare diseases
and their collection may help to gain insights into pathological
mechanisms. The interactions can be downloaded from the IntAct database
(https://www.ebi.ac.uk/intact/) and also from the RNA central website
(https://rnacentral.org/). . CC-BY 4.0 International license
made available under a
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bioRxiv preprint Figure 1. Methods commonly used to detect microRNA-mRNA
interactions. A) Luciferase assay comprises of the fusion of the luciferase
gene to the 3’ UTR of the microRNA target gene and the subsequent
transfection of the construct with or without the microRNA. The approach
demonstrates the direct interaction, compared to a mutated copy of the
predicted complementary region, which acts as a negative control. Introduction Quantification of the mRNA (qRT-PCR) or protein level (WB) helps to
distinguish between the mRNA degradation or the translation inhibition,
but are not proof of an interaction when used in isolation. B) CLIP and
CLASH. Cross-linking and immunoprecipitation of a RNA binding protein
(such as AGO2), followed by RNA sequencing, allows the determination
of all the RNAs bound to the protein. These approaches demonstrate
direct RNA-RNA binding, if the two interacting RNAs are ligated in a hybrid
before sequencing (CLASH, [9]). Data collection and deposition MicroRNA-mRNA interactions were manually annotated in the IntAct
database, according to the curation standards established by the IMEx
Consortium. We retrieved relevant papers by searching the literature with
text-mining seeking for the term “microRNA” (or synonymous) AND a
specific gene name in the abstract, and luciferase assay or RIP, or CLASH
in the methods. The collected papers were manually filtered to identify
those relevant for the curation process. More than 260 papers were
selected and annotated. Genes associated with rare diseases were prioritised for annotation. The
list of these genes was generated according to green panels from
GenomeEngland PanelApp for the following diseases: Aniridia, Anemia
Fanconi, Autism, Cakut, Cerebrak Folate Deficiency, Epidermolysis
Bullosa, Familial Hirschsprung Disease, Growth Failure in Early
Childhood, Early Onset Disease and Mitochondrial Disorders. These last
three presented a higher number of genes regulated by microRNAs and
were used in the subsequent analysis. The microRNAs are identified with
RNAcentral IDs and a short label, which specify the strand of the mature
microRNA [23].The database can be queried with Ensembl transcript IDs
or gene common names, (then selecting mRNA) or with mRNA common . CC-BY 4.0 International license
made available under a
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bioRxiv preprint names (mrna_name), as well as with the microRNA name or ID. To
retrieve information on mutagenesis analysis, in order to map the binding
nucleotides, from the Intact web page, click on the lens in the “Select”
column of the displayed interactions, then select “Features” in the
following web page. Data are also linked to RNA central [23] in the
microRNA entry. Network analysis and databases comparison The molecular interaction network of microRNA-mRNA interactions was
downloaded from IntAct using the IntAct app [24]. To build disease-
specific networks, the list of Ensembl transcript IDs was used. Networks
were built and analysed with Cytoscape [25]. For comparison with other databases, miRTarBase and Raid were chosen
to enable the selection of strong evidences of direct binding, comparable
to those annotated in IntAct. The data were downloaded, IDs were
uniformed, and the tables were uploaded in Cytoscape to build and
intersect the networks. Functional enrichment analysis For the “Biological Processes” enrichment analysis of microRNA targets,
each transcript ID was converted to the common gene name. The tables
were imported in Cytoscape to build the network, and BINGO tool [26] was
used to perform the enrichment with 0.003 as significance level for GFEC
and EOD related genes, while 0.05 was set for MD because there was no
enrichment at lower values. Statistical analysis Correlation analysis: A table was generated containing the number of
interacting microRNAs for each gene in each of the three databases. Pearson correlation coefficient was calculated with the ggpairs function in
the GGally R package [27,28] Venn Diagram: Intersections among
datasets were outlined by ggVennDiagram library [29] in R [28] and
Jaccard index was calculated as : J(A, B) =
! ∩ $
! ∪ $ [30] . CC-BY 4.0 International license
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bioRxiv preprint Building microRNA network in IntAct The main molecular function of microRNAs is to down-regulate the target
gene by binding to the mRNA through few complementary nucleotides
located at the 5’ of the microRNA and commonly (although not always) at
the 3’ UTR of the messenger RNA. As discussed in the introduction,
several experimental approaches have been developed to investigate
microRNA function, but only a subset provides clear evidence of
microRNA binding to mRNA, while discrimination between direct binding
and causal interaction is crucial for the design of interfering drugs. The partners of the IMEx consortium of interaction databases [18,19] have
agreed to consider luciferase assay as a proof of RNA-RNA interaction,
when validated with mutagenesis analysis (Figure 1). Papers describing
microRNA-mRNA interactions validated with the above-mentioned
methods, were selected from the literature and annotated in the IntAct
database. As the interaction with mRNA results in the down-regulation of
the expression, through the mRNA degradation, or in the inhibition of the
translation, the microRNA is featured as “regulator” and a causality
statement was used to annotate its effect. Most of the repositories which collect microRNA interactions, identify the
targets with the gene name or ID. However, microRNAs bind to
messenger RNAs and, in order to map the binding sites on the target, it is
necessary to refer to a specific transcript. To this aim, it is worth noting
that each gene produces several messengers by alternative splicing of
the precursor transcript. The number of the collected transcripts is
destined to increase as new sequences will be produced from different
cell-types and stages. To avoid ambiguities, we have therefore created
one full-detailed entry for each target gene, cross-referred it with its
Ensembl transcript ID [31], and, whenever possible, we have mapped all
the annotated interactions on this entry. Figure 2 shows this process and
its result. For human transcripts, among the possible transcripts listed in
the Ensembl database [31] we have selected the reference shown in the
GIFT curation tool (https://www.ebi.ac.uk/gifts/). . CC-BY 4.0 International license
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bioRxiv preprint . Building microRNA network in IntAct CC-BY 4.0 International license
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bioRxiv preprint Figure 2. MicroRNA binding sites on mRNA_hmga2 entry. A)
mRNA_hmga2 entry in IntAct is annotated with the sequence and ID of
the main transcript of the gene (identified by GIFT), together with the
Ensembl gene ID and a RefSeq. B) Each mutation analysis is mapped on
the transcript sequence. C) Hmga2-3’UTR regions identified as necessary
for the microRNA binding Figure 2. MicroRNA binding sites on mRNA_hmga2 entry. A)
mRNA_hmga2 entry in IntAct is annotated with the sequence and ID of
the main transcript of the gene (identified by GIFT), together with the
Ensembl gene ID and a RefSeq. B) Each mutation analysis is mapped on
the transcript sequence. C) Hmga2-3’UTR regions identified as necessary
for the microRNA binding We have mainly annotated human interactions, although mouse entries
are also represented. Figure 3A shows a subset of the microRNA-mRNA
human interaction network. Similarly to PPI networks, few nodes of both . CC-BY 4.0 International license
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bioRxiv preprint molecular types behave as hubs, presenting a number of interactions
above average. The network detail blown up in Fig3B shows two
connected hubs: the microRNA hsa-mir-17-5p and the mRNA_cdkn1a. The IntAct database contains thousands of human protein interactions,
each with a reliability score, so it is possible to investigate the protein
complexes affected by the microRNA regulation and the connections
between the protein products of co-regulated genes. Figure 3C shows
some high-reliable interactors of hsa-mir-17-5p targets, that may be
deregulated by mimics or inhibitors of the microRNA. Figure 3. Human microRNA Network in IntAct. Building microRNA network in IntAct A) Section of the
human microRNA Network, showing hubs of both entries type. B)
Enlargement of a detail to show two nodes and their interactors. C) PPI
network for the genes targeted by hsa-mir-17-5p was downloaded and
filtered to a MIscore ⋝0.7 and merged with microRNA-mRNA interactions. Figure 3. Human microRNA Network in IntAct. A) Section of the
human microRNA Network, showing hubs of both entries type. B)
Enlargement of a detail to show two nodes and their interactors. C) PPI
network for the genes targeted by hsa-mir-17-5p was downloaded and
filtered to a MIscore ⋝0.7 and merged with microRNA-mRNA interactions. . CC-BY 4.0 International license
made available under a
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bioRxiv preprint MicroRNA regulation of rare diseases associated genes While the amount of microRNA-interaction information is increasing
continuously, very little is known about microRNAs involved in rare
diseases. Until recently, there has been limited research on the molecular
mechanisms that underlie these pathologies, despite their global impact
on the population and the undeniable need for optimised treatment. Until recently, there has been limited research on the molecular
mechanisms that underlie these pathologies, despite their global impact
on the population and the undeniable need for optimised treatment. We collected in IntAct a list of interactions between microRNAs and genes
associated with the onset or progression of rare disorders. The list of
genes was retrieved from the Genome England PanelApp [32]. Among
the genes listed for each disease, few of them have been reported in the
literature to bind to microRNAs. We considered several rare diseases
(listed in material and methods) and, for the present study, we selected
three of them, since they have a considerable number of genes regulated
by microRNAs: Growth Failure in Early Childhood (GFEC), Mitochondrial
Disorders (MD) and Early Onset Dementia (EOD). Table 1 shows the list of the genes that were found to interact with
microRNAs and annotated in IntAct. Growth Failure in Early Childhood is
influenced by 48 genes, 17 of which were annotated in IntAct for mRNA-
microRNA interactions (Figure 4). Approximately 230 genes are
associated to Mitochondrial Disorders, (plus a few mitochondrial tRNA
genes we did not consider). We found microRNA interactions suitable to
be annotated in IntAct only for 14 of these. Finally, a list of 27 genes are
associated with Early Onset Dementia, 13 of which were annotated in
IntAct. We collected in IntAct a list of interactions between microRNAs and genes
associated with the onset or progression of rare disorders. The list of
genes was retrieved from the Genome England PanelApp [32]. Among
the genes listed for each disease, few of them have been reported in the
literature to bind to microRNAs. We considered several rare diseases
(listed in material and methods) and, for the present study, we selected
three of them, since they have a considerable number of genes regulated
by microRNAs: Growth Failure in Early Childhood (GFEC), Mitochondrial
Disorders (MD) and Early Onset Dementia (EOD). Table 1 shows the list of the genes that were found to interact with
microRNAs and annotated in IntAct. MicroRNA regulation of rare diseases associated genes Growth Failure in Early Childhood is
influenced by 48 genes, 17 of which were annotated in IntAct for mRNA-
microRNA interactions (Figure 4). Approximately 230 genes are
associated to Mitochondrial Disorders, (plus a few mitochondrial tRNA
genes we did not consider). We found microRNA interactions suitable to
be annotated in IntAct only for 14 of these. Finally, a list of 27 genes are
associated with Early Onset Dementia, 13 of which were annotated in
IntAct. . CC-BY 4.0 International license
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bioRxiv preprint Table 1: genes associated with the rare diseases and regulated by
microRNAs. Only interactions confirmed by mutagenesis analysis are
considered. Table 1: genes associated with the rare diseases and regulated by
microRNAs. Only interactions confirmed by mutagenesis analysis are
considered. Early Onset Dementia
Growth Failure in Early Childhood
Mitochondrial Disorders
APP
BLM
AIFM1
ATXN1
BRCA2
COX10
CSF1R
CBL
DMPK
DNMT1
CDKN1C
DNM2
GRN
FANCA
ISCU
HTT
FGFR3
MFN2
ITM2B
HMGA2
MPC1
MAPT
IGF1
NDUFA10
NOTCH3
IGF1R
PDHB
PRNP
IGF2
PDHX
PSEN1
KRAS
SLC25A1
TARDBP
MAP2K1 (MEK1 )
SLC25A12
TBK1
OBSL1
TAZ
PIK3R1
UQCC2
PTPN11
SOS1
UBE2T Early Onset Dementia
Growth Failure in Early Childhood
Mitochondrial Disor We used the BINGO tool to evaluate biological processes statistically
overrepresented in the microRNA regulated genes. We found an
enrichment of genes involved in positive regulation of cellular processes
and cell proliferation, as well as proteins involved in signalling pathways,
in genes associated with GFEC and EOD, in relation to non-annotated
genes. A small number of genes associated with mitochondrial dysfunctions (MD)
are regulated by microRNAs, and the enrichment analysis did not show
significant enrichment for any term. MicroRNA regulation of rare diseases associated genes Interestingly, however, there is a
strong enrichment of specific metabolic and biosynthetic processes, such
as: ATP synthesis coupled electron transport, carboxylic acid metabolic
process, generation of precursor metabolites and tRNA metabolic
processes, in genes associated with MD and not regulated by microRNAs. . CC-BY 4.0 International license
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bioRxiv preprint Figure 4. The Interaction Network of the genes associated to Growth
Failure in Early Childhood. Interactions within proteins associated to
GFEC merged into the microRNA-mRNA network. Proteins regulated by
microRNAs are highlighted in orange. The thickness of the edge between
proteins is proportional to the MI score (i.e. reliability) of the interaction
[37] Figure 4. The Interaction Network of the genes associated to Growth
Failure in Early Childhood. Interactions within proteins associated to
GFEC merged into the microRNA-mRNA network. Proteins regulated by
microRNAs are highlighted in orange. The thickness of the edge between
proteins is proportional to the MI score (i.e. reliability) of the interaction
[37] We compared our results with two other databases: miRTarBase
(https://mirtarbase.cuhk.edu.cn/~miRTarBase/miRTarBase_2022/php/in
dex.php interactions confirmed by “Luciferase assay”) and RAID
(https://www.rna-society.org/raid2/ interactions confirmed by “strong
evidence”). The low percentage of microRNA regulated genes was
confirmed in the two datasets. Supplementary Table 1 compares genes
found in each of the 3 databases to interact with microRNAs. Interestingly
there is a very high correlation among the number of interacting
microRNAs annotated in the 3 databases for each gene, and frequently
those with no interactions in one database are not annotated in the others
either (Figure 5A). This suggests that, although the coverage of annotated
microRNA interactions may still be low, the networks reflect what is
currently known in the literature, with more than half genes of each list not
affected by microRNA regulation, many involved in one or few
interactions, and a small number targeted by a huge number of . MicroRNA regulation of rare diseases associated genes CC-BY 4.0 International license
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bioRxiv preprint microRNAs. Despite the high correlation, the three datasets do not contain
the same interactions: the Venn diagram shows that only 8% of them
(calculated from the union of the three datasets) are present in all
databases, while each dataset contains a percentage of 21%, 15% or 13%
of interactions not annotated in the others (Figure 5B). This suggests that
a combined effort of different resources is necessary to get the full picture
and to help advance research in gene regulation (Supplementary Figure
1) . CC-BY 4.0 International license
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bioRxiv preprint Figure 5. Comparison of microRNA-mRNA interactions annotated in
IntAct, MirTarBase and RAID. MicroRNA regulation of rare diseases associated genes A) Correlation analysis: the number of
microRNAs regulating genes associated with the diseases in the 3
datasets was compared (see also Suppl. Table 1). B) Venn Diagram
comparing interacting pairs of microRNA-mRNA annotated in the 3
datasets. The Jaccard similarity coefficient (in parenthesis) is calculated
as intersection over union and indicates that only 8% of the interactions
are annotated in all the datasets, and that each resource contains some
exclusive information. Discussion ;
https://doi.org/10.1101/2023.06.20.545695
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bioRxiv preprint exhaustive information about microRNA binding could accelerate the
design of specific mimics or inhibitors, and to predict the effect of the
perturbation on the neighbourhood interactors. Since RNA-RNA
interactions occur through base pairing, the design and development of
interfering molecules is easier than protein inhibitors, as demonstrated by
the common use of complementary molecules in scientific papers. exhaustive information about microRNA binding could accelerate the
design of specific mimics or inhibitors, and to predict the effect of the
perturbation on the neighbourhood interactors. Since RNA-RNA
interactions occur through base pairing, the design and development of
interfering molecules is easier than protein inhibitors, as demonstrated by
the common use of complementary molecules in scientific papers. p
y
p p
We have prioritised the annotation of microRNAs regulating genes
involved in rare diseases, hopefully contributing to fill the gap of
knowledge in this field. It is worth mentioning that the IntAct database has
already collected a dataset of protein-protein interactions directly involved
in rare-diseases [35]. Some diseases are directly associated with genetic
mutations or variants: the disease is a direct consequence of the mutation,
or, more frequently, the mutation increases the probability of the onset or
progression. When the mutation directly affects a PPI interaction, the
information can be retrieved from the database [36]. Very few papers
describe the role of microRNA in rare diseases, and hopefully the list of
interactions that affect genes associated to the diseases may help in
elucidating their role. Although “rare” the “rare diseases” affect more than
30 million people in Europe, and are considered one of the major public
health issues. As the return of investment in research on each individual
disease may be limited, no treatment and diagnostic tests have been
developed for many of them. The annotation of genes involved in rare
diseases has been recognised as a crucial step to expedite the diagnosis
and to establish the appropriate treatment [32]. We have expanded this
knowledge by collecting existing data on the regulation of genes by
microRNAs. The selected interactions are evaluated by IntAct curators
and annotated according to approved CV terms. When more evidence
supports an edge, a score of reliability is generated which helps the user
to interpret the network [37]. Discussion Investigations of gene-disease associations typically focus on protein
variants, however, as the relevance of ncRNAs becomes more evident,
their involvement in many common disorders is conceivable. For an
accurate comprehension of microRNA functions, we need to assemble
the entire set of miRNA-mRNA interactions, possibly annotated using
standard procedures that are clearly explained to the user. Some
resources collect microRNA target interactions using natural language
processing to find co-occurrences of terms, followed by manual review. Despite the undeniable relevance and high coverage of these resources,
there is no general acceptance of what to consider a bona-fide interactor,
versus an indirect regulator. We have filtered the experimental data
according to high-quality standards provided by the IMEx consortium, to
collect a set of reliable microRNA targets. The IntAct database contains
approximately 900 interactions involving more than 200 microRNAs. Currently modest coverage, the data set is expected to increase, as new
interactions are continually being added. The target mRNAs were annotated with the Ensembl transcript ID as
identifier. In order to expedite the integration with other data identified by
gene identifiers, the entries are also linked to the Ensembl gene ID. Referring to a specific transcript sequence allows the user to
unequivocally annotate the interacting regions, confirmed by the
mutagenesis analysis. Recent advances in RNA molecules stabilisation and delivery methods
have renewed interest in RNA-based therapies. Chemical modifications
of the nucleotides increase their resistance to nucleases and
encapsulation of the microRNA mimics in neutral lipids allows the delivery
of the molecules into living cells or organisms [33]. In the last years
several microRNA-based therapeutics have been developed, and some
have entered phase II or III of clinical trials [34]. The availability of . CC-BY 4.0 International license
made available under a
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this version posted June 24, 2023. Discussion The collection of “true” interactors will simplify
procedures to assess the value and potential unrelated effects of a
therapeutic product, and will help the design of competitors. Data availability statement The data underlying this article are available in the IntAct database
(https://www.ebi.ac.uk/intact/) Funding This work was supported by European Molecular Biology Laboratory Core Funding,
National Human Genome Research Institute (NHGRI), Office of Director
(OD/DPCPSI/ODSS), National Institute of Allergy and Infectious Diseases (NIAID),
National Institute on Aging (NIA), National Institute of General Medical Sciences
(NIGMS), National Institute of Diabetes and Digestive and Kidney Diseases
(NIDDK), National Eye Institute (NEI), National Cancer Institute (NCI), National
Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health under
Award Number [U24HG007822] (the content is solely the responsibility of the
authors and does not necessarily represent the official views of the National
Institutes of Health), Open Targets; European Union's Horizon 2020 research and
innovation program 825575 (EJP RD). Key Points • Rare diseases are considered one of the main public health problems • Little is known about the involvement of microRNAs in rare diseases • We propose a method to identify microRNAs potentially involved in rare
diseases • We have collected the interactions in the IntAct database and compared
them with other collections . CC-BY 4.0 International license
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bioRxiv preprint Supplementary data Legends Supplementary figure 1. Interactions between microRNAs and disease-associated
genes. GFEC: Growth Failure in Early Childhood MD: Motochondrial Disorders EOD:
Early Onset Dementia. Integration of the interactions from the 3 datasets are shown
on the left side of the picture, Intersection on the right. Supplementary Table 1 Number of microRNAs interacting with the listed genes in
each of the 3 databases. For Raid database “strong evidence” interactions were
considered and for miRTarBase “Luciferase assay”. Data were downloaded in
November 2022. . CC-BY 4.0 International license
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Chronic Kidney Disease and Diabetic Retinopathy in Patients with Type 2 Diabetes
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RESEARCH ARTICLE OPEN ACCESS Citation: Rodríguez-Poncelas A, Mundet-Tudurí X,
Miravet-Jiménez S, Casellas A, Barrot-De la Puente
JF, Franch-Nadal J, et al. (2016) Chronic Kidney
Disease and Diabetic Retinopathy in Patients with
Type 2 Diabetes. PLoS ONE 11(2): e0149448. doi:10.1371/journal.pone.0149448 Chronic Kidney Disease and Diabetic
Retinopathy in Patients with Type 2 Diabetes Antonio Rodríguez-Poncelas1,2,3, Xavier Mundet-Tudurí3,4*, Sonia Miravet-Jiménez3,5,
Aina Casellas2, Joan F. Barrot-De la Puente2,6, Josep Franch-Nadal3,7,8, Gabriel Coll-de
Tuero1,2 1 Primary Health Care Center Anglès, Gerència Territorial Girona, Institut Català de la Salut, Girona, Spain,
2 Unitat de Suport a la Recerca Girona, Institut Universitari d’Investigació en Atenció Primària Jordi Gol
(IDIAP Jordi Gol), Barcelona, Spain, 3 Unitat de Suport a la Recerca Barcelona Ciutat, Institut Universitari
d'Investigació en Atenció Primària Jordi Gol (IDIAP Jordi Gol), Barcelona, Spain, 4 Universitat Autonoma de
Barcelona, Bellaterra, Spain, 5 Primary Health Care Center Martorell, Gerència Territorial Metropolitana
Sud, Institut Català de la Salut, l’Hospitalet de LLobregat, Barcelona, Spain, 6 Primary Health Care Center
Jordi Nadal (Salt), Gerència Territorial Girona, Institut Català de la Salut, Girona, Spain, 7 Primary Health
Care Center Raval Sud, Gerència d’Àmbit d’Atenció Primària Barcelona Ciutat, Institut Català de la Salut,
Barcelona, Spain, 8 CIBER of Diabetes and Associated Metabolic Diseases (CIBERDEM), Instituto de Salud
Carlos III (ISCIII), Madrid, Spain * xavier.mundet@uab.cat Methods This was a population-based, cross-sectional study. A total of 28,344 patients diagnosed
with DM2 who had recorded ophthalmologic and renal functional examinations were evalu-
ated. Data were obtained from a primary healthcare electronic database of medical records. CKD was defined as an estimated glomerular filtration ratio (eGFR) of <60 ml/min/1.73m2
and/or urine albumin to creatinine ratio (UACR) 30 mg/g. DR was categorized as non-
vision threatening diabetic retinopathy and vision threatening diabetic retinopathy. Copyright: © 2016 Rodríguez-Poncelas 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 ethical
restrictions, data are available upon request. The
data contain identifying human information and are
unsuitable for public deposition. Requests may be
made to the corresponding author. * xavier.mundet@uab.cat Purpose To explore the relationship between chronic kidney disease (CKD) and diabetic retinopathy
(DR) in a representative population of type 2 diabetes mellitus (DM2) patients in Catalonia
(Spain). Editor: Florian Kronenberg, Innsbruck Medical
University, AUSTRIA Kidney Disease and Diabetic Retinopathy in T2DM Conclusions Competing Interests: The authors have declared
that no competing interests exist. These results show that CKD, high UACR and/or low eGFR, appear to be associated with
DR in this DM2 population. Introduction Chronic kidney disease (CKD) and diabetes retinopathy (DR) are two major microvascular
complications found in long-standing type 1 (DM1) and type 2 diabetic (DM2) patients. In
addition to sharing risk factors, such as poor glycaemia control and systolic hypertension,
CKD and DR are reflected in the clinical manifestations of similar microvascular lesions in the
glomerular and retinal vessels [1–3]. UACR and eGFR are clinically markers for evaluate renal
function. Urine albumin to creatinine ratio (UACR) and low estimated glomerular filtration rate
(eGFR) are clinical markers of renal function. CKD is often associated with DR in DM1 [4, 5],
and in some studies in DM2 [6–8]. However, this relationship has not been properly investi-
gated in DM2, and there are few data on which of the two markers of CKD, eGFR or UACR, is
more closely related to DR. High UACR is a marker of endothelial dysfunction and may influence the microvasculature
of the kidney and retina. However some studies [9–10] found a relationship between higher
UACR levels and DR in DM1 patients but not in DM2 patients. Sabanayagam et al. [11]
observed that CKD was associated with DR only in the presence of albuminuria, suggesting
that CKD is more likely related to diabetes in the presence of albuminuria. Previous studies
have shown that UACR not only is an important clinical marker for CKD, but also is closely
associated with the progression of DR [12]. Moreover, the relationship between eGFR with DR
remains unclear, particularly in DM2. Man et al. [13] observed that lower levels of eGFR were
associated with the presence and severity of DR. A recent report showed that both an elevated
UACR and decreased eGFR predicted the development of DR in DM2 patients with the former
having a greater impact [14]. The aim of this study was to investigate the association between CKD, UACR and/or eGFR,
and DR in a representative Catalonian (Spain) DM2 population. Results CKD was associated with a higher rate of DR [OR], 95% confidence interval [CI], 1.5 (1.4–
1.7). When we analyzed the association between different levels of UACR and DR preva-
lence observed that DR prevalence rose with the increase of UACR levels, and this associa-
tion was significant from UACR values 10 mg/g, and increased considerably with UACR
values 300mg/g (Odds ratio [OR], 95% confidence interval [CI], 2.0 (1.6–2.5). This associ-
ation was lower in patients with eGFR levels 44 to 30 mL/min/1.73m2 [OR], 95% confidence
interval [CI], 1.3 (1.1–1.6). Funding: This study was supported by a grant from
the IDIAP J. Gol (SIDIAP-2013 from Fundacio J Gol i
Gurina, www.idiapjordigol.com) and the Education
and Research in Family Practice Chair (UAB-
Novartis). The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript. 1 / 10 PLOS ONE | DOI:10.1371/journal.pone.0149448
February 17, 2016 PLOS ONE | DOI:10.1371/journal.pone.0149448
February 17, 2016 Assessment of Retinopathy In a large, community-based screening programmer, such as that employed in our study, fun-
dus photography is considered to be the preferred option [19]. Photographs are captured from
each eye and the severity of DR is categorized according to the international clinical diabetic
retinopathy severity scales recommended by the Global Diabetic Retinopathy Project Group
[20] as: non-vision threatening DR (non-VTDR) and vision threatening DR (VTDR). The for-
mer includes no apparent retinopathy (no DR); mild non-proliferative DR (mild NPDR); and
moderate non-proliferative DR (moderate NPDR). The latter is composed of severe non-prolif-
erative DR (severe NPDR); proliferative DR (PDR); and diabetic macular edema (DME). In
our study, retinal photography was performed by skilled personnel using a non-midriatic cam-
era. Subsequently, in the primary health care center, a family physician trained in reading eye
fundus photographs registered the result in the patient’s medical records. Each eye was given a
DR grade according to the worst result. In the case of patients having more than one fundus
photograph during this period, the most recent was employed for analysis. In the study we
only included retinal photographs from patients undergoing diabetic retinopathy screening
performed at primary health care centers. Measures of Kidney Diabetic Disease Serum creatinine levels and UACR were determined. CKD was defined as UACR >30 mg/g
and/or eGFR <60 mL/min/1.73m2. Normoalbuminuria was defined as UACR <30 mg/g; microalbuminuria as UACR 30–299
mg/g; and macroalbuminuria as UACR 300 mg/g. The Chronic Kidney Disease Epidemiol-
ogy Collaboration equation was employed to measure eGFR [18]. Clinical variables The following data were obtained from each patient: age, age at diagnosis of diabetes, duration
of diabetes, gender, and glycated hemoglobin levels (A1C). Cardiovascular risk factors includ-
ing body mass index (BMI), total cholesterol, low-density (LDL) cholesterol, high density lipo-
protein (HDL) cholesterol, non-HDL cholesterol, systolic and diastolic blood pressure (SBP
and DBP), pulse pressure (PP), and smoking status according to the last condition registered
before the index date were collected. Data for clinical variables were gathered from the 15
months prior to the index date with the exception of blood pressure, PP, and BMI, which were
obtained from the previous 12 months. Additional data were gathered on medication. The study was approved by the Ethics Committee of the Primary Health Care University
Research Institute Jordi Gol (protocol number 13/137). All patient records and information
was anonymized and de-identified prior to analysis. Materials and Methods A population-based, cross-sectional study was performed in Catalonia (Spain) with patients
aged between 30 and 90 years (at 31st December, 2012), diagnosed with DM2 prior to retinal
photography screening, and whose DR category was recorded in their medical records. The
most recent retinal photography registered was selected and employed as the index date. The
criteria for the diagnosis of type DM2 were those established by the American Diabetes Associ-
ation [15] at the time of registration in the electronic database. Data were obtained from the SIDIAP (System for Research and Development in Primary
Care) electronic database. The SIDIAP includes data from the primary healthcare electronic
medical records (e-CAP) on demographic information, appointment dates with doctors and
nurses, clinical diagnoses, clinical variables, prescriptions written, referrals to specialists and
hospitals, results from laboratory tests, and medication sold by pharmacies. The quality of the
SIDIAP data has been previously documented, and the database has been widely used to study
the epidemiology of a number of health outcomes [16, 17]. 2 / 10 Kidney Disease and Diabetic Retinopathy in T2DM Results Prior to retinography 28,344 DM2 patients had their eGFR and UACR values registered. From
the total sample, 14.6% had an eGFR <60 mL/min/1.73m2, women had lower rates than men
(p<0.001). As can be observed in Table 1, diabetes duration, hypertension, cardiovascular dis-
ease (CVD), and treatment with insulin displayed a direct association with decreased eGFR. In contrast, an inverse relationship between eGFR and non-HDL cholesterol, plasma hemo-
globin, heart rate, and DBP was reported. With respect to UACR values (Table 2), 16.0% were
30 mg/g with higher levels being found in men (p<0.001). DM2 duration, poor glycemic
control (glycated hemoglobin; A1C >7%), SBP, increased heart rate, hypertension, CVD, and
treatment with insulin showed a direct relationship with an increase in UACR levels. An
inverse relationship, however, was observed between UACR and plasma hemoglobin levels. In Table 3 the characteristics of the patients with respect to the presence or absence of DR
can be observed. Duration of diabetes, A1C levels, SBP, heart rate, CVD, and insulin treatment
all displayed a direct relationship with DR. An inverse relationship, however, was observed for
DR with non-HDL cholesterol, plasma hemoglobin, and DBP. Table 4 shows that CKD was associated with a higher rate of DR (Odds ratio [OR], 1.5; 95%
confidence interval [CI], 1.4–1.7). Patients with elevated UACR had higher rates of DR, partic-
ularly if UACR values were 300 mg/g (OR, 2.0; 95% CI, 1.6–2.5); this rate was lower in
patients with eGFR levels 44 to 30 mL/min/1.73m2 (OR, 1.3; 95% CI, 1.1–1.6). Patients with Table 1. Patients’ characteristics with respect to glomerular filtration rate (eGFR). Statistical analysis A descriptive analysis stratified by renal function (according to eGFR and UACR) and retino-
graphy result was performed. The absolute and relative frequencies of the qualitative variables,
and their mean and standard deviation (or median and interquartile range according to the
characteristics of the variable) were calculated. In order to study whether the clinical variables of interest depended on renal function, Pear-
son’s chi square test and analysis of variance (ANOVA) were employed for means and propor-
tions, respectively. The hypothesis contrast was bilateral in all cases with a 0.05 level of
significance. 3 / 10 PLOS ONE | DOI:10.1371/journal.pone.0149448
February 17, 2016 Kidney Disease and Diabetic Retinopathy in T2DM In addition, the relationship between renal function values (according to eGFR and UACR)
and the presence of retinopathy was assessed by logistic regression models adjusted by age, sex,
age at retinography, duration of DM2, systolic blood pressure, A1C 7%, smoking, BMI, and
cardiovascular disease. Analyses were performed with Stata/SE version 13 for Windows (Stata Corp., College Sta-
tion, Texas, USA). y
y
A1C, glycated hemoglobin; bpm, beats per minute; DBP, Diastolic Blood Pressure; DR: diabetic retinopathy. aP value for comparison of groups by glomerular filtration rate with Pearson’s chi-square test for qualitative variables and t-test for quantitative ones.
bAny cardiovascular disease includes coronary heart disease and/or stroke doi:10.1371/journal.pone.0149448.t001 y
y
A1C, glycated hemoglobin; bpm, beats per minute; DBP, Diastolic Blood Pressure; DR: diabetic retinopathy. value for comparison of groups by glomerular filtration rate with Pearson s chi square test for qualitative variables
y cardiovascular disease includes coronary heart disease and/or stroke g
p
y g
q
q
q
se includes coronary heart disease and/or stroke s by glomerular filtration rate with Pearson’s chi-square test for qualitative variables and t-test for quantitative ones y
obin; bpm, beats per minute; DBP, Diastolic Blood Pressure; DR: diabetic retinopathy. onary heart disease and/or stroke P value for comparison of groups by glomerular filtration rate with Pearson s chi square test for qualitative variables and t test for quantitative ones.
bAny cardiovascular disease includes coronary heart disease and/or stroke y
er minute; DBP, Diastolic Blood Pressure; DR: diabetic retinopathy. Results Variable
Global
eGFR 60
eGFR 59–45
eGFR 44–30
eGFR <30
p-valuea
Female, n (%)
12,520 (44.2)
10,363 (42.8)
1,478(51.2)
568 (53.9)
111 (52.6)
<0.001
Hypertension, n (%)
22,971 (81.0)
19,001 (78.5)
2,737 (94.8)
1,026 (97.4)
207 (98.1)
<0.001
Any cardiovascular diseaseb, n (%)
3,969 (14.0)
3,026 (12.5)
590 (20.4)
286 (27.2)
67 (31.8)
<0.001
Coronary Heart disease, n (%)
3,048 (10.8)
2,326 (9.6)
446 (15.5)
221 (21.0)
55 (26.1)
<0.001
Stroke, n (%)
1,148 (4.1)
854 (3.5)
186 (6.4)
90 (8.5)
18 (8.5)
<0.001
Insulin treatment, n (%)
4,737 (16.7)
3,781 (15,6)
567 (19.6)
302 (28.7)
87 (41.2)
<0.001
Age T2DM (years), mean (SD)
58.8(10.5)
57.5(10.2)
65.6 (9.0)
67.2 (9.7)
65.0(10.1)
<0.001
Age at retinography (years), mean (SD)
65.7(10.7)
64.3(10.4)
73.7(7.9)
76.1 (7.8)
74.6(8.9)
<0.001
Diabetes duration (years), mean (SD)
7.0 (5.2)
6.8 (5.0)
8.2 (5.6)
9.0 (6.0)
9.7(6.1)
<0.001
A1C (%), mean (SD)
7.4 (1.4)
7.4(1.4)
7.2 (1.2)
7.3 (1.4)
7.2 (1.3)
<0.001
Non-HDL cholesterol (mg/dL), mean (SD)
137.3(35.2)
137.8(35.2)
135.4(34.6)
132.5(35.2)
132.5(38.2)
<0.001
Hemoglobin (g/dL), mean (SD)
14.0 (1.5)
14.1 (1.4)
13.3 (1.6)
12.7 (1.6)
12.0 (1.5)
<0.001
SBP (mmHg), mean (SD)
134.9(12.6)
134.8(12.5)
136.1(13.1)
135.8(13.5)
133.9(14.0)
<0.001
DBP (mmHg), mean (SD)
76.4 (8.3)
76.9 (8.2)
74.2 (8.2)
71.9 (8.3)
70.4 (8.3)
<0.001
Heart Rate (bpm), mean (SD)
76.1 (12.1)
76.3 (12.0)
75 (12.5)
74.2 (12.5)
72.5 (12.0)
<0.001
aP value for comparison of groups by glomerular filtration rate with Pearson’s chi-square test for qualitative variables and t-test for quantitative ones Patients’ characteristics with respect to glomerular filtration rate (eGFR). PLOS ONE | DOI:10.1371/journal.pone.0149448
February 17, 2016 4 / 10 y
y
A1C, glycated hemoglobin; DBP, diastolic blood pressure; DR: diabetic retinopathy; SBP, systolic blood pressure; VTDR, Vision Threatening Diabetic a P value for comparison of groups by glomerular filtration rate with Pearson’s chi-square test for qualitative variables and t-test for the quantitative ones.
b Any cardiovascular disease includes coronary heart disease and/or stroke doi:10.1371/journal.pone.0149448.t003 y ca d o ascu a d sease
c udes co o a y
ea t d sease a d/o st o e
A1C, glycated hemoglobin; DBP, diastolic blood pressure; DR: diabetic retinopathy; SBP, systolic blood pressure; VTDR, Vision Threatening Diabetic
Retinopathy. a P value for comparison of groups by glomerular filtration rate with Pearson’s chi-square test for qualitative variables and t-test for the quantitative ones.
b y
stolic blood pressure; DR: diabetic retinopathy; SBP, systolic blood pressure; VTDR, Vision Threatening Diabetic aP value for comparison of groups by urine albumin to creatinine ratio (ACR) with Pearson’s chi-square test for qualitative variables and t-test for the
quantitative ones. Kidney Disease and Diabetic Retinopathy in T2DM Table 2. Patients’ characteristics with respect to urine albumin to creatinine ratio (UACR). Table 2. Patients’ characteristics with respect to urine albumin to creatinine ratio (UACR). Variable
Global
UACR <30 mg/g
UACR = 30–299 mg/g
UACR 300 mg/g
p-valuea
Female, n (%)
12,520 (44.2)
10,886 (45.7)
1,443 (36.4)
191 (33.7)
<0.001
Hypertension, n (%)
22,971 (81.0)
18,857 (79.2)
3,573 (90.2)
541 (95.4)
<0.001
Any cardiovascular diseaseb, n (%)
3,969 (14.0)
2,996 (12.6)
830 (21.0)
143 (25.2)
<0.001
Coronary Heart disease, n (%)
3,048 (10.8)
2,321 (9.7)
616 (15.6)
111 (19.6)
<0.001
Stroke, n (%)
1,148 (4.1)
842 (3.5)
268 (6.8)
38 (6.7)
<0.001
Insulin treatment, n (%)
4,737 (20.0)
3,512 (17,9)
1,028 (29.0)
197 (39.4)
<0.001
Age T2DM (years), mean (SD)
58.8(10.5)
58.6(10.4)
59.8(10.9)
59.1(10.5)
<0.001
Age at retinography (years), mean (SD)
65.7(10.7)
65.4(10.6)
67.6(11.0)
67.6(11.0)
<0.001
Diabetes duration (years), mean (SD)
7.0 (5.2)
6.8 (5.1)
7.8 (5.4)
8.6 (5.8)
<0.001
A1C (%), mean (SD)
7.4 (1.4)
7.3(1.3)
7.7 (1.6)
7.8 (1.7)
<0.001
Non-HDL cholesterol (mg/dL), mean (SD)
137.3(35.2)
137.4 (34.7)
136.5(37.6)
136.6(37.3)
0.320
Hemoglobin (g/dL), mean (SD)
14.0 (1.5)
14.0 (1.5)
13.8 (1.7)
13.5 (1.9)
<0.001
SBP (mmHg), mean (SD)
134.9(12.6)
134.2(12.2)
138.3(13.7)
141.8(15.8)
<0.001
DBP (mmHg), mean (SD)
76.4 (8.3)
76.4 (8.1)
76.4 (9.0)
76.7 (10.0)
0.736
Heart Rate (bpm), mean (SD)
76.1 (12.1)
76.0(12.0)
76.6 (12.8)
77.5 (13.2)
0.002
a aP value for comparison of groups by urine albumin to creatinine ratio (ACR) with Pearson’s chi-square test for qualitative variables and t-test for the
quantitative ones UACR values 30 to 299 mg/gr had DR rate slightly higher than those with eGFR values of
30 to 44 mL/min/1.73m2 (OR, 1.5; 95% CI, 1.4–1.7, and OR, 1.3; 95% CI 1.1–1.6, respectively). When we analyzed the association between different levels of UACR and DR prevalence
Any cardiovascular disease includes coronary heart disease and/or stroke
A1C, glycated hemoglobin; DBP, diastolic blood pressure; SBP, systolic blood pressure. doi:10.1371/journal.pone.0149448.t002 doi:10.1371/journal.pone.0149448.t002 UACR values 30 to 299 mg/gr had DR rate slightly higher than those with eGFR values of
30 to 44 mL/min/1.73m2 (OR, 1.5; 95% CI, 1.4–1.7, and OR, 1.3; 95% CI 1.1–1.6, respectively). a P value for comparison of groups by glomerular filtration rate with Pearson’s chi-square test for qualitative variab
b Any cardiovascular disease includes coronary heart disease and/or stroke moglobin; DBP, diastolic blood pressure; DR: diabetic retinopathy; SBP, systolic blood pressure; VTDR, Vision Thre y
y
C, glycated hemoglobin; DBP, diastolic blood pressure; DR: diabetic retinopathy; SBP, systolic blood pressure; VT
inopathy. PLOS ONE | DOI:10.1371/journal.pone.0149448
February 17, 2016 q
bAny cardiovascular disease includes coronary heart disease and/or stroke When we analyzed the association between different levels of UACR and DR prevalence UACR values 30 to 299 mg/gr had DR rate slightly higher than those with eGFR values of
30 to 44 mL/min/1.73m2 (OR, 1.5; 95% CI, 1.4–1.7, and OR, 1.3; 95% CI 1.1–1.6, respectively). When we analyzed the association between different levels of UACR and DR prevalence Table 3. Patients’ characteristics with respect to diabetic retinopathy. Variable
Global
Non DR
Non-VTDR
VTDR
p-valuea
Female, n (%)
12,520 (44.2)
11,089 (44.3)
1,316 (43.8)
115 (40.2)
0.345
Hypertension, n (%)
22,971 (81.0)
20,141 (80.4)
2,589 (86.1)
241 (84.3)
<0.001
Any cardiovascular disease, b n (%)
3,969 (14.0)
3,347 (13.4)
553 (18.4)
69 (24.1)
<0.001
Coronary heart disease, n (%)
3,048 (10.8)
2,602 (10.4)
396 (13.2)
50 (17.5)
<0.001
Stroke, n (%)
1,148 (4.1)
935 (3.7)
187 (6.2)
26 (9.1)
<0.001
Insulin treatment, n (%)
4,737 (20.0)
3,529 (17.1)
1,067 (38.8)
141 (52.8)
<0.001
Age DM2 (years), mean (SD)
58.8(10.5)
58.8 (10.4)
58.1 (11.2)
58.4 (11.6)
<0.001
Age at retinography (years), mean (SD)
65.7(10.7)
65.6 (10.6)
67.1 (10.8)
67.9 (10.9)
<0.001
Diabetes duration (years), mean (SD)
7.0 (5.2)
6.7 (5.0)
9.0 (5.8)
9.5 (5.8)
<0.001
A1C (%), mean (SD)
7.4 (1.4)
7.3 (1.3)
7.9 (1.6)
8.2 (1.7)
<0.001
Non-HDL cholesterol (mg/dL), mean (SD)
137.3(35.2)
137.8 (35.0)
133.0 (35.5)
133.9 (40.1)
<0.001
Hemoglobin (g/dL), mean (SD)
14.0 (1.5)
14.0 (1.5)
13.7 (1.6)
13.7 (1.8)
<0.001
SBP (mmHg), mean (SD)
134.9(12.6)
134.6 (12.5)
137.4 (13.6)
138.2 (12.7)
<0.001
DBP (mmHg), mean (SD)
76.4 (8.3)
76.5 (8.3)
75.7 (8.5)
75.0 (8.3)
<0.001
Heart Rate (bpm), mean (SD)
76.1 (12.1)
76.0 (12.1)
76.9 (12.1)
78.2 (12.3)
<0.001 Table 3. Patients’ characteristics with respect to diabetic retinopathy. PLOS ONE | DOI:10.1371/journal.pone.0149448
February 17, 2016 5 / 10 Kidney Disease and Diabetic Retinopathy in T2DM Table 4. Types of chronic kidney disease and diabetic retinopathy. OR
(95% CI)
p-value
UACR (mg/g)a
<30
1.0
30 to 299
1.5
(1.4, 1.7)
<0.001
300
2.0
(1.6, 2.5)
<0.001
eGFR (mL/min / 1.73m2)a
90
1.0
[60, 89]
1.0
(0.9, 1.1)
0.674
[45, 59]
1.2
(1.0, 1.3)
0.142
[30, 44]
1.3
(1.1, 1.6)
0.009
<30
1.3
(0.9, 1.9)
0.193
aAdjusted by age, sex, age at retinography, duration type 2 diabetes, systolic blood pressure <140 mmHg,
A1C 7%, smoking, cardiovascular disease, BMI, UACR and/or eGFR. Model Diabetic Retinopathy (DR): without DR vs. doi:10.1371/journal.pone.0149448.g001 (Fig 1) we observed that DR prevalence rose with the increase of UACR levels, and this associa-
tion was significant from UACR values 10 to 29 mg/g, (OR, 1.2; CI, 1.1–1.4), and increased
considerably with UACR values 300 mg/g (OR, 2.0; 95% CI, 1.5–2.5). Discussion Our study showed that high UACR level significantly increases the prevalence of diabetic reti-
nopathy in type 2 diabetic patients, even after adjustment for variables, and this association
was significant from UACR values 10 mg/g. These findings concur with the RIACE study
cohort [21]. We observed that individuals with elevated UACR had a higher DR prevalence
than those with decreased eGFR. A number of factors could have contributed to the renal and
retinal tissue injury observed; including A1C levels, hypertension, dyslipidemia, diabetes dura-
tion, age at onset of DM2, smoking, and UACR levels [6, 10, 22, 23]. Patients with CKD are
more likely to present with DR VTDR [24]. High UACR doubles the possibility of developing
DR with respect to normal UACR levels, a risk that increases considerably with macroalbumi-
nuria, even after adjusting for other factors [7, 14, 25]. In addition, an association was observed
between low eGFR and greater prevalence of DR. Lu et al. [26] showed that a decrease in eGFR
was significantly correlated with DR, after controlling for sex, age, and albuminuria staging. Wu J et al. [27] observed that, after the adjustment of the variables affecting the relationship
between eGFR and DR, results of a univariate analysis suggested that eGFR remained signifi-
cantly associated with DR. In our study we observed that eGFR levels <45 ml/min/1.73m2
were associated with higher DR prevalence, but we did not find this association with eGFR lev-
els 45/ml/min/1.73 m2. This association was not significant with eGFR levels <30 mL/min/
1.73m2, probably because there were few patients in this group. Penno et al. [28] reported an association between UACR 300 mg/g and DR (OR 2.9; 95%
CI, 2.1–4.0) higher than that observed in our work. Such a variation could be due to the differ-
ent grouping of DR types. Nevertheless, in both studies elevated UACR had a greater associa-
tion with DR than decreased eGFR. CKD in DM2 is more heterogeneous than in type 1 diabetes, retinopathy and albuminuria
were both absent in 30% of adults with DM2 and chronic renal insufficiency according to the
Third National Health and Nutrition Survey [29]. Patients with diabetes are also susceptible to
non diabetic renal disease [30]. A decrease in eGFR with normoalbuminuria in such patients
sometimes may be linked to the presence of arteriosclerosis. Recently, Lee et al. [31] showed a direct association between DR and CKD. Kidney Disease and Diabetic Retinopathy in T2DM Fig 2. Association of chronic kidney disease and diabetic retinopathy. *Adjusted by sex, age at digital photography, duration of type 2 diabetes, systolic
blood pressure <140 mmHg, and A1C 7%; DR, diabetic retinopathy; eGFR, estimated glomerular filtration rate; UACR, urine albumin to creatinine ratio;
VTDR, vision threatening diabetic retinopathy (severe non proliferative DR, proliferative DR, and diabetic macular edema); non-VTDR, mild non-proliferative
DR and moderate non-proliferative DR. Fig 2. Association of chronic kidney disease and diabetic retinopathy. *Adjusted by sex, age at digital photography, duration of type 2 diabetes, systolic
blood pressure <140 mmHg, and A1C 7%; DR, diabetic retinopathy; eGFR, estimated glomerular filtration rate; UACR, urine albumin to creatinine ratio;
VTDR, vision threatening diabetic retinopathy (severe non proliferative DR, proliferative DR, and diabetic macular edema); non-VTDR, mild non-proliferative
DR and moderate non-proliferative DR. Fig 2. Association of chronic kidney disease and diabetic retinopathy. *Adjusted by sex, age at digital photography, duration of type 2 diabetes, systolic
blood pressure <140 mmHg, and A1C 7%; DR, diabetic retinopathy; eGFR, estimated glomerular filtration rate; UACR, urine albumin to creatinine ratio;
VTDR, vision threatening diabetic retinopathy (severe non proliferative DR, proliferative DR, and diabetic macular edema); non-VTDR, mild non-proliferative
DR and moderate non-proliferative DR. Fig 2. Association of chronic kidney disease and diabetic retinopathy. *Adjusted by sex, age at digital photography, duration of type 2 diabetes, systolic
blood pressure <140 mmHg, and A1C 7%; DR, diabetic retinopathy; eGFR, estimated glomerular filtration rate; UACR, urine albumin to creatinine ratio;
VTDR, vision threatening diabetic retinopathy (severe non proliferative DR, proliferative DR, and diabetic macular edema); non-VTDR, mild non-proliferative
DR and moderate non-proliferative DR. doi:10.1371/journal.pone.0149448.g002 any type DR (n = 26,897 patients)
DR, diabetic retinopathy; eGFR, estimated glomerular filtration rate; OR, odds ratio; UACR, urine albumin
to creatinine ratio. doi:10 1371/journal pone 0149448 t004 Table 4. Types of chronic kidney disease and diabetic retinopathy. p-value doi:10.1371/journal.pone.0149448.t004 (Fig 1) we observed that DR prevalence rose with the increase of UACR levels, and this associa-
tion was significant from UACR values 10 to 29 mg/g, (OR, 1.2; CI, 1.1–1.4), and increased
considerably with UACR values 300 mg/g (OR, 2.0; 95% CI, 1.5–2.5). The prevalence of DR using the KDIGO combination of UACR and eGFR categories is
shown in S1 Table. Moreover, Fig 2 shows the association between CKD and DR severity. Patients with eGFR <60 mL/min/1.73m2 and UACR <30 mg/g had an increased non-VTDR
rate (OR 1.3; 95% CI 1.1–1.5) and a non-significant increase in VTDR (OR 1.4; 95% CI 1.0–
2.1; p = 0.082). In individuals with UACR 30 mg/g and eGFR 60 mL/min/1.73m2 the rate
of VTDR (OR 2.6; 95% CI 1.9–3.5) was higher than non-VTDR (OR 1.6; 95% CI 1.4–1.7). Fig 1. Urine albumin to creatinine ratio and prevalence of diabetic retinopathy. *Adjusted by age, sex, age at retinography, duration type 2 diabetes,
systolic blood pressure <140 mmHg, A1C 7%, smoking, cardiovascular disease, BMI, and eGFR. DR, diabetic retinopathy; OR, odds ratio; UACR, urine
albumin to creatinine ratio. Fig 1. Urine albumin to creatinine ratio and prevalence of diabetic retinopathy. *Adjusted by age, sex, age at retinography, duration type 2 diabetes,
systolic blood pressure <140 mmHg, A1C 7%, smoking, cardiovascular disease, BMI, and eGFR. DR, diabetic retinopathy; OR, odds ratio; UACR, urine Fig 1. Urine albumin to creatinine ratio and prevalence of diabetic retinopathy. *Adjusted by age, sex, age at retinography, duration type 2 diabetes,
systolic blood pressure <140 mmHg, A1C 7%, smoking, cardiovascular disease, BMI, and eGFR. DR, diabetic retinopathy; OR, odds ratio; UACR, urine
albumin to creatinine ratio. PLOS ONE | DOI:10.1371/journal.pone.0149448
February 17, 2016 6 / 10 PLOS ONE | DOI:10.1371/journal.pone.0149448
February 17, 2016 Acknowledgments This study was supported by a grant from the IDIAP J. Gol and the Education and Research in
Family Practice Chair (UAB-Novartis). We acknowledge Mònica Gratacòs and Amanda
Prowse for providing support in the manuscript preparation and editing. CIBER of Diabetes
and Associated Metabolic Diseases (CIBERDEM) is an initiative from Instituto de Salud Carlos
III. Supporting Information S1 Table. Prevalence of diabetic retinopathy using KDIGO combinations of UACR and
eGFR categories. (DOCX) S1 Table. Prevalence of diabetic retinopathy using KDIGO combinations of UACR and
eGFR categories. Author Contributions Conceived and designed the experiments: AR-P XM-T GC-T. Performed the experiments: AR-
P XM-T SM-J JFB-D JF-N. Analyzed the data: AC. Contributed reagents/materials/analysis
tools: AR-P XM-T SM-J JFB-D JF-N. Wrote the paper: AR-P XM-T GC-T. Conceived and designed the experiments: AR-P XM-T GC-T. Performed the experiments: AR-
P XM-T SM-J JFB-D JF-N. Analyzed the data: AC. Contributed reagents/materials/analysis
tools: AR-P XM-T SM-J JFB-D JF-N. Wrote the paper: AR-P XM-T GC-T. Conclusions CKD appears to be associated with DR in this DM2 population. Elevated UACR had a greater
association with DR prevalence than decreased eGFR, but both were associated with DR in
DM2 patients. Discussion The presence of
CKD identified a group of DM2 patients of being at a greater risk of presenting macro and
microvascular complications. Moreover, the presence of CKD and DR was associated with a
more rapid reduction in renal function and greater mortality in this group of patients who
might benefit from more aggressive treatment. 7 / 10 Kidney Disease and Diabetic Retinopathy in T2DM Our study should be interpreted with consideration of the following limitations. First, this
study was a cross-sectional analysis; therefore, the data were recorded from an electronic data-
base and diabetic patients with DR-VTDR, who were attended by an ophthalmologist or endo-
crinologist, could have been poorly represented. Second, because of the small number of cases
in some categories, we could not carry out the final analysis using all KDIGO categories. Despite these limitations, this study used a nationally representative sample of DM2 patients. Moreover, to the best of our knowledge, this is the first large population based study to examine
the association between CKD, decreased eGFR and high UACR, and DR among a representa-
tive Catalonian, Spain, DM2 population. PLOS ONE | DOI:10.1371/journal.pone.0149448
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PMID: 24624891 22. Adler AI, Stratton IM, Neil HA, Yudkin JS, Matthews DR, Cull CA, et al. Association of systolic blood
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spective observational study. BMJ. 2000; 321(7258): 412–419. PMID: 10938049 23. References 1. Mathiesen ER, Ronn B, Storm B, Foght H, Deckert T. The natural course of microalbuminuria in insulin-
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development and progression of diabetic nephropathy in the Diabetes Control and Complications Trial. Kidney Int. 1995; 47(6): 1703–1720. PMID: 7643540 3. UK Prospective Diabetes Study (UKPDS) Group. Effect of intensive blood-glucose control with metfor-
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retinopathy in juvenile-onset type I diabetes: a 40-yr follow-up study. Diabetes Care. 1986; 9(5): 443–
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minuria, arterial hypertension, retinopathy and neuropathy in patients with insulin dependent diabetes. Br Med J (Clin Res Ed). 1988; 296(6616): 156–160. 6. Stratton IM, Kohner EM, Aldington SJ, Turner RC, Holman RR, Manley SE, et al. UKPDS 50: risk fac-
tors for incidence and progression of retinopathy in Type II diabetes over 6 years from diagnosis. Diabe-
tologia. 2001; 44(2): 156–163. PMID: 11270671 8 / 10 PLOS ONE | DOI:10.1371/journal.pone.0149448
February 17, 2016 Kidney Disease and Diabetic Retinopathy in T2DM 7. Manaviat MR, Afkhami M, Shoja MR. Retinopathy and microalbuminuria in type II diabetic patients. BMC Ophthalmol. 2004; 4: 9. PMID: 15228626 8. Estacio RO, McFarling E, Biggerstaff S, Jeffers BW, Johnson D, Schrier RW. Overt albuminuria pre-
dicts diabetic retinopathy in Hispanics with NIDDM. Am J Kidney Dis. 1998; 31(6): 947–953. PMID:
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patients with types 1 and 2 diabetes mellitus. Clin Nephrol. 2007; 68: 81–6. PMID: 17722706 10. Pedro RA, Ramon SA, Marc BB, Juan FB, Isabel MM. Prevalence and relationship between diabetic
retinopathy and nephropathy, and its risk factors in the North-East of Spain, a population-based study. Ophthalmic Epidemiol. 2010; 17(4): 251–265. doi: 10.3109/09286586.2010.498661 PMID: 20642348 11. Sabanayagam C, Foo VH, Ikram MK, Huang H, Lim SC, Lamoureux EL, et al. Is chronic kidney disease
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Penno G, Solini A, Zoppini G, Orsi E, Zerbini G, Trevisan R, et al. Rate and determinants of association
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Lee WJ, Sobrin L, Kang MH, Seong M, Kim YJ, Yi JH, et al. Ischemic diabetic retinopathy as a possible
prognostic factor for chronic kidney disease progression. Eye (Lond). 2014; 28(9): 1119–1125. References Wong TY, Cheung N, Tay WT, Wang JJ, Aung T, Saw SM, et al. Prevalence and risk factors for diabetic
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j.ophtha.2008.05.014 PMID: 18584872 24. Harris Nwanyanwu K, Talwar N, Gardner TW, Wrobel JS, Herman WH, Stein JD. Predicting develop-
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buminuric people with type 2 diabetes mellitus. Diabetol Metab Syndr. 2012; 4(1): 29. doi: 10.1186/
1758-5996-4-29 PMID: 22747972 26. Lu B, Song X, Dong X, Yang Y, Zhang Z, Wen J, et al. High prevalence of chronic kidney disease in
population-based patients diagnosed with type 2 diabetes in downtown Shanghai. J Diabetes Compli-
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Rate and Diabetic Retinopathy. J Ophthalmol. 2015; 2015: 326209. doi: 10.1155/2015/326209 PMID:
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February 17, 2016 Kidney Disease and Diabetic Retinopathy in T2DM 28. Penno G, Solini A, Zoppini G, Orsi E, Zerbini G, Trevisan R, et al. Rate and determinants of association
between advanced retinopathy and chronic kidney disease in patients with type 2 diabetes: the Renal
Insufficiency And Cardiovascular Events (RIACE) Italian multicenter study. Diabetes Care. 2012; 35
(11): 2317–2323. doi: 10.2337/dc12-0628 PMID: 23093684 29. Kramer HJ, Nguyen QD, Curhan G, Hsu CY. Renal insufficiency in the absence of albuminuria and reti-
nopathy among adults with type 2 diabetes mellitus. JAMA. 2003; 289(24): 3273–3277. PMID:
12824208 30. Tone A, Shikata K, Matsuda M, Usui H, Okada S, Ogawa D, et al. Clinical features of non-diabetic renal
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16098920 31. Lee WJ, Sobrin L, Kang MH, Seong M, Kim YJ, Yi JH, et al. Ischemic diabetic retinopathy as a possible
prognostic factor for chronic kidney disease progression. Eye (Lond). 2014; 28(9): 1119–1125. 10 / 10
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TWAKASARATA: ACKNOWLEDGE THE POWER OF FIRST IMPRESSION.
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International journal of advanced research
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TWAKASARATA: ACKNOWLEDGE THE POWER OF FIRST IMPRESSION. Sunil P. Nikhate1, Renuka B. Ikkar2, Narendra D.Yadnik 3, Vijay V. Bhagat4 and Vaidehi V. Raole5. 1
PhD (Scholar) Associate Professor Dept of Kriya Sharir Parul Institute of Ayurved Parul University; Sunil P. Nikhate1, Renuka B. Ikkar2, Narendra D.Yadnik 3, Vijay V. Bhagat4 and Vaidehi V. Raole5. Sunil P. Nikhate1, Renuka B. Ikkar2, Narendra D.Yadnik 3, Vijay V. Bhagat4 and Vaidehi V. Raole5. (
j
)
te Professor, Dept of Kriya Sharir, SSAC &H, Panchavati ,Nashik (Maharashtra). j
2. Associate Professor, Dept of Kriya Sharir, SSAC &H, Panchavati ,Nashik (Maharashtra). 3. PhD (Scholar), Assistant Professor, Dept of Kriya Sharir, Parul Institute of Ayurved, Parul University;
Limda, Vadodara. (Gujarat) 3. PhD (Scholar), Assistant Professor, Dept of Kriya Sharir, Parul Institute of Ayurved, Parul University;
Limda, Vadodara. (Gujarat) j
4. Associate professor, Dept of Samhita & Siddhanta, Parul Institute of Ayurved, Parul University; Limda,
Vadodara. (Gujarat). 4. Associate professor, Dept of Samhita & Siddhanta, Parul Institute of Ayurved, Parul University; Limda,
Vadodara. (Gujarat). 5. PhD Guide; Professor, PG Dept of Kriya Sharir, Parul Institute of Ayurved, Parul University; Limda,
Vadodara. (Gujarat). …………………………………………………………………………………………………….... Manuscript Info
Abstract
……………………. ………………………………………………………………
Manuscript History
Received: 03 May 2019
Final Accepted: 05 June 2019
Published: July 2019
Key words:-
Dhatusara,
Types,
Twakasara-
Characteristics, Importance. Sara is unique concept illuminated in Ayurveda. It is core or excellence
part of Dhatu (tissues). Sara Parikshana (Examination) is very
important to rule out Bala (strength) of Dhatu. Skin is one of the largest organs of the body. Because of this, caring for
skin can directly affect our overall health. Skin acts as a protective
shield and is most vulnerable to outside elements. It’s affected by more
factors than we may think. So it is necessary to take care of the skin. Dalhanacharya is described Rasasara as a Twakasara because of
functions of the Rasa Dhatu is best carried out through Skin. Physical
Characteristics of Twakasara is totally concerned with skin on body
hair. Physical attractiveness creates a powerful first impression on the mind,
so in fact that we may go much beyond looks and simply start
generating assumptions about a person's success, status, parenting, and
intelligence, even if they prove not to be true. To understand strength or weakness of our look, we need to
acknowledge the power of first impressions i.e Twakasarata. It
influences the way we see and treat others. TWAKASARATA: ACKNOWLEDGE THE POWER OF FIRST IMPRESSION. Once an impression is
entrenched, it takes a lot to change our attitude about a person. By
virtue of Knowledge of skin and Twakasarata we would be able to
maintain healthy status of the skin. Copy Right, IJAR, 2019,. All rights reserved. …………………………………………………………………………………………………….... Introduction:-
Dosha-Dhatu-Mala is the basic concept of Ayurveda as they are prime constituents of the human body1. They are
considered as base of the body like root for the plant. Each and every person is having same elementary entities as
mentioned above. Beside that we observed, there are structural and functional variation in components of Sharira. Sara is unique concept illuminated in Ayurveda. It is core or excellence
part of Dhatu (tissues). Sara Parikshana (Examination) is very
important to rule out Bala (strength) of Dhatu. Skin is one of the largest organs of the body. Because of this, caring for
skin can directly affect our overall health. Skin acts as a protective
shield and is most vulnerable to outside elements. It’s affected by more
factors than we may think. So it is necessary to take care of the skin. Dalhanacharya is described Rasasara as a Twakasara because of
functions of the Rasa Dhatu is best carried out through Skin. Physical
Characteristics of Twakasara is totally concerned with skin on body
hair. Physical attractiveness creates a powerful first impression on the mind,
so in fact that we may go much beyond looks and simply start
generating assumptions about a person's success, status, parenting, and
intelligence, even if they prove not to be true. To understand strength or weakness of our look, we need to
acknowledge the power of first impressions i.e Twakasarata. It
influences the way we see and treat others. Once an impression is
entrenched, it takes a lot to change our attitude about a person. By
virtue of Knowledge of skin and Twakasarata we would be able to
maintain healthy status of the skin. Copy Right, IJAR, 2019,. All rights reserved. Copy Right, IJAR, 2019,. All rights reserved. Dosha-Dhatu-Mala is the basic concept of Ayurveda as they are prime constituents of the human body1. They are
considered as base of the body like root for the plant. Each and every person is having same elementary entities as
mentioned above. Beside that we observed, there are structural and functional variation in components of Sharira. 124
Corresponding Author:-Sunil p. Nikhate. ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 7(7), 124-129 Journal Homepage: -www.journalijar.com
Article DOI:10.21474/IJAR01/ 9334
DOI URL: http://dx.doi.org/10.21474/IJAR01/9334 Types: yp
Individuals have been classified in various categories, depending upon the predominance of particular Dhatu Sara in
the body by virtue of its quantity, quality and functions. Acharya Charak5, Sushruta6 and Vagbhata7 have mentioned
eight type of Sara. These are Twak, Rakta, Mansa, Meda, Asthi, Majja, Shukra, and Satvasara. Kashyapa has added another type of Sara i.e. Oja Sara in this way, he mentioned nine types of Sara8. Dhatusara Purusha is classified into three viz. Pravara, Madhyama and Hina Sarata on the basis of Bala (strength)9. Dhatusara Purusha is classified into three viz. Pravara, Madhyama and Hina Sarata on the basis of B TWAKASARATA: ACKNOWLEDGE THE POWER OF FIRST IMPRESSION. Address:- PhD (Scholar), Associate Professor , Dept of Kriya Sharir, Parul Institute of Ayurved, Parul
University; Limda, Vadodara.(Gujarat). . 124
Corresponding Author:-Sunil p. Nikhate. Address:- PhD (Scholar), Associate Professor , Dept of Kriya Sharir, Parul Institute of Ayurved, Parul
University; Limda, Vadodara.(Gujarat). . 124
Corresponding Author:-Sunil p. Nikhate. Address:- PhD (Scholar), Associate Professor , Dept of Kriya Sharir, Parul Institute of Ayurved, Parul
University; Limda, Vadodara.(Gujarat). 124 ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 7(7), 124-129 Int. J. Adv. Res. 7(7), 124-129 Int. J. Adv. Res. 7(7), 124-129 The state of Dosha Dhatu Mala is dynamic. There equilibrium is health and vitiations lead to development of
diseases. Among these entities Dhatu are very important, which provides support to the body. They perform functions of
Dharana and Poshana2. It is necessary to know the strength of these important body supporters. In order to evaluate
Bala of these Dhatu, Charakacharya mentioned tenfold examination viz; Prakruti, Sara, Samhana, etc3. Out of these,
Sara Parikshana is one. By examining Dhatusarata, we can be evaluated Dhatu for its qualitative and functional
state. As per Dhatu Poshana, Rasa Dhatu is important as it is foremost one and it gives origin to all Dhatu. In other way
we can say it provides nourishment to all remaining Dhatu. Aim & Objectives:- 1. To study Sara & Its Types. 1. To study Sara & Its Types. 2. To study influence of Rasadhatu on Twakasara. 3. To understand characteristics of Twakasara in different samhita. 4. To study importance of Twakasara. SARA Sara is Sthiransha & Bala (strength). In Ayurveda Sara ‘‘word is put for the Vishuddhatara Dhatu. Sara is
excellence, essence of part or best part, the supreme quality, superb functional aspect of Dhatu4. Little waning or
vitiations of Dhatu is not hampered functions immediately if Dhatu is Uttama Sara. Twaksara Purusha Vivechana No
Characteristics
Charaka-Samhita
Sushruta-Samhita
Kashyapa-
Samhita
1
Sukha
+
-
-
2
Saubhagya
+
-
-
3
Aishwarya
+
-
-
4
Upbhoga
+
-
+
5
Buddhi
+
-
-
6
Vidya
+
-
-
7
Arogya
+
-
-
8
Praharsha
+
-
- (Body Hair)
Alpa
+
-
-
Gambhira
+
-
-
Mridu
+
+
-
Prasanna
+
+
-
Other characteristics:-
Sr. No
Characteristics
Charaka-Samhita
Sushruta-Samhita
Kashyapa-
Samhita
1
Sukha
+
-
-
2
Saubhagya
+
-
-
3
Aishwarya
+
-
-
4
Upbhoga
+
-
+
5
Buddhi
+
-
-
6
Vidya
+
-
-
7
Arogya
+
-
-
8
Praharsha
+
-
- Other characteristics:- Twak (Skin)
1. Snigdha: Snigdhata provides moistening and smoothening. Its results into unctuous, oily skin and having
soothing look. Snigdha Guna is composed of Apya Mahabhut. It raises Bala (strength) of the body and produces
softness in whole body. (
)
1. Snigdha: Snigdhata provides moistening and smoothening. Its results into unctuous, oily skin and having
soothing look. Snigdha Guna is composed of Apya Mahabhut. It raises Bala (strength) of the body and produces
softness in whole body. y
2. Slakshana: Mean the skin which possesses the property of smoothness. It has feeling of talcum powder. The
smoothness property has power of wound healing (Ropana). p
p
y
p
g
p
3. Mridu: Mridu mean softness. Person is having a soft skin which gives a feeling like a velvety cloth. This
characteristic (Mrudu) is due to predominance Jala & Akash Mahabhuta. 3. Mridu: Mridu mean softness. Person is having a soft skin which gives a feeling like a velvety cloth. This
characteristic (Mrudu) is due to predominance Jala & Akash Mahabhuta. 4. Prasanna: Means blooming; Person who has a healthy, energetic, attractive look and creates a feeling of joy to
the observer. E.g. good looking male/female models. They are having freshness which observes for long lasting
persists for a whole day. 4. Prasanna: Means blooming; Person who has a healthy, energetic, attractive look and creates a feeling of joy to
the observer. E.g. good looking male/female models. They are having freshness which observes for long lasting
persists for a whole day. p
y
5. Sukshma: Person is having thin skin. Since the thickness of skin depends on its site as well as on the amount of
subcutaneous fat. Twaksara Purusha Vivechana Twaksara individual are having Sukshma Twak as compare to other. p
y
5. Sukshma: Person is having thin skin. Since the thickness of skin depends on its site as well as on the amount of
subcutaneous fat. Twaksara individual are having Sukshma Twak as compare to other. 6. Saprabhev Twak: Skin having a natural glow over it. Rasadhatu is Apya, Dravyanusari (fluid in nature). Due
to fluidity, it provides nourishment to peripheral tissue and enhances its Prinana Karma. Function of Rasadhatu
are physically expressed over the skin that why skin look likes Saprabev without any artificial cosmetic make-
up. 7. Twak-Rogarahito: It means free from skin diseases. Rasa Dhatu provides strength, growth and nutrition to the
skin (body). Rasa Dhatu Sara person are having Vyadhi Kshamatva (good resistance) against skin disease. By
chance disease occurs it may possibly short lasting and easily curable as compare to Hina Rasadhatu sara
person. 7. Twak-Rogarahito: It means free from skin diseases. Rasa Dhatu provides strength, growth and nutrition to the
skin (body). Rasa Dhatu Sara person are having Vyadhi Kshamatva (good resistance) against skin disease. By
chance disease occurs it may possibly short lasting and easily curable as compare to Hina Rasadhatu sara
person. p
8. Sadyahchhatpraroh: It means a person having good wound healing power. Whenever Twaksara individual
come across the wound or trauma, it‘s duration of healing is earlier as comparison to Rasa- Asara person. It‘s
due to Slakshna property15 &Prinana Karma of Rasadhatu. Twaksara Purusha Vivechana ‘Twak-Sara’ means Persons possessing an excellent quality of Rasa Dhatu residing in the skin and Loma i.e. the
persons having well-nourished attractive skin and Loma are known as Twaksara Purusha. During the description of
Dhatu Sara types, the term ‘Rasa Sara’ is not used in any of the Samhita; it is described as Twak-Sara. Dalhana
clarified that in the term ―Twak-Sara’’ the word ‘Twak’ means the Rasa underneath the Twacha10. Probably it is
due to the fact that the functions of the Rasa Dhatu i.e. the Prinana11, is best revealed through Twak. ‘Twak-Sara’ means Persons possessing an excellent quality of Rasa Dhatu residing in the skin and Loma i.e. the
persons having well-nourished attractive skin and Loma are known as Twaksara Purusha. During the description of
Dhatu Sara types, the term ‘Rasa Sara’ is not used in any of the Samhita; it is described as Twak-Sara. Dalhana
clarified that in the term ―Twak-Sara’’ the word ‘Twak’ means the Rasa underneath the Twacha10. Probably it is
due to the fact that the functions of the Rasa Dhatu i.e. the Prinana11, is best revealed through Twak. The characteristics of Twaksara person are well described in different Ayurveda Samhita-Charaka Samhita,
Sushruta Samhita and Kashyapa Samhita. These characteristics can be grouped in to two as-
h
i
l h
i i The characteristics of Twaksara person are well described in different Ayurveda Samhita-Charaka Samhita,
Sushruta Samhita and Kashyapa Samhita. These characteristics can be grouped in to two as-
1. Physical characteristics y
2. Other characteristics i.e. psycho-somatic characteristics 2. Other characteristics i.e. psycho-somatic characteristics
Physical Characteristics:-
Body Part
Lakshnani
Charaka-
Samhita12
Sushruta-
Samhita13
Kashyapa-
Samhita14
Twaka
(Skin)
Snigdha
+
-
-
Slakshana
+
-
-
Mridu
+
+
-
Prasanna
+
+
+
Sukshma
+
-
-
Saprabha
+
-
+
Twak-Rog-Rahito
-
-
+
Sadyah-Kshata-Praroh
-
-
+
Loma
Sukshma
+
-
- 125 Int. J. Adv. Res. 7(7), 124-129 ISSN: 2320-5407 (Body Hair)
Alpa
+
-
-
Gambhira
+
-
-
Mridu
+
+
-
Prasanna
+
+
-
Other characteristics:-
Sr. Loma (Body Hair) Happiness in Rasa Sara person might be
in dietary substances as well as for the substances used for improving the quality of skin. They usually become
very happy by taking a delicious food or by getting a good quality of makeup material. As all these activities
going to improve Prinana Karma of Twaka. p
ya: It means person bless with good luck/ fortune. It also means beauty, charm and grace. g
g
p
2. Saubhagya: It means person bless with good luck/ fortune. It also means beauty, charm and grace. 3. Aishwarya: It means prosperity. In Twaksara person, Aishwarya flourishes due to his/her attractiveness and
good looking (skin and hairs). 3. Aishwarya: It means prosperity. In Twaksara person, Aishwarya flourishes due to his/her attractiveness and
good looking (skin and hairs). g
g (
)
4. Upbhoga: It means enjoyment of available stuffs. Twak Sara person enjoys the pleasure of different delicious
diet and different cosmetics. 5. Buddhi: It means intelligence (power of understanding). It is a measure of decision making power of the person
during tough situations. they can easily take good decision within short period of time. Rasasara person
endowed with intelligence and can be assessed with intelligence quotient.(I.Q.test) 5. Buddhi: It means intelligence (power of understanding). It is a measure of decision making power of the person
during tough situations. they can easily take good decision within short period of time. Rasasara person
endowed with intelligence and can be assessed with intelligence quotient.(I.Q.test) 6. Viddya: It means knowledge of the text. Rasasara person may have knowledge of the different fields like
sports, economics, politics etc. 6. Viddya: It means knowledge of the text. Rasasara person may have knowledge of the different fields like
sports, economics, politics etc. 7. Arogya: It means health. Twaksara persons are having a good quality, quantity, and functions of Rasa Dhatu as
well as Twak. So, they are more resistance against the diseases shared by these two. They generally do not
suffer with skin disease. They usually not affected by Rasapradoshaj Vyadhi i.e. Pandu, Hridroga etc .If disease
occurs, it will improve faster and cure easily. A Twaksara person can easily tolerate the condition of
Rasakshaya or Vriddhi. In such individual the symptoms of the same will be delayed. They can tolerate louder
sound; they will be not exhausted easily and can tolerate the thirst and fast easily. Loma (Body Hair) Loma (Body Hair) 1. Sukshma: It means very fine hairs. It is silky in appearance. 2. Alpa: It means such a person will have numerous hair over the body. Since the number of hairs depends on age,
sex, and the anatomical site. 2. Alpa: It means such a person will have numerous hair over the body. Since the number of hairs depends on age,
sex, and the anatomical site. 3. Gambheer: It means deeply rooted hairs. Rasadhatu nourishes the skin and its appendages (hairs and hair
follicles). Twaksara person do not have history of Lomashatana (hair fall) as compare to Hina Rasa sara person. 4. Mridu: Rasasara person possesses Mridu Loma i.e. soft and silky appearance of hair that gives velvet like
feelings. 3. Gambheer: It means deeply rooted hairs. Rasadhatu nourishes the skin and its appendages (hairs and hair
follicles). Twaksara person do not have history of Lomashatana (hair fall) as compare to Hina Rasa sara person. 3. Gambheer: It means deeply rooted hairs. Rasadhatu nourishes the skin and its appendages (hairs and hair
follicles). Twaksara person do not have history of Lomashatana (hair fall) as compare to Hina Rasa sara person. 4. Mridu: Rasasara person possesses Mridu Loma i.e. soft and silky appearance of hair that gives velvet like
f
li 4. Mridu: Rasasara person possesses Mridu Loma i.e. soft and silky appearance of hair that give
feelings. g
5. Prasanna: Person is having lustrous hairs, lucidity in hair & might be dandruff free hairs. 126 Int. J. Adv. Res. 7(7), 124-129 ISSN: 2320-5407 Int. J. Adv. Res. 7(7), 124-129 Int. J. Adv. Res. 7(7), 124-129 Other Characteristics:- Other Characteristics:
Only Acharya Charaka has mentioned about other characteristics including physio-psychological characteristics. Ot e C a acte st cs:
Only Acharya Charaka has mentioned about other characteristics including physio-psychological characteristics. 1. Sukha: It means person feels happiness after doing some act or work. Happiness in Rasa Sara person might be
in dietary substances as well as for the substances used for improving the quality of skin. They usually become
very happy by taking a delicious food or by getting a good quality of makeup material. As all these activities
going to improve Prinana Karma of Twaka. 1. Sukha: It means person feels happiness after doing some act or work. Importance of Twakasara Parikshana:- Sara examination is important in various aspects. Some are elaborated here Sara examination is important in various aspects. Some are elaborated here 1. For evaluation of Bala- We cannot conclude Rasa Sara person just looking externally. For both aims of
Ayurved science, namely, safeguarding of health and if diseased, to cure the disease and come back to
homeostatic condition, one needs to know his Bala. Sara examination gives us idea about types of Bala whether
it is Pravara, Madhayama or Hina. 1. For evaluation of Bala- We cannot conclude Rasa Sara person just looking externally. For both aims of
Ayurved science, namely, safeguarding of health and if diseased, to cure the disease and come back to
homeostatic condition, one needs to know his Bala. Sara examination gives us idea about types of Bala whether
it is Pravara, Madhayama or Hina. 2. In Chikitsa- Knowledge of Sarata is helpful to decide severity of the disease, prognosis of the disease, pattern
of treatment and the drug dose in disease. e.g. Twaka Hina Dhatusara individuals are more susceptible to
Rasadhatu Kshaya, & Twaka Vikara. Rasayana therapy can be adapted to improve Dhatusara. In Ayurveda,
Rasayana therapy is mentioned to enhance quality of Dhatu. Rasa Dhatu is foremost Dhatu and responsible for
Poshana and Prinana (nutrition) of body. 3. In Vyadhikshamatva- Sara is three dimensional Concept of the Dhatu i.e. it put forward qualitative,
quantitative as well as functional status of the Dhatu. Twakasara individuals are more resist for any abnormal
Vridhi (increase), Kshaya (decrease) or Rasadhatu-Pradushtas Vikara (vitiation of that Dhatu). Person with
Uttam Rasadhatusarata can easily tolerate the symptoms of vitiated Dhatu and also easily cure from the same. Rasa Dhatu Sara persons can easily tolerate frequent fasting (Upavasa) without symptoms of Rasadhatukshaya. 4. For better progeny-In gestational periods, Sarata of progeny is influenced by many factors. So that, parent
should be examined for their Dhatu Sarata to issue better progeny. After examination we can advise treatment in
Hina dhatu sarata for its enhancement. In this way Sara examination is also useful in prevention of hereditary
disorders. 5. In occupation- Sara examination is also essential for selection of particular occupation. Each occupation must
need well qualified persons with physical and psychological health. e.g. Loma (Body Hair) Hence from the above
discussion it is clear that a Twaksara person is comparatively immune for Rasa Kshaya, Rasa Vriddhi, or Rasa
Pradoshaj Vyadhi as well as for skin disease. j
y
8. Praharsha: It means excessive joyful condition of mind. It is due to Prinana karma of Rasa. It nourishes not
only body but also mind of an individual. As Prinana Karma of Rasa Dhatu is expressed in the form of
freshness and happiness, an individual looks fresh and happy. 8. Praharsha: It means excessive joyful condition of mind. It is due to Prinana karma of Rasa. It nourishes not
only body but also mind of an individual. As Prinana Karma of Rasa Dhatu is expressed in the form of
freshness and happiness, an individual looks fresh and happy. Importance of Twakasara Parikshana:- Rasa and Rakta Sara individuals are
having beautiful look, and intolerance for heat and physical strain so that, they are not able to hard work & will
be best for modeling and so on. In day to day life it is observed that, whenever a person got a job according
their physical and psychological capability, He / She do better in their field. By considering all this fact, we
understand how much Sara examination is essential for selection of particular occupation. 127 ISSN: 2320-5407 Int. J. Adv. Res. 7(7), 124-129 Int. J. Adv. Res. 7(7), 124-129 Conclusion:- 1. Sara is excellence, superb functional aspect of Dhatu. 1. Sara is excellence, superb functional aspect of Dhatu. 2. Acharya Charak, Sushruta and Vagbhata have mentioned eight type of Sara. These are Twak, Rakta, Mansa,
Meda, Asthi, Majja, Shukra, and Satvasara. Acharya Kashyapa has added another type of Sara i.e. Oja Sara. 8 2. Acharya Charak, Sushruta and Vagbhata have mentioned eight type of Sara. These are Twak, Rakta, Mansa,
Meda, Asthi, Majja, Shukra, and Satvasara. Acharya Kashyapa has added another type of Sara i.e. Oja Sara. 8 3. In Twakaaara’’ the word ‘Twak’ means the Rasa underneath the Twacha8. Probably it is due to the fact that the
functions of the Rasa Dhatu i.e. the Prinana8, is best revealed through Twak. 3. In Twakaaara’’ the word ‘Twak’ means the Rasa underneath the Twacha8. Probably it is due to the fact that the
functions of the Rasa Dhatu i.e. the Prinana8, is best revealed through Twak. 4. Examination of Twakasara is mainly advantageous for evaluation of Bala of Rasadhatu and also valuable for
planning of Chikitsa, to know Vyadhikshamatva, for getting better progeny and for selection of occupation. 4. Examination of Twakasara is mainly advantageous for evaluation of Bala of Rasadhatu and also valuable for
planning of Chikitsa, to know Vyadhikshamatva, for getting better progeny and for selection of occupation. References/ Bibliography:- 1. Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya and ‘Nyayacandrika’
Commentary by Gayadasa: edited by Yadavaji Trikamaji Aacharya, Sutrasthana Chapter15; Verse no. 3; page
no.67, published by Chaukhamba Sanskrit Sansthana Varanasi, reprint 2009. 1. Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya and ‘Nyayacandrika’
Commentary by Gayadasa: edited by Yadavaji Trikamaji Aacharya, Sutrasthana Chapter15; Verse no. 3; page
no.67, published by Chaukhamba Sanskrit Sansthana Varanasi, reprint 2009. 2. Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya and Nyayacandrika
Commentary by Gayadasa: edited by Yadavaji Trikamaji Aacharya, Chapter15; Verse no. 12; page no.62,
published by Chaukhamba Sanskrit Sansthana Varanasi, reprint 2009. 2. Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya and Nyayacandrika
Commentary by Gayadasa: edited by Yadavaji Trikamaji Aacharya, Chapter15; Verse no. 12; page no.62,
published by Chaukhamba Sanskrit Sansthana Varanasi, reprint 2009. p
y
p
3. Charak Samhita of Agnivesha elaborated by Charaka & Drudhabala with ‘Ayurveda-Dipika’ commentary by
Chakrapanidatta, edited by Vaidya Yadavaji Trikamji Aacharya, Vimansthana Chapter 8;Verse No-94. Page
no.276, Published by Chaukhamba Surbharati Prakashan Varanasi. Reprint 2011. 3. Charak Samhita of Agnivesha elaborated by Charaka & Drudhabala with ‘Ayurveda-Dipika’ commentary by
Chakrapanidatta, edited by Vaidya Yadavaji Trikamji Aacharya, Vimansthana Chapter 8;Verse No-94. Page
no.276, Published by Chaukhamba Surbharati Prakashan Varanasi. Reprint 2011. 4. Charak Samhita of Agnivesha elaborated by Charaka & Drudhabala with ‘Ayurveda-Dipika’ commentary by
Chakrapanidatta, edited by Vaidya Yadavaji Trikamji Aacharya, Vimansthana Chapter 8;Verse No 102 , page
no. 278, Published by Chaukhamba Surbharati Prakashan Varanasi.Reprint 2011. 4. Charak Samhita of Agnivesha elaborated by Charaka & Drudhabala with ‘Ayurveda-Dipika’ commentary by
Chakrapanidatta, edited by Vaidya Yadavaji Trikamji Aacharya, Vimansthana Chapter 8;Verse No 102 , page
no. 278, Published by Chaukhamba Surbharati Prakashan Varanasi.Reprint 2011. y
p
5. Charak Samhita of Agnivesha elaborated by Charaka & Drudhabala with ‘Ayurveda-Dipika’ commentary by
Chakrapanidatta, edited by Vaidya Yadavaji Trikamji Aacharya, Vimansthana Chapter 8;Verse No 103. , page
no. 278, Published by Chaukhamba Surbharati Prakashan Varanasi.Reprint2011. 5. Charak Samhita of Agnivesha elaborated by Charaka & Drudhabala with ‘Ayurveda-Dipika’ commentary by
Chakrapanidatta, edited by Vaidya Yadavaji Trikamji Aacharya, Vimansthana Chapter 8;Verse No 103. , page
no. 278, Published by Chaukhamba Surbharati Prakashan Varanasi.Reprint2011. 6. Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya, edited by Yadavaji
Trikamaji Aacharya, Chapter35; Verse no. 16. Page no.152, published by Chaukhamba Sanskrit
SansthanaVaranasi, reprint2009. 6. Discussion:- A healthy skin reflects the healthy physiological state of the body which in turn depends on excellence state of Rasa
Dhatu16. The main function of Rasa Dhatu is Prinana and responsible for the healthy skin and its appendage. It
further helps to maintain nutritional state of the body. Any disturbance in Rasdhatua Formation & its Samhana
(metabolism) leads to abnormal changes over Twak. Hence Rasasara and Twaksara are used as synonyms of each
other. Twakarasarata is result of superb function of Rasa Dhatu and it reflects in the form of its Characteristics. Strength of
Rasa Dhatu is assessed by critical examination of characteristic features mentioned in Twakasara. In the ancient text
the characteristics are described in very concise manner. Each and every word has many meaning. Here
characteristics are described depending upon reference to context. 12. Charak Samhita of Agnivesha elaborated by Charaka & Drudhabala with ‘Ayurveda-Dipika’ commentary by
Chakrapanidatta, edited by Vaidya Yadavaji Trikamji Aacharya, Vimansthana Chapter 8;Verse No 103. , page
no. 278, Published by Chaukhamba Surbharati Prakashan Varanasi.Reprint2011. ,
p
14. Kashyapa Samhita or Vriddhajivakatantra, Preached by Maharshi Marich Kashyapa; Summarized by Acahraya
Vriddhajivaka; redacted by vatsya with English commentatory ; edited by Prof. Premvati Tiwari, Sutrasthana
chapter 28 verse 37 page no.86; Chaukhamba Vivshvabharati Varanasi reprint 2013 13. Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya, edited by Yadavaji
Trikamaji Aacharya, Chapter35; Verse no. 16. Page no.152, published by Chaukhamba Sanskrit
SansthanaVaranasi, reprint2009. p
p g
p
15. Ashtanga Hridaya of Vagbhata with the commentaries Sarvangasudara of Arundatta & Ayurvedarasayana of
Hemadri; Annotated by Dr.Anna Kunte & Krishna Navre, edited by Pt.Hari Sadashiva Shastri Paradkar;
Sutrasthana chapter 1, verse no.18, page no.12 Chaukhamba Sanskrit Sansthan Varanasi Reprinted 2009. 16. Nikhate Sunil P, Sant Sampada S; The comparative study of skin sebum content in Twaksara-asarta; Ayurlog:
National Journal of Research in Ayurved Science-2015; 3(3): 67-72. References/ Bibliography:- Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya, edited by Yadavaji
Trikamaji Aacharya, Chapter35; Verse no. 16. Page no.152, published by Chaukhamba Sanskrit
SansthanaVaranasi, reprint2009. p
7. Ashtanga Hridaya of Vagbhata with the commentaries Sarvangasudara of Arundatta & Ayurvedarasayana of
Hemadri; Annotated by Dr.Anna Kunte & Krishna Navre, edited by Pt.Hari Sadashiva Shastri Paradkar
Sharirasthana chapter 3, verse no.117, page no.407 Chaukhamba Sanskrit Sansthan Varanasi Reprinted 2009. p
7. Ashtanga Hridaya of Vagbhata with the commentaries Sarvangasudara of Arundatta & Ayurvedarasayana of
Hemadri; Annotated by Dr.Anna Kunte & Krishna Navre, edited by Pt.Hari Sadashiva Shastri Paradkar
Sharirasthana chapter 3, verse no.117, page no.407 Chaukhamba Sanskrit Sansthan Varanasi Reprinted 2009. 8. Kashyapa Samhita or Vriddhajivakatantra, Preached by Maharshi Marich Kashyapa; Summarized by Acahraya
Vriddhajivaka; redacted by vatsya with English commentatory ; edited by Prof. Premvati Tiwari, Sutrasthana
chapter 28 verse 36 page no.86; Chaukhamba Vivshvabharati Varanasi reprint 2013 p
p g
p
9. Charak Samhita of Agnivesha elaborated by Charaka & Drudhabala with ‘Ayurveda-Dipika’ commentary by
Chakrapanidatta, edited by Vaidya Yadavaji Trikamji Aacharya, Vimansthana Chapter 8;Verse No 111-113. ,
page no. 278, Published by Chaukhamba Surbharati Prakashan Varanasi.Reprint 2011. 10. Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya, edited by Yadavaji
Trikamaji Aacharya, Chapter35; Verse no. 16. Page no.152, published by Chaukhamba Sanskrit
SansthanaVaranasi, reprint 2009. 11. Ashtanga Hridaya of Vagbhata with the commentaries Sarvangasudara of Arundatta & Ayurvedarasayana of
Hemadri; Annotated by Dr.Anna Kunte & Krishna Navre, edited by Pt.Hari Sadashiva Shastri Paradkar;
Sutrasthana chapter 11, verse no.4, page no.183 Chaukhamba Sanskrit Sansthan Varanasi Reprinted 2009. 128 Int. J. Adv. Res. 7(7), 124-129 Int. J. Adv. Res. 7(7), 124-129 ISSN: 2320-5407 Int. J. Adv. Res. 7(7), 124-129 12. Charak Samhita of Agnivesha elaborated by Charaka & Drudhabala with ‘Ayurveda-Dipika’ commentary by
Chakrapanidatta, edited by Vaidya Yadavaji Trikamji Aacharya, Vimansthana Chapter 8;Verse No 103. , page
no. 278, Published by Chaukhamba Surbharati Prakashan Varanasi.Reprint2011. y
p
13. Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya, edited by Yadavaji
Trikamaji Aacharya, Chapter35; Verse no. 16. Page no.152, published by Chaukhamba Sanskrit
SansthanaVaranasi, reprint2009. 13. Sushruta Samhita of Sushruta with ‘Nibandhasangraha’ commentary by Dalhanacharya, edited by Yadavaji
Trikamaji Aacharya, Chapter35; Verse no. 16. Page no.152, published by Chaukhamba Sanskrit
SansthanaVaranasi, reprint2009. ,
p
14. Kashyapa Samhita or Vriddhajivakatantra, Preached by Maharshi Marich Kashyapa; Summarized by Acahraya
Vriddhajivaka; redacted by vatsya with English commentatory ; edited by Prof. References/ Bibliography:- Premvati Tiwari, Sutrasthana
chapter 28 verse 37 page no.86; Chaukhamba Vivshvabharati Varanasi reprint 2013 p
p g
p
15. Ashtanga Hridaya of Vagbhata with the commentaries Sarvangasudara of Arundatta & Ayurvedarasayana of
Hemadri; Annotated by Dr.Anna Kunte & Krishna Navre, edited by Pt.Hari Sadashiva Shastri Paradkar;
Sutrasthana chapter 1, verse no.18, page no.12 Chaukhamba Sanskrit Sansthan Varanasi Reprinted 2009. p
p g
p
15. Ashtanga Hridaya of Vagbhata with the commentaries Sarvangasudara of Arundatta & Ayurvedarasayana of
Hemadri; Annotated by Dr.Anna Kunte & Krishna Navre, edited by Pt.Hari Sadashiva Shastri Paradkar;
Sutrasthana chapter 1, verse no.18, page no.12 Chaukhamba Sanskrit Sansthan Varanasi Reprinted 2009. 16. Nikhate Sunil P, Sant Sampada S; The comparative study of skin sebum content in Twaksara-asarta; Ayurlog:
National Journal of Research in Ayurved Science-2015; 3(3): 67-72. 16. Nikhate Sunil P, Sant Sampada S; The comparative study of skin sebum content in Twaksara-asarta; Ayurlog:
National Journal of Research in Ayurved Science-2015; 3(3): 67-72. 129
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Insurance approval rates for collagenase clostridium histolyticum prior to discontinuation: a Canada-wide analysis
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Research Square (Research Square)
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Introduction Peyronie’s disease (PD) is a condition consisting of an abnormal wound healing response of the tunica albuginea that results in excess
scar formation [1]. The resulting signs and symptoms include penile pain, shortening, curvature, narrowing, hinge deformity, palpable
plaques, erectile dysfunction, and negative effects on a patient’s emotional state [2, 3]. Though initially thought to be a rare condition, it is
now estimated to affect 9% [1, 4] of the male population. Unfortunately, although many patients ultimately experience stabilization of
their disease, spontaneous resolution of the condition is rare [1, 2]. While surgical management of PD is associated with high success rates, effective non-surgical management strategies remain limited. Non-surgical treatment options include penile traction, oral medications, and intralesional therapy [4, 5]. However, there are no oral
pharmacologic options currently recommended by Canadian Urologic Association (CUA) or American Urologic Association (AUA)
guidelines due to a lack of evidence regarding their efficacy [5, 6]. Intralesional therapies such as verapamil are potential options for
patients, however the use of these agents remains off-label [5]. Surgical correction remains a viable option for many PD patients, though
is associated with its own host of potential complications. In 2013, collagenase clostridium histolyticum (CCh) became the first
pharmacologic treatment for PD to be formally approved by the Food and Drug Administration following publication of the landmark
IMPRESS I and II trials, which were able to demonstrate the utility of CCh as a safe and efficacious, non-surgical treatment for Peyronie’s
Disease [7, 8]. Subsequently, CCh or Xiaflex® (Endo Pharmaceuticals) was included as a treatment option in the most recent CUA and
AUA guidelines for the management of PD. Despite the findings from the IMPRESS trials and further studies confirming the efficacy of alternative treatment protocols to reduce the
cost of therapy, CCh was discontinued in Australia and Asia in 2019, followed by Europe and Canada in 2020. The primary reason for
discontinuation cited by the producer, Endo Pharmaceuticals, was that “The product did not receive government reimbursement approval
and demand for the product has remained unsustainably low.” [9] This discontinuation represents a loss to the PD patient, for whom a
safe, effective, and relatively non-invasive treatment option is no longer available. A study by Mann et al. 2021 investigating Canadian
provider perspectives on CCh found that most providers were proponents of CCh, with the majority of their patients being satisfied with
their outcomes [9]. Introduction Moreover, 96% of respondents in this survey study felt that the discontinuation of CCh represented a loss for
Canadian patients [10]. Thus, despite its demonstrated efficacy and both patient and provider satisfaction, the United States is now the only market in which
intralesional CCh remains available. Given the impact that its discontinuation has left on multiple countries including Canada, we sought
to assess insurance approval rates and ultimately usage of CCh across Canada to better understand the factors that led to its
withdrawal. Article Version of Record: A version of this preprint was published at International Journal of Impotence Research on August 25th, 2023. See the
published version at https://doi.org/10.1038/s41443-023-00749-7. Page 1/13 Abstract Collagenase Clostridium histolyticum (CCh), the first approved non-surgical treatment for Peyronie’s disease (PD), was withdrawn from
the European, Canadian, and Asian markets due to poor demand and lack of government reimbursement options. We sought to assess
insurance approval rates and usage of CCh across Canada to understand the factors that led to its withdrawal. Data on patients
prescribed CCh for PD or Dupuytren's contracture was obtained through collaboration with BioScript Solutions to assess the association
of variables with insurance approval and prescription filling. We identified 3297 insurance coverage applications for Xiaflex from April
2018 to June 2020. Of all applications for PD, 92.9% applications were approved while 7.1% were rejected. Despite the withdrawal of CCh
from Canadian markets, coverage application approval rates for 2018, 2019, and 2020 were 86.5%, 90.1%, and 89.1%, respectively. Of
2921 approved applications, 88.8% prescriptions were filled. For the 376 rejected applications, 66.4% of prescriptions were filled. Overall,
90% of the cost of Xiaflex was covered in Canada among those with extended health benefits, with an out-of-pocket expense of $210.4. Insurance coverage requests for Xiaflex were approved at a high rate in Canada with approved patients being very likely to proceed with
therapy, despite interprovincial variation. Materials and Methods Data regarding all patients who had been prescribed CCh for either PD or Dupuytren’s contracture (DC) were obtained from BioScript
Solutions, which served the overwhelming majority of patients (> 95%) being considered for CCh treatment in Canada via the Xiaflex
Access Program. BioScript Solutions is a specialty healthcare company that supports a range of complex drug therapies and provides a
variety of services including wholesale pharmaceutical distribution as well as clinics and pharmacies. Patients who were deemed
potential candidates for CCh by their physicians were forwarded to the Xiaflex Access Program, which would then assist with obtaining Page 2/13 Page 2/13 insurance coverage, navigating reimbursement, dispensing the product itself, and overall patient support. In this role, Bioscript Solutions
would obtain information regarding insurance coverage, insurance provider, insurance approval or rejection, coverage amount, and more. For the purposes of this study, Bioscript Solutions was able to provide this data for all patients enrolled in the Xiaflex Access Program
from April 2018 to June 2020. Research ethics board approval as per TCPS2 was not required in this study as all data from BioScript
Solutions is previously anonymized and no confidential patient information was accessible. insurance coverage, navigating reimbursement, dispensing the product itself, and overall patient support. In this role, Bioscript Solutions
would obtain information regarding insurance coverage, insurance provider, insurance approval or rejection, coverage amount, and more. For the purposes of this study, Bioscript Solutions was able to provide this data for all patients enrolled in the Xiaflex Access Program
from April 2018 to June 2020. Research ethics board approval as per TCPS2 was not required in this study as all data from BioScript
Solutions is previously anonymized and no confidential patient information was accessible. Statistical analysis was performed with SPSS 24 software (Armonk, NY: IBM Corp.). Continuous variables were presented as medians
and interquartile ranges [25th − 75th ] in accordance with data distribution on the normality test, and a comparison of continuous
variables was performed using the Kruskal-Wallis test. Categorical variables were presented as absolute variables and frequencies and
analyzed with Chi-square test. Then, a univariable and multivariable-adjusted logistic regression analysis was performed to determine
the association of variables with insurance approval and prescription filling. A p-value < 0.05 was considered statistically significant. Results From April 2018 to June 2020, 3297 insurance coverage applications for Xiaflex were identified. This included prescriptions for both PD
and DC. Of these, 2535 (76.9%) of applications were for DC, 748 (22.7%) for PD, and 14 (0.04%) for both conditions. Of all 2921 approved applications, 2594 (88.8%) prescriptions were filled. However, for the 376 rejected applications, 250 (66.4%) of
prescriptions were filled. Overall, 90% (80–100) of the cost of Xiaflex was covered in Canada, with an out-of-pocket expense of $210.4 (0-
283.3). Table 1 demonstrates baseline characteristics stratified by applications per Canada Province. Significant differences were seen
with respect to application approvals, applications submitted in each calendar year (2018–2020), insurance company and coverage,
prescriber volume, out of pocket expense, and whether the prescription was filled. Page 3/13 Table 1
Overall characteristics of the patients that underwent insurance application for xiaflex in accordance with the different provinces in
Canada. Results Overall
n =
3297
(100%)
AB
n = 338
(10.3%)
BC
n = 102
(3.1%)
MB
n = 14
(0.4%)
NB
n = 114
(3.5%)
NFLD
n = 187
(5.7%)
NS
n = 88
(2.7%)
ON
n =
1728
(52.4%)
PQ
n = 715
(21.7%)
SK
n = 11
(0.3%)
p-
value
Condition
Dupuytren's
contracture
2535
(76.9%)
253
(74.9%)
84
(82.4%)
14
(100%)
114
(100%)
174
(93%)
88
(100%)
1246
(72.1%)
551
(77.1%)
11
(100%)
Peyronie's
disease
748
(22.7%)
85
(25.1%)
18
(17.6%)
0
0
13 (7%)
0
477
(27.6%)
155
(21.7%)
0
Both
14
(0.4%)
0
0
0
0
0
0
5
(0.3%)
9
(1.3%)
0
<
0.001
Initial
application
approval
Rejected
383
(11.6%)
68
(20.1%)
3
(2.9%)
5
(35.7%)
7
(6.1%)
25
(13.4%)
1
(1.1%)
38
(2.2%)
233
(32.6%)
3
(27.3%)
Approved
2914
(88.4%)
270
(79.9%)
99
(97.1%)
9
(64.3%)
107
(93.9%)
162
(86.6%)
87
(98.9%)
1690
(97.8%)
482
(67.4%)
8
(72.7%)
<
0.001
Subsequent
application
aproval (n =
383)
Rejected
376
(98.2%)
68
(100%)
3
(100%)
4 (80%)
7
(100%)
25
(100%)
1
(100%)
36
(94.7%)
229
(98.3%)
3
(100%)
Approved
7
(1.8%)
0
0
1 (20%)
0
0
0
2
(5.3%)
4
(1.7%)
0
0.090
Overall
approval
Rejected
376
(11.4%)
68
(20.1%)
3
(2.9%)
4
(28.6%)
7
(6.1%)
25
(13.4%)
1
(1.1%)
36
(2.1%)
229
(32%)
3
(27.3%)
Approved
2921
(88.6%)
270
(79.9%)
99
(97.1%)
10
(71.4%)
107
(93.9%)
162
(86.6%)
87
(98.9%)
1692
(97.9%)
486
(68%)
8
(72.7%)
<
0.001
Year
2018
1246
(37.8%)
100
(29.6%)
25
(24.5%)
9
(64.3%)
40
(35.1%)
102
(54.5%)
39
(44.3%)
680
(39.4%)
251
(35.1%)
0
2019
1564
(47.4%)
164
(48.5%)
57
(55.9%)
5
(35.7%)
56
(49.1%)
78
(41.7%)
38
(43.2%)
789
(45.7%)
366
(51.2%)
11
(100%)
2020
487
(14.8%)
74
(21.9%)
20
(19.6%)
0
18
(15.8%)
7
(3.7%)
11
(12.5%)
259
(15%)
98
(13.7%)
0
<
0.001
Insurance
Blue Cross
294
(8.9%)
99
(29.3%)
28
(27.5%)
6
(42.9%)
44
(38.6%)
35
(18.7%)
21
(23.9%)
23
(1.3%)
34
(4.8%)
4
(36.4%)
Canada Life
407
(12.3%)
44
(13%)
9
(8.8%)
4
(28.6%)
7
(6.1%)
35
(18.7%)
7 (8%)
270
(15.6%)
29
(4.1%)
2
(18.2%)
Median [Interquartile range 25th – 75th ]. * 2921 patients with insurance approval including patients approved after subsequent
applications. Results AB (Alberta) BC (British Columbia) MB (Manitoba) NB (New Brunswick) NFLD (Newfoundland) NS (Nova Scotia) ON (Ontario) PQ Page 4/13 Page 4/13 Overall
n =
3297
(100%)
AB
n = 338
(10.3%)
BC
n = 102
(3.1%)
MB
n = 14
(0.4%)
NB
n = 114
(3.5%)
NFLD
n = 187
(5.7%)
NS
n = 88
(2.7%)
ON
n =
1728
(52.4%)
PQ
n = 715
(21.7%)
SK
n = 11
(0.3%)
p-
value
Condition
ClaimSecure
62
(1.9%)
7
(2.1%)
7
(6.9%)
1
(7.1%)
3
(2.6%)
3
(1.6%)
3
(3.4%)
32
(1.9%)
6
(0.8%)
0
Combination
159
(4.8%)
12
(3.6%)
5
(4.9%)
0
4
(3.5%)
7
(3.7%)
1
(1.1%)
115
(6.7%)
14 (2%)
1
(9.1%)
Desjardins
87
(2.6%)
2
(0.6%)
1 (1%)
0
0
3
(1.6%)
2
(2.3%)
24
(1.4%)
55
(7.7%)
0
Express
Scripts
67 (2%)
1
(0.3%)
4
(3.9%)
0
4
(3.5%)
2
(1.1%)
0
56
(3.2%)
0
0
Green Shield
273
(8.3%)
15
(4.4%)
13
(12.7%)
1
(7.1%)
0
5
(2.7%)
3
(3.4%)
223
(12.9%)
12
(1.7%)
1
(9.1%)
Johnson
Insurance
76
(2.3%)
0
2 (2%)
0
3
(2.6%)
22
(11.8%)
1
(1.1%)
47
(2.7%)
1
(0.1%)
0
La Capital
59
(1.8%)
0
0
0
0
0
0
0
59
(8.3%)
0
Manulife
434
(13.2%)
28
(8.3%)
14
(13.7%)
0
8 (7%)
16
(8.6%)
7 (8%)
286
(16.6%)
73
(10.2%)
2
(18.2%)
PSHCP
264
(8%)
6
(1.8%)
1 (1%)
1
(7.1%)
12
(10.5%)
30
(16%)
20
(22.7%)
167
(9.7%)
27
(3.8%)
0
RAMQ
(Government
Plan)
185
(5.6%)
1
(0.3%)
0
0
0
0
0
0
184
(25.7%)
0
SSQ
74
(2.2%)
1
(0.3%)
0
0
0
0
0
8
(0.5%)
65
(9.1%)
0
Sun Life
485
(14.7%)
75
(22.2%)
7
(6.9%)
0
13
(11.4%)
12
(6.4%)
11
(12.5%)
287
(16.6%)
80
(11.2%)
0
Telus
58
(1.8%)
1
(0.3%)
4
(3.9%)
0
4
(3.5%)
0
0
47
(2.7%)
2
(0.3%)
0
Other
313
(9.5%)
46
(13.6%)
7
(6.9%)
1
(7.1%)
12
(10.5%)
17
(9.1%)
12
(13.6%)
143
(8.3%)
74
(10.3%)
1
(9.1%)
<
0.001
Prescriptions
by provider
1–20
prescriptions
1345
(40.8%)
152
(45%)
102
(100%)
14
(100%)
30
(26.3%)
47
(25.1%)
63
(71.6%)
637
(36.9%)
289
(40.4%)
11
(100%)
21–40
prescriptions
706
(21.4%)
140
(41.4%)
0
0
21
(18.4%)
29
(15.5%)
25
(28.4%)
423
(24.5%)
68
(9.5%)
0
> 40
prescriptions
1246
(37.8%)
46
(13.6%)
0
0
63
(55.3%)
111
(59.4%)
0
668
(38.7%)
358
(50.1%)
0
<
0.001
Out of
pocket
expense*
210.4
[0 -
283.3]
144.7
[0–
286]
261.7
[0–
300.3]
270.7
[16.5–
564.9]
50
[15–
267.3]
140.6
[9.2–
275.1]
269
[10–
283.5]
139.1
[0–
278.2]
283.3
[244.9–
494.4]
140
[0–
263.1]
<
0.001
Median [Interquartile range 25th – 75th ] * 2921 patients with insurance approval including patients approved after subsequent P
5 13
Median [Interquartile range 25th – 75th ]. Results * 2921 patients with insurance approval including patients approved after subsequent
applications. AB (Alberta), BC (British Columbia), MB (Manitoba), NB (New Brunswick), NFLD (Newfoundland), NS (Nova Scotia), ON (Ontario), PQ
(Quebec), SK (Saskatchewan) Page 5/13 Page 5/13 Overall
n =
3297
(100%)
AB
n = 338
(10.3%)
BC
n = 102
(3.1%)
MB
n = 14
(0.4%)
NB
n = 114
(3.5%)
NFLD
n = 187
(5.7%)
NS
n = 88
(2.7%)
ON
n =
1728
(52.4%)
PQ
n = 715
(21.7%)
SK
n = 11
(0.3%)
p-
value
Condition
Coverage*
90
[80–
100]
90
[80–
100]
80
[80–
100]
80
[57.8–
85]
95
[80–
100]
80
[80–
99]
80
[80–
100]
90
[80–
100]
80 [80–
90]
80
[52.5–
80]
<
0.001
Prescription
filled
No
453
(13.7%)
71
(21%)
5
(4.9%)
0
20
(17.5%)
44
(23.5%)
8
(9.1%)
189
(10.9%)
114
(15.9%)
2
(18.2%)
Yes
2844
(86.3%)
267
(79%)
97
(95.1%)
14
(100%)
94
(82.5%)
143
(76.5%)
80
(90.9%)
1539
(89.1%)
601
(84.1%)
9
(81.8%)
<
0.001
Median [Interquartile range 25th – 75th ]. * 2921 patients with insurance approval including patients approved after subsequent
applications. AB (Alberta), BC (British Columbia), MB (Manitoba), NB (New Brunswick), NFLD (Newfoundland), NS (Nova Scotia), ON (Ontario), PQ
(Quebec), SK (Saskatchewan) AB (Alberta), BC (British Columbia), MB (Manitoba), NB (New Brunswick), NFLD (Newfoundland), NS (Nova Scotia), ON (Ontario), PQ
(Quebec), SK (Saskatchewan) Table 2 compares approved and rejected applications. Of all insurance coverage applications for DC, 2214 (87.3%) applications were
approved, while 321 (12.7%) were rejected. Of all applications for PD, 695 (92.9%) applications were approved while 53 (7.1%) were
rejected. Despite the withdrawal of CCh from Canadian markets in 2021, coverage application approval rates for 2018, 2019, and 2020
were 86.5%, 90.1%, and 89.1% respectively. There was a statistically significant difference in the likelihood of approval by condition (DP
vs. PD) and which insurance company was utilized, ultimately affecting whether the prescription was filled. No difference was noted by
each subsequent year (2018–2020) or provider volume. Table 2 compares approved and rejected applications. Of all insurance coverage applications for DC, 2214 (87.3%) applications were
approved, while 321 (12.7%) were rejected. Of all applications for PD, 695 (92.9%) applications were approved while 53 (7.1%) were
rejected. Despite the withdrawal of CCh from Canadian markets in 2021, coverage application approval rates for 2018, 2019, and 2020
were 86.5%, 90.1%, and 89.1% respectively. Results Significant interprovincial variation was present, however overall insurance approval rates remained high in Canada. Higher
volume prescribers were more likely to ascertain insurance approval for their patients and patients were more likely to fill the prescription
if insurance approval was accepted. In Table 4, multivariate logistic regression analysis was performed to test for associations between
variables and whether the prescription was filled. This demonstrated that patients with PD were more likely to fill their prescriptions for
CCh than patients with DC (OR 0.57, p < 0.001). Significant interprovincial variation was present, however overall insurance approval
rates remained high in Canada. Patients were more likely to fill their prescription with insurance approval (OR 4.31, p < 0.001). Rejected
n = 376
Approved
n = 2921
p-value
Condition
Yes
250 (8.8%)
2594 (91.2%)
< 0.001
Median [Interquartile range 25th – 75th ]. In Table 3, multivariate logistic regression analysis was performed to test for associations between variables and insurance approval. This demonstrated that patients with PD were more likely to receive insurance coverage for CCh than patients with DC (OR 2.44, p <
0.001). Significant interprovincial variation was present, however overall insurance approval rates remained high in Canada. Higher
volume prescribers were more likely to ascertain insurance approval for their patients and patients were more likely to fill the prescription
if insurance approval was accepted. In Table 4, multivariate logistic regression analysis was performed to test for associations between
variables and whether the prescription was filled. This demonstrated that patients with PD were more likely to fill their prescriptions for
CCh than patients with DC (OR 0.57, p < 0.001). Significant interprovincial variation was present, however overall insurance approval
rates remained high in Canada. Patients were more likely to fill their prescription with insurance approval (OR 4.31, p < 0.001). In Table 3, multivariate logistic regression analysis was performed to test for associations between variables and insurance approval. This demonstrated that patients with PD were more likely to receive insurance coverage for CCh than patients with DC (OR 2.44, p <
0.001). Significant interprovincial variation was present, however overall insurance approval rates remained high in Canada. Higher
volume prescribers were more likely to ascertain insurance approval for their patients and patients were more likely to fill the prescription
if insurance approval was accepted. Results There was a statistically significant difference in the likelihood of approval by condition (DP
vs. PD) and which insurance company was utilized, ultimately affecting whether the prescription was filled. No difference was noted by
each subsequent year (2018–2020) or provider volume. Table 2 compares approved and rejected applications. Of all insurance coverage applications for DC, 2214 (87.3%) applications were
approved, while 321 (12.7%) were rejected. Of all applications for PD, 695 (92.9%) applications were approved while 53 (7.1%) were
rejected. Despite the withdrawal of CCh from Canadian markets in 2021, coverage application approval rates for 2018, 2019, and 2020
were 86.5%, 90.1%, and 89.1% respectively. There was a statistically significant difference in the likelihood of approval by condition (DP
vs. PD) and which insurance company was utilized, ultimately affecting whether the prescription was filled. No difference was noted by
each subsequent year (2018–2020) or provider volume. Page 6/13 Page 6/13 Table 2 approval and rejection. Rejected
n = 376
Approved
n = 2921
p-value
Condition
Dupuytren's contracture
321 (12.7%)
2214 (87.3%)
Peyronie's disease
53 (7.1%)
695 (92.9%)
Both
2 (14.3%)
12 (85.7%)
< 0.001
Year
2018
168 (13.5%)
1078 (86.5%)
2019
155 (9.9%)
1409 (90.1%)
2020
53 (10.9%)
434 (89.1%)
0.012
Insurance
Blue Cross
65 (22.1%)
229 (77.9%)
Canada Life
35 (8.6%)
372 (91.4%)
ClaimSecure
0
62 (100%)
Combination
6 (3.8%)
153 (96.2%)
Desjardins
0
87 (100%)
Express Scripts
0
67 (100%)
Green Shield
0
273 (100%)
Johnson Insurance
0
76 (100%)
La Capital
0
59 (100%)
Manulife
11 (2.5%)
423 (97.5%)
PSHCP
0
264 (100%)
RAMQ (Government Plan)
182 (98.4%)
3 (1.6%)
SSQ
14 (18.9%)
60 (81.1%)
Sun Life
0
485 (100%)
Telus
0
58 (100%)
Other
63 (20.1%)
250 (88.6%)
< 0.001
Prescriptions by provider
1–20 prescriptions
160 (11.9%)
1185 (88.1%)
21–40 prescriptions
65 (9.2%)
641 (90.8%)
> 40 prescriptions
151 (12.1%)
1095 (87.9%)
0.115
Out of pocket expense
NA
210.4 [0–283.3]
---
Coverage
NA
90 [80–100]
---
Prescription filled
No
126 (27.8%)
327 (72.2%)
Median [Interquartile range 25th – 75th ]. Rejected
n = 376
Approved
n = 2921
p-value
Condition
Yes
250 (8.8%)
2594 (91.2%)
< 0.001
Median [Interquartile range 25th – 75th ]. In Table 3, multivariate logistic regression analysis was performed to test for associations between variables and insurance approval. This demonstrated that patients with PD were more likely to receive insurance coverage for CCh than patients with DC (OR 2.44, p <
0.001). Results In Table 4, multivariate logistic regression analysis was performed to test for associations between
variables and whether the prescription was filled. This demonstrated that patients with PD were more likely to fill their prescriptions for
CCh than patients with DC (OR 0.57, p < 0.001). Significant interprovincial variation was present, however overall insurance approval
rates remained high in Canada. Patients were more likely to fill their prescription with insurance approval (OR 4.31, p < 0.001). Page 8/13 Page 8/13 Table 3
Univariable and multivariable adjusted logistic regression analysis to insurance approval and analyzed
variables. Univariable
Multivariable
OR
95% CI
p-value
OR
95% CI
p-value
Condition
Dupuytren's contracture
1
1
Peyronie's disease
1.90
1.40–2.57
< 0.001
2.44
1.69–3.35
< 0.001
Both
0.87
0.19–3.91
0.856
2.73
0.52–14.34
0.236
Year
2018
1
1
2019
1.42
1.12–1.79
0.003
1.70
1.30–2.23
< 0.001
2020
1.28
0.92–1.77
0.145
1.33
0.91–1.95
0.145
Provinces in Canada
Alberta
0.61
0.37–1.01
0.054
0.44
0.26–0.77
0.003
British Columbia
5.09
1.50–17.31
0.009
2.71
0.77 - 9.56
0.121
Manitoba
0.39
0.11–1.33
0.130
0.23
0.07–0.83
0.025
New Brunswick
2.36
0.99–5.65
0.054
2.18
0.89–5.37
0.088
Newfoundland and Labrador
1
1
Nova Scotia
13.43
1.79–100.77
0.012
10.63
1.39–81.36
0.023
Ontario
7.25
4.25–12.39
< 0.001
5.03
2.87–8.82
< 0.001
Quebec
0.33
0.21–0.51
< 0.001
0.20
0.12–0.33
< 0.001
Saskatchewan
0.41
0.10–1.66
0.211
0.25
0.06–1.08
0.064
Prescription by provider
1–20 prescriptions
1
1
21–40 prescriptions
1.33
0.98–1.80
0.065
0.85
0.59–1.21
0.363
> 40 prescriptions
0.98
0.77–1.24
0.862
0.93
0.70–1.25
< 0.001
Prescription filled
No
1
1
Yes
4.00
3.14–5.10
< 0.001
4.67
3.50–6.27
< 0.001
CI: Confidence interval; OR: Odds ratio. Page 9/13 Page 9/13 Page 9/13 Table 4
Univariable and multivariable adjusted logistic regression analysis to prescription filled and analyzed
variables. Results Univariable
Multivariable
OR
95% CI
p-value
OR
95% CI
p-value
Condition
Dupuytren's contracture
1
1
Peyronie's disease
0.73
0.58–0.91
0.005
0.57
0.44–0.73
< 0.001
Both
0.88
0.20–3.96
0.870
0.67
0.14–3.17
0.614
Year
2018
1
1
2019
1.19
0.96–1.48
0.122
1.12
0.89–1.41
0.336
2020
0.76
0.57–1.002
0.052
0.75
0.56–1.02
0.066
Provinces in Canada
Alberta
1.16
0.76–1.77
0.503
1.71
1.07–2.74
0.026
British Columbia
5.97
2.28–15.59
< 0.001
6.51
2.42–17.47
< 0.001
Manitoba*
--
---
New Brunswick
1.45
0.80–2.61
0.220
1.26
0.69–2.31
0.452
Newfoundland and Labrador
1
1
Nova Scotia
3.08
1.38–6.86
0.006
2.75
1.21–6.25
0.016
Ontario
2.51
1.73–3.63
< 0.001
2.51
1.70–3.73
< 0.001
Quebec
1.62
1.10–2.40
0.016
2.70
1.76–4.14
< 0.001
Saskatchewan
1.39
0.29–6.65
0.684
1.88
0.36–9.80
0.453
Prescription by provider
1–20 prescriptions
1
1
21–40 prescriptions
0.94
0.72–1.22
0.629
1.07
0.81–1.42
0.626
> 40 prescriptions
0.99
0.79–1.24
0.934
1.27
0.98–1.64
0.067
Overall approval
Rejected
1
1
Approved
4.00
3.14–5.10
< 0.001
4.31
3.23–5.73
< 0.001
CI: Confidence interval; OR: Odds ratio. * All patients in Manitoba got their prescription filled. Discussion With the publication of the IMPRESS I and II trials demonstrating the efficacy of CCh and subsequent approval of this medication for the
treatment of PD by a variety of national health regulatory bodies, CCh became a popular and important non-surgical treatment option for
patients diagnosed with PD. Despite CCh being recommended as a first line therapy by the CUA and AUA [5, 6], Xiaflex® has been
removed from Australian, Asian, European, and Canadian European markets. Our study aimed to assess insurance approval rates and
usage of CCh in Canada as a potential factor for discontinuation of the medication. Page 10/13
Overall, our study demonstrated high insurance approval rates for use of CCh in Peyronie’s disease, with 92.9% of requests being
approved in Canada. Applications were more likely to be accepted for PD compared to DP, however prescriptions for PD patients were less likely to be filled. A potential explanation is the out-of-pocket expense associated with Xiaflex may act as a barrier for patients to
access this medication, even with insurance approval. Interestingly, higher volume prescribers were more likely to have accepted
applications. However, it is possible that this is due to the fact that providers who have received prior acceptances are more likely to
submit more applications and know how to appropriately complete the required application. Although significant interprovincial variation
was present, overall insurance coverage application approvals were high for the country as a whole. Once approved, patients were likely
to proceed with CCh treatment, with 91.2% of all approved prescriptions being filled. less likely to be filled. A potential explanation is the out-of-pocket expense associated with Xiaflex may act as a barrier for patients to
access this medication, even with insurance approval. Interestingly, higher volume prescribers were more likely to have accepted
applications. However, it is possible that this is due to the fact that providers who have received prior acceptances are more likely to
submit more applications and know how to appropriately complete the required application. Although significant interprovincial variation
was present, overall insurance coverage application approvals were high for the country as a whole. Once approved, patients were likely
to proceed with CCh treatment, with 91.2% of all approved prescriptions being filled. Despite CCh being the only approved medical therapy for PD, it was unfortunately removed from multiple countries including Canada. Discussion With a lack of effective non-surgical options, this will surely lead to increased surgical intervention rates as the authors of this paper have
anecdotally noted. Although a cost-effectiveness analysis by Cordon et al. demonstrated that the cost of penile plication was
significantly less than that of the CCh protocol utilized in IMPRESS I and II [11], other considerations should be taken into account. The
primary outcome assessed in their study was improvement in curvature [11], and CCh still has utility as a primary non-surgical option not
only for achieving a functionally straight penis, but also as a tool to help improve patient satisfaction. Further investigation of alternative
CCh protocols were able to demonstrate significantly improved cost effectiveness, with cost being comparable to primary surgery [12]. Additionally, limited operating room time in a single-payer system such as Canada is another consideration that was not accounted for in
the US and UK [11, 13] cost effectiveness studies. Aside from the additional stress on the already limited resources of a universal
healthcare system, there are several patient factors to consider. Not all patients may be ideal candidates for surgery, and regardless of
candidacy, there are inherent risks of surgery as well as post-operative complications that must be discussed with the patient. Among
surgical candidates, secondary effects of surgery, such as approximately 2cm of penile shortening associated with penile plication. In
contrast, a recent study combining CCh with RestoreX traction device demonstrated 1.9cm improvement in length in addition to curvature
improvements [14]. This ~ 4cm difference in penile length between correcting a curve with plication versus CCh + RestoreX traction is
dramatic for many patients and must be considered when evaluating patient outcomes and cost-effectiveness. For other techniques
such as plaque incision and grafting, a 50% [15] risk of worsening erectile dysfunction is also considered as a non-acceptable risk for
many men and leads to a lifetime of adjuvant ED therapies, accruing further expenses. Ultimately, although immediate outcomes for
surgical repair are good, patients may express concerns in the long term regarding their quality of life such as satisfaction with penile
cosmesis, off-target effects of surgery and new onset ED [16, 17]. The potential effects of the discontinuation of CCh in the Canadian healthcare system was addressed in our prior study, which sought to
understand the perception of Canadian providers regarding this issue [9]. Discussion A large majority of the survey participants felt that the
discontinuation of CCh would be a significant loss for Canadian PD patients. In addition, few Canadian providers would offer alternative
intralesional therapies in the future and would be more likely to offer surgical management for PD. The sentiment among Canadian
providers to not offer alternative intralesional therapy may result in a large proportion of PD patients who are not suitable for surgical
management to remain untreated. Furthermore, many patients who are unwilling to proceed with surgery due to personal preferences will
also remain untreated. This has significant implications given the prevalence of PD as well as its negative impact on mental health [18,
19], with approximately one third of men with PD may suffer from clinical depression [19]. Additionally, PD has been associated with
significant emotional and psychosocial distress in domains such as quality of life, self-esteem, social isolation, relationship strain, and
stigmatization [18]. Given the mental health difficulties faced by many PD patients, the lack of non-surgical options may further dissuade
patients from proceeding with therapy for their condition. This will unfortunately lead to further worsening of both the patient’s physical
and mental health. Intralesional interferon has also been discontinued for treatment of Peyronie’s disease in Canada, leaving traction therapy and
intralesional verapamil as the only recommended non-surgical options. However, intralesional verapamil has heterogeneous results in the
literature [20] and lacks high level data as well as a well-established protocol. Mechanical traction devices may be undesirable to many
patients as well, as many treatment regimens recommend use for multiple hours daily. The significant limitations on the efficacy of non-
surgical treatments are an important consideration when treating a population where depression is a significant issue [18, 19]. In the
future, individuals with Peyronie’s disease may be further dissuaded from proceeding with therapy, putting patients at further risk for
worsening of their condition and psychosocial status. This study is not without limitations. Our data was limited to what could be provided by the Canadian distributer, Bioscript Solutions. While relatively granular data was obtained regarding insurance application, acceptance, amount of coverage, and more, data regarding
certain baseline information was lacking including age, comorbidities, socioeconomic status, and employment status. Most importantly,
detailed explanations for insurance coverage rejection were not available. Conclusion The discontinuation of CCh in Australia, Asia, Europe, and most recently Canada, has further limited the non-surgical options for the
treatment of PD. Low rates of government reimbursement approvals and inadequate demand for the product was cited as a reason for
withdrawal from these markets. Despite the limitations, this study was able to demonstrate that in Canada, insurance coverage requests
for Xiaflex were approved at a high rate, with approved patients very likely to proceed with therapy. Going forward, the lack of
conservative management for PD will be to the detriment of PD patients and may act as another barrier for individuals to proceed with
therapy. Key Messages - Insurance coverage requests for Xiaflex were approved at a high rate, with approved patients very likely to - proceed with therapy
- Discontinuation of CCh has further limited non-surgical options for treatment of PD - Insurance coverage requests for Xiaflex were approved at a high rate, with approved patients very likely to - proceed with therapy
Di
ti
ti
f CCh h
f
th
li
it d
i
l
ti
f
t
t
t f PD - Insurance coverage requests for Xiaflex were approved at a high rate, with approved patients very likely to - proceed with therap - Insurance coverage requests for Xiaflex were approved at a high rate, with approved patients very likely to - proceed with therapy Discontinuation of CCh has further limited non-surgical options for treatment of PD - Discontinuation of CCh has further limited non-surgical options for treatment of PD - Lack of conservative management strategies for PD may act as another barrier for individuals w - Lack of conservative management strategies for PD may act as another barrier for individuals wanting to proceed with therapy
l
i Funding No financial assistance was received in support of this study. Ethical Approval Research ethics board approval as per TCPS2 was not required in this study as all data from BioScript Solutions is previously
anonymized and no confidential patient information was accessible. Conflicts of Interest RF has received speaking honoraria and an education grant from Boston Scientific and speaking honoraria from Paladin Labs. PP is a
consultant for Boston Scientific. The remaining authors have no conflicts of interest to declare. Discussion Additionally, this data is likely biased towards Canadian
individuals with extended health coverage, and it is possible that a significant number of patients without extended health benefits did Page 11/13 Page 11/13 not attempt applying for Xiaflex approval. Given that 0.5% of men seek assessment and management of their PD with a healthcare
professional, and 11–13% of men likely have PD [21], the total number of prescriptions over a ~ 2-year period of 748 suggests that a
significant number of Canadian men suffering with PD are not seeking healthcare evaluation or are not choosing CCH as a treatment
method. This reinforces the notion that better public awareness for PD is necessary to engage men suffering in silence and reducing
barriers to treatment. not attempt applying for Xiaflex approval. Given that 0.5% of men seek assessment and management of their PD with a healthcare
professional, and 11–13% of men likely have PD [21], the total number of prescriptions over a ~ 2-year period of 748 suggests that a
significant number of Canadian men suffering with PD are not seeking healthcare evaluation or are not choosing CCH as a treatment
method. This reinforces the notion that better public awareness for PD is necessary to engage men suffering in silence and reducing
barriers to treatment. Author Contribution Statement DC, BS, DSB, TS, RBB, MG, RF, PP each contributed to the study design, manuscript drafting, approval of the final version, and agree to be
accountable for the accuracy of the work. Data Availability Statement The data that support the findings of this study are available from BioScript Solutions but restrictions apply to the availability of this
data, which was used under license for the current study, and not publicly available. Data are however available from the authors upon
reasonable request and with permission of BioScript Solutions. Declarations Data Availability Statement References Page 12/13 Page 12/13 1. Mulhall JP, Creech SD, Boorjian SA, et al. Subjective and objective analysis of the prevalence of Peyronie’s disease in a population of
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doi:10.1016/j.juro.2015.05.098 6. Gelbard M, Goldstein I, Hellstrom WJG, et al. Clinical efficacy, safety and tolerability of collagenase clostridium histolyticum for the
treatment of peyronie disease in 2 large double-blind, randomized, placebo controlled phase 3 studies. J Urol. 2013;190(1):199-207. doi:10.1016/j.juro.2013.01.087 6. Gelbard M, Goldstein I, Hellstrom WJG, et al. Clinical efficacy, safety and tolerability of collagenase clostridium histolyticum for the
treatment of peyronie disease in 2 large double-blind, randomized, placebo controlled phase 3 studies. J Urol. 2013;190(1):199-207. doi:10.1016/j.juro.2013.01.087 7. Hellstrom WJG, Tue Nguyen HM, Alzweri L, et al. Intralesional Collagenase Clostridium histolyticum Causes Meaningful
Improvement in Men with Peyronie’s Disease: Results of a Multi-Institutional Analysis. J Urol. 2019;201(4):777-782. doi:10.1097/JU.0000000000000032 7. Hellstrom WJG, Tue Nguyen HM, Alzweri L, et al. Intralesional Collagenase Clostridium histolyticum Causes Meaningful
Improvement in Men with Peyronie’s Disease: Results of a Multi-Institutional Analysis. J Urol. 2019;201(4):777-782. doi:10.1097/JU.0000000000000032 8. Paladin Labs Inc. Healthcare Professional Information regarding discontinuation of XIAFLEX®. References Published April 30, 2020. https://dupuytrencanada.ca/wp-content/uploads/2020/05/60841-002-Xiaflex_Discontinuation_Notice_04302020.pdf 8. Paladin Labs Inc. Healthcare Professional Information regarding discontinuation of XIAFLEX®. Published April 30, 2020. https://dupuytrencanada.ca/wp-content/uploads/2020/05/60841-002-Xiaflex_Discontinuation_Notice_04302020.pdf 9. Mann U, Shiff B, Jain K, Flannigan R, Elterman D, Patel P. Canadian provider perspectives on Collagenase Clostridium histolyticum
for the treatment of Peyronie’s disease and the impact of its discontinuation. Int J Impot Res. 2021. doi:10.1038/s41443-021-00458-
z 9. Mann U, Shiff B, Jain K, Flannigan R, Elterman D, Patel P. Canadian provider perspectives on Collagenase Clostridium histolyticum
for the treatment of Peyronie’s disease and the impact of its discontinuation. Int J Impot Res. 2021. doi:10.1038/s41443-021-00458-
z 10. Cordon BH, Hofer MD, Hutchinson RC, Broderick GA, Lotan Y, Morey AF. Superior Cost Effectiveness of Penile Plication vs
Intralesional Collagenase Injection for Treatment of Peyronie’s Disease Deformities. Urol Pract. 2017;4(2):118-125. doi:10.1016/j.urpr.2016.04.005 10. Cordon BH, Hofer MD, Hutchinson RC, Broderick GA, Lotan Y, Morey AF. Superior Cost Effectiveness of Penile Plication vs
Intralesional Collagenase Injection for Treatment of Peyronie’s Disease Deformities. Urol Pract. 2017;4(2):118-125. doi:10.1016/j.urpr.2016.04.005 11. Harvey N, Pearce I. At what cost is collagenase clostridium histolyticum viable for treating Peyronie’s disease in a public healthcare
system? Andrology. 2020;8(5):1304-1311. doi:10.1111/andr.12830 11. Harvey N, Pearce I. At what cost is collagenase clostridium histolyticum viable for treating Peyronie’s disease in a public healthcare
system? Andrology. 2020;8(5):1304-1311. doi:10.1111/andr.12830 12. Wymer K, Hebert K, Kohler T, Trost L. MP65-05 COMPARATIVE COST-EFFECTIVENESS OF SURGERY, COLLAGENASE CLOSTRIDIUM
HISTOLYTICUM, AND PENILE TRACTION THERAPY IN THE TREATMENT OF PEYRONIES DISEASE. J Urol. 2019;201(Supplement 4). doi:10.1097/01.ju.0000556917.78697.44 12. Wymer K, Hebert K, Kohler T, Trost L. MP65-05 COMPARATIVE COST-EFFECTIVENESS OF SURGERY, COLLAGENASE CLOSTRIDIUM
HISTOLYTICUM, AND PENILE TRACTION THERAPY IN THE TREATMENT OF PEYRONIES DISEASE. J Urol. 2019;201(Supplement 4). doi:10.1097/01.ju.0000556917.78697.44 13. Gamidov S, Shatylko T, Gasanov N, Scherbakov D, Li K, Sukhikh G. Long-term outcomes of surgery for Peyronie’s disease: focus on
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patient satisfaction. Int J Impot Res. 2021;33(3):332-338. doi:10.1038/s41443-020-0297-6 14. Alom M, Sharma KL, Toussi A, Kohler T, Trost L. Efficacy of Combined Collagenase Clostridium histolyticum and RestoreX Penile
Traction Therapy in Men with Peyronie’s Disease. J Sex Med. 2019;16(6):891-900. doi:10.1016/j.jsxm.2019.03.007 14. Alom M, Sharma KL, Toussi A, Kohler T, Trost L. Efficacy of Combined Collagenase Clostridium histolyticum and RestoreX Penile
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predictors. J Sex Med. 2011;8(7):2031-2037. doi:10.1111/j.1743-6109.2011.02299.x 15. Flores S, Choi J, Alex B, Mulhall JP. Erectile dysfunction after plaque incision and grafting: Short-term assessment of incidence and
predictors. J Sex Med. 2011;8(7):2031-2037. doi:10.1111/j.1743-6109.2011.02299.x 16. Chung E, Clendinning E, Lessard L, Brock G. Five-Year Follow-Up of Peyronie’s Graft Surgery: Outcomes and Patient Satisfaction. J
Sex Med. 2011;8(2):594-600. doi:10.1111/j.1743-6109.2010.02102.x 17. Punjani N, Nascimento B, Salter C, et al. Predictors of Depression in Men With Peyronie’s Disease Seeking Evaluation. J Sex Med. 2021;18(4):783-788. doi:10.1016/j.jsxm.2021.02.002 18. Nelson CJ, Mulhall JP. Psychological Impact of Peyronie’s Disease: A Review. J Sex Med. 2013;10(3):653-660. doi:10.1111/j.1743-
6109.2012.02999.x 19. Shirazi M, Haghpanah AR, Badiee M, Afrasiabi MA, Haghpanah S. Effect of intralesional verapamil for treatment of Peyronie’s
disease: A randomized single-blind, placebo-controlled study. Int Urol Nephrol. 2009;41(3):467-471. doi:10.1007/s11255-009-9522-4 20. Russo GI, Milenkovic U, Hellstrom W, Levine LA, Ralph D, Albersen M. Clinical Efficacy of Injection and Mechanical Therapy for
Peyronie’s Disease: A Systematic Review of the Literature[Figure presented]. Eur Urol. 2018;74(6):767-781. doi:10.1016/j.eururo.2018.07.005 Page 13/13
21. Stuntz M, Perlaky A, Des Vignes F, Kyriakides T, Glass D. The prevalence of Peyronie’s disease in the United States: A population-
based study. PLoS One. 2016;11(2). doi:10.1371/journal.pone.0150157 Page 13/13
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Detection of attractors of large Boolean networks via exhaustive enumeration of appropriate subspaces of the state space
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BMC bioinformatics
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* Correspondence: nikolaos.berntenis@roche.com
Non-Clinical-Safety, F. Hoffmann – La Roche AG, Grenzacherstrasse 124, 4070,
Basel, Switzerland SOFTWARE Open Access Open Access © 2013 Berntenis and Ebeling; 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. Detection of attractors of large Boolean networks
via exhaustive enumeration of appropriate
subspaces of the state space Nikolaos Berntenis* and Martin Ebeling Nikolaos Berntenis* and Martin Ebeling Abstract It is further argued that these
detectable cycles are also the biologically more important ones. Furthermore, lnet also provides standard Boolean
analysis features such as node loop detection. inherent in cycle detection is a classification scheme based on the number of non-frozen nodes of the cycle member
states, with cycles characterized by fewer non-frozen nodes being easier to detect. It is further argued that these
detectable cycles are also the biologically more important ones. Furthermore, lnet also provides standard Boolean
analysis features such as node loop detection. Conclusions: lnet is a software package that facilitates the analysis of large Boolean networks. Its intuitive
approach helps to better understand the network in question. Keywords: Boolean network, Attractor, Fixed state, Cycle, Regulatory network, State space Berntenis and Ebeling BMC Bioinformatics 2013, 14:361
http://www.biomedcentral.com/1471-2105/14/361 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361
http://www.biomedcentral.com/1471-2105/14/361 Abstract Background: Boolean models are increasingly used to study biological signaling networks. In a Boolean network,
nodes represent biological entities such as genes, proteins or protein complexes, and edges indicate activating or
inhibiting influences of one node towards another. Depending on the input of activators or inhibitors, Boolean
networks categorize nodes as either active or inactive. The formalism is appealing because for many biological
relationships, we lack quantitative information about binding constants or kinetic parameters and can only rely
on a qualitative description of the type “A activates (or inhibits) B”. A central aim of Boolean network analysis is
the determination of attractors (steady states and/or cycles). This problem is known to be computationally complex,
its most important parameter being the number of network nodes. Various algorithms tackle it with considerable
success. In this paper we present an algorithm, which extends the size of analyzable networks thanks to simple and
intuitive arguments. Results: We present lnet, a software package which, in fully asynchronous updating mode and without any
network reduction, detects the fixed states of Boolean networks with up to 150 nodes and a good part of any
present cycles for networks with up to half the above number of nodes. The algorithm goes through a complete
enumeration of the states of appropriately selected subspaces of the entire network state space. The size of these
relevant subspaces is small compared to the full network state space, allowing the analysis of large networks. The
subspaces scanned for the analyses of cycles are larger, reducing the size of accessible networks. Importantly, Results: We present lnet, a software package which, in fully asynchronous updating mode and without any
network reduction, detects the fixed states of Boolean networks with up to 150 nodes and a good part of any
present cycles for networks with up to half the above number of nodes. The algorithm goes through a complete
enumeration of the states of appropriately selected subspaces of the entire network state space. The size of these
relevant subspaces is small compared to the full network state space, allowing the analysis of large networks. The
subspaces scanned for the analyses of cycles are larger, reducing the size of accessible networks. Importantly,
inherent in cycle detection is a classification scheme based on the number of non-frozen nodes of the cycle member
states, with cycles characterized by fewer non-frozen nodes being easier to detect. Background the activation state, or value, of any node in a network
as a function of its activating and/or inhibiting inputs. In
general, for a target node with k different input nodes,
each of which can again be either “active” or “inactive”,
the logical functions assign the resulting values of the
target node for each of the 2k possible input patterns. The use of Boolean models I n the study of biological
networks was proposed and worked out already in the
1970s [1-5]. A Boolean network model is characterized
by the topology of a biological interaction network and a
set of qualitative parameters termed “logical functions”
by Thomas and D’Ari [6]. Logical functions determine Boolean networks are best suited to analyze and de-
scribe steady states of systems (which are independent
of kinetic parameters). As demonstrated by Thomas,
non-trivial steady states are determined by the presence
of negative or positive feedback loops, with the former Page 2 of 10 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361
http://www.biomedcentral.com/1471-2105/14/361 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361
http://www.biomedcentral.com/1471-2105/14/361 characterizing homeostatic or oscillatory processes, and
the
latter
leading
to
switch-like
or
differentiation
behaviour. the number of activation patterns grows even faster. We
refer to this number as the “state complexity” of a
network. For every node in a network, the logical parameters
determine how it reacts to the input it receives from
other nodes, more specifically, to which value (0 or 1) a
node will tend under any given input pattern, also re-
ferred to as the “image” of the node for this input pat-
tern. In general, for a node with k inputs (activating or
inhibiting), there are 2k possible input patterns and cor-
responding logical parameters. We refer to this as “par-
ameter complexity” of a network. For the choice of
logical parameters, there are some obvious constraints,
e.g., adding an activating input to an already active node
should not lead to its inactivation. For simplicity rea-
sons, Boolean network analyses often adopt the conven-
tion that a node under the influence of at least one
inhibitor always tends towards being inactive irrespective
of the presence of any activators; in the absence of any
inhibiting influences, a single activator will be sufficient
to activate it. This effectively eliminates the parameter
complexity of the problem. Background In the case of multi-valued Boolean networks,
where some of the nodes can have more than 2 values, TF
Rep
CA1
CA2
T1
Ex1
T2
Ex2
0
0
0
0
K R,CA2
0
KR
0
0
0
0
1
K R
0
KR,CA2
0
0
0
1
0
K R,CA1,CA2
0
KR,CA1
0
0
0
1
1
K R,CA1
0
KR,CA1,CA2
0
0
1
0
0
KCA2
0
K = 0
0
0
1
0
1
K = 0
0
KCA2
0
0
1
1
0
KCA1,CA2
0
KCA1
0
0
1
1
1
KCA1
0
KCA1,CA2
0
1
0
0
0
K TF,R,CA2
0
KTF,R
0
1
0
0
1
K TF,R
0
KTF,R,CA2
1
1
0
1
0
K TF,R,CA1,CA2 = 1 1
KTF,R,CA1
0
1
0
1
1
K TF,R,CA1
0
KTF,R,CA1,CA2 = 1 1
1
1
0
0
K TF,CA2
0
KTF
0
1
1
0
1
K TF
0
KTF,CA2
0
1
1
1
0
K TF,CA1,CA2
0
KTF,CA1
0
1
1
1
1
K TF,CA1
0
KTF,CA1,CA2
0
A
C
B
Figure 1 State complexity and parameter complexity. A: Schematic representation of a hypothetical regulatory biological network. A
transcription factor TF (striped circle) interacts with either one of two competing co-activators (CA1, CA2) to drive expression of one of two target
genes (triangles T1, T2). Presence of an inhibitor (grey, rounded rectangle) blocks TF. B: In the absence of detailed knowledge of the underlying
interactions, the network from A can be simplified into this diagram, with the state complexity translated into parameter complexity as shown in
C. C: For the 24 possible activation patterns of the 4 input nodes in B, logical functions for the two targets T1 and T2 are specified. The only
knowledge is about the activating or inhibiting influence of the input nodes on the targets. For the logical functions, we use the convention
introduced by Thomas et al. [6], i.e. parameter KTF,R,CA2 stands for the value (either 0 or 1) to which the target node tends under the positive
influence (i.e., presence of activators, absence of inhibitors) of the input nodes mentioned as subscripts. For lnet, only two of the logical parameters
for every node are pre-defined (values assigned in bold). Two additional columns, Ex1 and Ex2, specify the concrete values of all parameters for
the system as depicted in A. Background Here, we deviate from this
convention, motivated by biological examples where, for
example, both a transcription factor and a co-activator
are required for a certain function, or where co-
activators and co-repressors compete for a target tran-
scription factor (Figure 1). The only assumption we The analysis of Boolean networks comprised of more
than a few nodes is feasible thanks to tools developed by
various groups. Garg et al. [7] introduced the concept of
binary decision diagrams and developed SQUAD [8]. GINsim, a tool implementing Thomas’ program was pro-
vided by Gonzalez et al. [9]. Himkelman et al. [10] used
algebraic methods to develop ADAM, Helikar et al. [11]
developed the simulation platform ChemicalChains and
Müssel et al. developed BoolNet [12]. Algorithms were also developed that simplify network
architectures without affecting the steady state proper-
ties. They eliminate iteratively single nodes that: do not
regulate their own function [13,14] or: have one incom-
ing and one outgoing edge (simple mediator nodes) or
have the same value in all attractors [15,16]. Both ap-
proaches preserve the fixed point structure of the net-
work. The latter preserves also the cycle attractors,
while the former may, in certain cases, introduce spuri-
ous ones. For a binary Boolean network with n nodes, there are
2n possible activation patterns that form the state space:
every state is represented by an n-dimensional binary
vector. Background TF
Rep
CA1
CA2
T1
Ex1
T2
Ex2
0
0
0
0
K R,CA2
0
KR
0
0
0
0
1
K R
0
KR,CA2
0
0
0
1
0
K R,CA1,CA2
0
KR,CA1
0
0
0
1
1
K R,CA1
0
KR,CA1,CA2
0
0
1
0
0
KCA2
0
K = 0
0
0
1
0
1
K = 0
0
KCA2
0
0
1
1
0
KCA1,CA2
0
KCA1
0
0
1
1
1
KCA1
0
KCA1,CA2
0
1
0
0
0
K TF,R,CA2
0
KTF,R
0
1
0
0
1
K TF,R
0
KTF,R,CA2
1
1
0
1
0
K TF,R,CA1,CA2 = 1 1
KTF,R,CA1
0
1
0
1
1
K TF,R,CA1
0
KTF,R,CA1,CA2 = 1 1
1
1
0
0
K TF,CA2
0
KTF
0
1
1
0
1
K TF
0
KTF,CA2
0
1
1
1
0
K TF,CA1,CA2
0
KTF,CA1
0
1
1
1
1
K TF,CA1
0
KTF,CA1,CA2
0
A
C
B
Fi
1 S
l
i
d
l
i
A S h
i
i
f
h
h i
l
l
bi l
i
l
k A A
B TF
Rep
CA1
CA2
T1
Ex1
T2
Ex2
0
0
0
0
K R,CA2
0
KR
0
0
0
0
1
K R
0
KR,CA2
0
0
0
1
0
K R,CA1,CA2
0
KR,CA1
0
0
0
1
1
K R,CA1
0
KR,CA1,CA2
0
0
1
0
0
KCA2
0
K = 0
0
0
1
0
1
K = 0
0
KCA2
0
0
1
1
0
KCA1,CA2
0
KCA1
0
0
1
1
1
KCA1
0
KCA1,CA2
0
1
0
0
0
K TF,R,CA2
0
KTF,R
0
1
0
0
1
K TF,R
0
KTF,R,CA2
1
1
0
1
0
K TF,R,CA1,CA2 = 1 1
KTF,R,CA1
0
1
0
1
1
K TF,R,CA1
0
KTF,R,CA1,CA2 = 1 1
1
1
0
0
K TF,CA2
0
KTF
0
1
1
0
1
K TF
0
KTF,CA2
0
1
1
1
0
K TF,CA1,CA2
0
KTF,CA1
0
1
1
1
1
K TF,CA1
0
KTF,CA1,CA2
0
C A C B Figure 1 State complexity and parameter complexity. A: Schematic representation of a hypothetical regulatory biological network. A
transcription factor TF (striped circle) interacts with either one of two competing co-activators (CA1, CA2) to drive expression of one of two target
genes (triangles T1, T2). Presence of an inhibitor (grey, rounded rectangle) blocks TF. Background TF
Rep
CA1
CA2
T1
Ex1
T2
Ex2
0
0
0
0
K R,CA2
0
KR
0
0
0
0
1
K R
0
KR,CA2
0
0
0
1
0
K R,CA1,CA2
0
KR,CA1
0
0
0
1
1
K R,CA1
0
KR,CA1,CA2
0
0
1
0
0
KCA2
0
K = 0
0
0
1
0
1
K = 0
0
KCA2
0
0
1
1
0
KCA1,CA2
0
KCA1
0
0
1
1
1
KCA1
0
KCA1,CA2
0
1
0
0
0
K TF,R,CA2
0
KTF,R
0
1
0
0
1
K TF,R
0
KTF,R,CA2
1
1
0
1
0
K TF,R,CA1,CA2 = 1 1
KTF,R,CA1
0
1
0
1
1
K TF,R,CA1
0
KTF,R,CA1,CA2 = 1 1
1
1
0
0
K TF,CA2
0
KTF
0
1
1
0
1
K TF
0
KTF,CA2
0
1
1
1
0
K TF,CA1,CA2
0
KTF,CA1
0
1
1
1
1
K TF,CA1
0
KTF,CA1,CA2
0
A
C
B
Figure 1 State complexity and parameter complexity. A: Schematic representation of a hypothetical regulatory biological network. A
transcription factor TF (striped circle) interacts with either one of two competing co-activators (CA1, CA2) to drive expression of one of two target
genes (triangles T1, T2). Presence of an inhibitor (grey, rounded rectangle) blocks TF. B: In the absence of detailed knowledge of the underlying
interactions, the network from A can be simplified into this diagram, with the state complexity translated into parameter complexity as shown in
C. C: For the 24 possible activation patterns of the 4 input nodes in B, logical functions for the two targets T1 and T2 are specified. The only
knowledge is about the activating or inhibiting influence of the input nodes on the targets. For the logical functions, we use the convention
introduced by Thomas et al. [6], i.e. parameter KTF,R,CA2 stands for the value (either 0 or 1) to which the target node tends under the positive
influence (i.e., presence of activators, absence of inhibitors) of the input nodes mentioned as subscripts. For lnet, only two of the logical parameters
for every node are pre-defined (values assigned in bold). Two additional columns, Ex1 and Ex2, specify the concrete values of all parameters for
the system as depicted in A. Fixed states detection Crucial for the development of the algorithm is the
realization that the discrete nature of Boolean networks
allows us to restrict the search to selected subspaces of
the state space in which the fixed states (if any exist) res-
ide. These subspaces turn out to be orders of magnitude
smaller than the actual state space rendering possible an
exact enumeration. This is achieved via a two-step
process (Figure 2). For any given network and network state, it is not ob-
vious if all nodes satisfy the logical functions, i.e., have
values in agreement with their inputs; in fact, it is not
even obvious if such a state exists. Node values can be
updated by switching them in agreement with the input
pattern. Whenever stated that in a given state a node
satisfies the equations, it is meant that the node will re-
tain its value should we choose to update it (i.e., the
value is identical to the image for the current input pat-
tern). For simplicity, we refer to such a node as a “con-
tent” node. A fixed state of the network is, by definition,
a state in which all the node equations are satisfied, i.e. all the nodes are content. In contrast, a node whose
current value is in disagreement with its current input,
and which will be modified when chosen for an update,
is called a “discontent” node. Assume a Boolean network of n nodes. For k of them
(typically, k around 10), generate the 2k × k matrix
representing all their 2k possible states. Then for each
one of the remaining n-k nodes go through the following
iterative process: Extend the matrix by first duplicating it and then add-
ing to it an extra column with the values of an appropri-
ately chosen (k + 1)st node (0 and 1 for each pair of
identical rows). The outcome is a 2k+1 × (k + 1) matrix. The choice of the new node is crucial: All its incoming
nodes must be present in the initial k-member group
(see remark below). Then, for each row, we check
whether the value of the added node satisfies its node
equation. If it violates it then we remove the row, for it
cannot lead to a fixed state. By doing so, all its 2n-(k+1)
downstream successors are effectively removed once and
for all. Background B: In the absence of detailed knowledge of the underlying
interactions, the network from A can be simplified into this diagram, with the state complexity translated into parameter complexity as shown in
C. C: For the 24 possible activation patterns of the 4 input nodes in B, logical functions for the two targets T1 and T2 are specified. The only
knowledge is about the activating or inhibiting influence of the input nodes on the targets. For the logical functions, we use the convention
introduced by Thomas et al. [6], i.e. parameter KTF,R,CA2 stands for the value (either 0 or 1) to which the target node tends under the positive
influence (i.e., presence of activators, absence of inhibitors) of the input nodes mentioned as subscripts. For lnet, only two of the logical parameters
for every node are pre-defined (values assigned in bold). Two additional columns, Ex1 and Ex2, specify the concrete values of all parameters for
the system as depicted in A. Figure 1 State complexity and parameter complexity. A: Schematic representation of a hypothetical regulatory biological network. A
transcription factor TF (striped circle) interacts with either one of two competing co-activators (CA1, CA2) to drive expression of one of two target
genes (triangles T1, T2). Presence of an inhibitor (grey, rounded rectangle) blocks TF. B: In the absence of detailed knowledge of the underlying
interactions, the network from A can be simplified into this diagram, with the state complexity translated into parameter complexity as shown in
C. C: For the 24 possible activation patterns of the 4 input nodes in B, logical functions for the two targets T1 and T2 are specified. The only
knowledge is about the activating or inhibiting influence of the input nodes on the targets. For the logical functions, we use the convention
introduced by Thomas et al. [6], i.e. parameter KTF,R,CA2 stands for the value (either 0 or 1) to which the target node tends under the positive
influence (i.e., presence of activators, absence of inhibitors) of the input nodes mentioned as subscripts. For lnet, only two of the logical parameters
for every node are pre-defined (values assigned in bold). Two additional columns, Ex1 and Ex2, specify the concrete values of all parameters for
the system as depicted in A. Berntenis and Ebeling BMC Bioinformatics 2013, 14:361
http://www.biomedcentral.com/1471-2105/14/361 Page 3 of 10 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361
http://www.biomedcentral.com/1471-2105/14/361 format also used in [7]. Background In the input text file each line
corresponds to either an activation A →B or to an in-
hibition C −| D. make here is that under “optimal” conditions, i.e., pres-
ence of all activators and absence of all inhibitors, a
node must be activated, and likewise, in the presence of
all inhibitors and absence of any activators, it must be
inactivated. These minimum assumptions ensure that
every node must in principle be able to switch between
its two values. We note that state and parameter com-
plexity are related and it is often possible to reduce par-
ameter complexity by adding symbolic nodes to the
network that represent complexes or intermediate steps
along a process; however, this comes at the expense of
higher state complexity. The source code is freely available from the authors. Fixed states detection The same procedure is followed for any of the k
initially selected nodes, if all its incoming nodes are in
the matrix. Boolean networks have often been studied using “syn-
chronous” update strategies, where the values of all dis-
content nodes are switched simultaneously. (Note that
this switching can lead to the generation of new discon-
tent nodes.) While this approach is technically conveni-
ent, it does not properly reflect the characteristics of
biological networks, and it may even introduce artifacts
in their behavior. “Asynchronous” updates proceed with
one node at a time, often selecting them in random
order. This method, referred to as “general asynchron-
ous” in [15], is the one we choose. Then a (k + 2)nd node is added, further extending the
matrix, its equation is tested against all rows, and the
process is repeated until all the nodes have been taken
into account. At each iteration step, if no remaining
node is found having all its incoming nodes already
present in the matrix, the one with the maximal out-
degree is chosen. The algorithm is simply trying to in-
crease the probability that at each step at least one node
having all its incoming nodes already present will be
available. The choice of the first k nodes is based on this
reasoning too: the ones with the highest out-degree
values are selected. In the following, we are referring to random asyn-
chronous updating and to binary Boolean networks
(although the algorithm is also valid for multiple discrete-
valued ones). Furthermore, all results were generated
from testing on networks having: (i) values of the ratio
of edges to nodes between 2 to 3 and (ii) all their nodes
with non-zero in- and out-degrees and with at least one
of them larger than one (i.e., without simple mediator
nodes). The successive removal of equation-violating states
(i.e., states containing at least one discontent node) leads
to a dramatic decrease of the number of states exam-
ined. It can simplify the problem by many orders of
magnitude, depending on network size and complexity. In a typical network the number of examined states
reaches its maximum value at about or just after the
time half of the nodes have been included in the matrix. Implementation We start with
the full listing of all value combinations for nodes 1 and 2 in a 2 x 22 matrix, then add node 3 as described to obtain a (2 + 1) x 22+1 matrix. For
this, we also specify the images 1′, 2′, 3′ to which the three nodes tend for any input combination, using logical parameters. In this simple
example, only node 3 receives multiple inputs and thus has non-trivial logical functions. Removing the states which cannot match their image
state for any valid choice of the logical functions, we obtain two candidates for fixed states. Comparing the states to their image states, we realize
that state (1, 1, 0) is a fixed state if 31 equals 0 (i.e., node 3 is not activated by the presence of its activator 1 alone), whereas state (0, 0, 1) is fixed
if 32 equals 1 (i.e., node 3 is already activated by the absence of its inhibitor 2 alone). Setting both 31 = 0 and 32 = 1 renders both states fixed. Experimental data in agreement with state (1, 1, 0) but not with (0, 0, 1) thus help in estimating the logical functions. cycles, i.e., sets of states out of which the system cannot
escape once it reaches them. Once a system enters a
basin of attraction then the equations drive it deeper
into it, gradually increasing the number of content
nodes. However, for any basin of attraction, it is not triv-
ial to find out if it can be reached from any given
current state. Then it decreases rapidly as many of the node equations
are taken into account during the evaluation. When the
full set of nodes has been processed, only the fixed states
remain in the final matrix. We note that the approach described here simultan-
eously copes with both state and parameter complexity. When testing if the actual value of a node is compatible
with the node equations, we accept any states for which
this condition holds for at least one choice of the logical
parameters. Thus, the algorithm does not only yield
fixed states for a concrete combination of logical param-
eters, but for all possible ones. The conditions on the lo-
gical parameters needed to realize a fixed point can then
be read from the final matrix (Figure 2B). This aspect is
of key importance for reverse-engineering problems. Implementation We consider a state to be the more unstable/disordered
the higher the number of its discontent nodes, and we
refer to the state as being 1-, 2- up to n-discontent. Intuitively, the system equations tend to drive the sys-
tem towards the more stable/ordered regions of the
state space. We note that most of the interesting
stable cycles reside in sufficiently ordered space state
regions, where a good part of the node values of their
members satisfy the equations. Stable cycles including
states with a large number of discontent nodes are less
relevant which is apparent from the following consi-
derations (compare Figure 3). The generalization to multiple-valued case is straight-
forward: At each iterative step, instead of just duplicat-
ing the rows, the algorithm adds m-1 replicates of the
previous matrix when adding a node with m levels, with
the corresponding level (from 1 to m-1) appended. Then
it proceeds as previously described, namely by removing
all equation-violating states. If a state of a system with n nodes is k-discontent, it
has k successor states, because each one of the k discon-
tent nodes could be updated, and the equations do not
change the values of content nodes. A cycle is stable when all its member states flow
within the cycle. A single cycle member having a succes-
sor that is not part of the cycle renders the cycle meta-
stable (Figure 3A). Implementation The software application has been written in ANSI C
and it is single-threaded. The only hardware require-
ments concern available random access memory. The
network topology can be loaded using the straightforward Berntenis and Ebeling BMC Bioinformatics 2013, 14:361
Page 4 of 10
http://www.biomedcentral.com/1471-2105/14/361 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361
http://www.biomedcentral.com/1471-2105/14/361 Page 4 of 10 A
B
Figure 2 For this simplified account of the algorithm described in the text, we analyze a 3-node network (A). We start with the full listing
of all value combinations for nodes 1 and 2 in a 2 x 22 matrix, then add node 3 as described to obtain a (2 + 1) x 22+1 matrix (B). We start with
the full listing of all value combinations for nodes 1 and 2 in a 2 x 22 matrix, then add node 3 as described to obtain a (2 + 1) x 22+1 matrix. For
this, we also specify the images 1′, 2′, 3′ to which the three nodes tend for any input combination, using logical parameters. In this simple
example, only node 3 receives multiple inputs and thus has non-trivial logical functions. Removing the states which cannot match their image
state for any valid choice of the logical functions, we obtain two candidates for fixed states. Comparing the states to their image states, we realize
that state (1, 1, 0) is a fixed state if 31 equals 0 (i.e., node 3 is not activated by the presence of its activator 1 alone), whereas state (0, 0, 1) is fixed
if 32 equals 1 (i.e., node 3 is already activated by the absence of its inhibitor 2 alone). Setting both 31 = 0 and 32 = 1 renders both states fixed. Experimental data in agreement with state (1, 1, 0) but not with (0, 0, 1) thus help in estimating the logical functions. A
B A
B A A B Figure 2 For this simplified account of the algorithm described in the text, we analyze a 3-node network (A). We start with the full listing
of all value combinations for nodes 1 and 2 in a 2 x 22 matrix, then add node 3 as described to obtain a (2 + 1) x 22+1 matrix (B). Cycle detection
Preliminaries The system equations direct each state to flow to neigh-
boring states and (in asynchronous mode) neighboring
states differ by the value of at most a single node. The
changing node is set to a value that satisfies the system
equations, which increases the number of content nodes
by one; however, this change will typically have effects
on other nodes, and other discontent nodes can result. If
the state space contains basins of attraction then the sys-
tem will eventually flow towards them. Basins of attrac-
tion signal the existence of fixed states or of stable A stable cycle containing a state with even a moderate
number of discontent nodes is necessarily a long cycle
(state “c” in Figure 3B is 3-discontent); this state has
multiple successors and all of them must be members of
the cycle as well as all their successors, etc. On the other
hand, cycles whose members have very few discontent
nodes are often short. We refer to a k-cycle to indicate
that
its
most
discontent
member
state(s)
is
(are) Page 5 of 10 Page 5 of 10 Page 5 of 10 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361
http://www.biomedcentral.com/1471-2105/14/361 a
b
c
d
e
g
f
a
b
c
e
f
h
g
d
A
B
Figure 3 Analysis of simple and complex cycles. Filled circles
represent states of the system, not to individual nodes. In
asynchronous update mode, two neighboring states differ by the
value of exactly one node. Thus, the shortest possible cycle (0, 0) →
(0, 1) →(1, 1) →(1, 0) →(0, 0) →…, composed of two changing
nodes, has length 4 in state space. A: The cycle “abcd” is fixed only
in the absence of the dashed transition a →g. In the presence of
this transition, the cycle leaks into state g, and the system has only a
single fixed point, g. B: In the cycle “abcd”, here state c is
3-discontent and, hence, can transition to three different successor
states d, e, and f. For the cycle to be fixed, as shown here, all the
successor states of d, e, and f have to return to the cycle. As a
consequence, complex fixed cycles are expected to be rare in
biologically motivated networks. line with the “standard” graph-theoretical approaches,
first finds all parent–child state relationships and then
searches for stable cycles. Cycle detection
Preliminaries A 1-cycle, in which each state mem-
ber has a single successor, is also referred to as a simple
cycle (Figure 3A, in the absence of the a →g transition). Otherwise it is a mixed cycle and in such cycles “branch-
ing” is present (Figure 3B). Simple cycles are necessarily
stable. A typical example is: a 1-cycle that consists of 4
states distinguishable by the 4 combinations (0–0, 0–1,
1–0, and 1–1) of the values of two nodes. The remaining
k-2 nodes in the network are all content and shared by
all the 4 cycle states. k-discontent states. A 1-cycle, in which each state mem-
ber has a single successor, is also referred to as a simple
cycle (Figure 3A, in the absence of the a →g transition). Otherwise it is a mixed cycle and in such cycles “branch-
ing” is present (Figure 3B). Simple cycles are necessarily
stable. A typical example is: a 1-cycle that consists of 4
states distinguishable by the 4 combinations (0–0, 0–1,
1–0, and 1–1) of the values of two nodes. The remaining
k-2 nodes in the network are all content and shared by
all the 4 cycle states. (ii) Search for cycles within the selected set of states:
a. First, identify the basin of attraction of each fixed
state. This is done by “walking uphill” the
collected information on successor/predecessor
relationships, labeling and counting along the way
all states belonging to the current basin. Any
remaining, non-labeled states belong exclusively
to basins of cyclic attractors. In subsequent steps,
the search is restricted to these states (apart from
the basin size calculations). Absence of non-
labeled states clearly implies the absence of cycles
in the selected state set; the search is effectively
terminated at this point. We note that, in principle, there can be cycles that are
long despite the fact that all their states have few discon-
tent nodes. b. Starting from state(s) with the lowest k-value,
look for directed cycles using again the successor/
predecessor data structures, by creating all di-
rected walks in the graph of selected states. If any
such cycle is found then it is checked whether
any of its member states belong to any of the cur-
rently detected basins. If this is the case then the
cycle is discarded as unstable. Cycle detection
Preliminaries Otherwise, check
whether the cycle is “closed”, i.e., whether the
children of all its members are also members of
the cycle. If this is not the case, the cycle either
has members (or successors) belonging to a basin
that was not yet found or it is part of a larger
mixed cycle. At the current step, such a cycle is
simply registered; it will be dealt with at a later
stage. If the cycle is found to be closed, then it is
stable and constitutes an attractor. All its basin b. Starting from state(s) with the lowest k-value,
look for directed cycles using again the successor/
predecessor data structures, by creating all di-
rected walks in the graph of selected states. If any
such cycle is found then it is checked whether
any of its member states belong to any of the cur-
rently detected basins. If this is the case then the
cycle is discarded as unstable. Otherwise, check
whether the cycle is “closed”, i.e., whether the
children of all its members are also members of
the cycle. If this is not the case, the cycle either
has members (or successors) belonging to a basin
that was not yet found or it is part of a larger
mixed cycle. At the current step, such a cycle is
simply registered; it will be dealt with at a later
stage. If the cycle is found to be closed, then it is
stable and constitutes an attractor. All its basin Cycle detection
Preliminaries If and when one is found, it
“creates”
its
basin
of
attraction
via
a
bottom-up
approach. a
b
c
d
e
g
f
a
b
c
e
f
h
g
d
A
B
Figure 3 Analysis of simple and complex cycles Filled circles a
b
c
d
e
g
f
A A B The algorithm goes through two main stages: (i) Determine the appropriate subset of the state space. (i) Determine the appropriate subset of the state space. a. As in the fixed state case, go through iterations
adding a node at a time and keeping at each step
all states up to and including k-discontent states. Each state is labeled by its number of discontent
nodes (in {0, 1, …, k}). The end result is the set of
all these states. The current implementation can
readily handle sets with up to tens of millions
states. Note that all network fixed states are in-
cluded and labeled by zero. Figure 3 Analysis of simple and complex cycles. Filled circles Figure 3 Analysis of simple and complex cycles. Filled circles
represent states of the system, not to individual nodes. In
asynchronous update mode, two neighboring states differ by the
value of exactly one node. Thus, the shortest possible cycle (0, 0) →
(0, 1) →(1, 1) →(1, 0) →(0, 0) →…, composed of two changing
nodes, has length 4 in state space. A: The cycle “abcd” is fixed only
in the absence of the dashed transition a →g. In the presence of
this transition, the cycle leaks into state g, and the system has only a
single fixed point, g. B: In the cycle “abcd”, here state c is
3-discontent and, hence, can transition to three different successor
states d, e, and f. For the cycle to be fixed, as shown here, all the
successor states of d, e, and f have to return to the cycle. As a
consequence, complex fixed cycles are expected to be rare in
biologically motivated networks. b. The predecessors and successors of each selected
state are recorded in appropriate data structures. All states that do not have any of their successors
in the selected set of states (i.e., they flow
“outside”) cannot be assessed and are from now
on excluded. k-discontent states. Benchmarking
F
b
h For benchmarking purposes we generated programmat-
ically a number of sufficiently complex Boolean net-
works that have features similar to known, biologically
relevant examples. As mentioned above, nodes with a
single incoming and a single outgoing edge were ex-
cluded. The ratio of edges to nodes was around 2.5 for
most of the networks (when not then it is stated so) and
no more than 6 incoming edges per node were allowed. We chose to compare lnet to GenYsis and GINsim be-
cause these two packages support both true asynchron-
ous updating and guarantee detection of all cycles (for The tests were performed on a Linux computer with a
Xeon 2.4 GHz processor and 96 GB of physical memory. The GINsim version was 2.4 and it ran on Java 1.5. 40
50
60
70
80
90
100
110
0
5
10
15
20
25
Network nodes (Log2( all states ))
Log2( Enumerated states )
Figure 6 Enumerated states versus the total number of states. This log2-log2 plot clearly demonstrates the significant reduction in the
number of analyzed states computed to all possible network states. 40
50
60
70
80
90
100
110
0
5
10
15
20
25
Network nodes (Log2( all states ))
Log2( Enumerated states ) ous updating and guarantee detection of all cycles (for
Discontent nodes
0
1
8
120
80
30
0
Network nodes
Grey:
lnet + GINsim + GenYsis
Green:
lnet+ GINsim
Burgundy: lnet + GenYsis
Blue:
lnet
Red:
GenYsis
Figure 4 Schematic comparison of the three algorithms. Colored regions indicate attractors that can be analyzed using any
of the three tools GenYsis, GINsim and lnet. For the region in blue,
attractors having at least one discontent node and more than 30
nodes overall, only lnet are applicable. 40
50
60
70
80
90
100
110
0
5
10
15
20
25
Network nodes (Log2( all states ))
Log2( Enumerated states )
Figure 6 Enumerated states versus the total number of states. This log2-log2 plot clearly demonstrates the significant reduction in the
number of analyzed states computed to all possible network states. Discontent nodes
0
1
8
120
80
30
0
Network nodes
Grey:
lnet + GINsim + GenYsis
Green:
lnet+ GINsim
Burgundy: lnet + GenYsis
Blue:
lnet
Red:
GenYsis
Figure 4 Schematic comparison of the three algorithms. Stable cycle finding An iteration process is called which, starting from
an appropriately chosen “seed” cycle, keeps
enlarging it by adding any new states from cycles
with states flowing into it. The iterations continue
until this enlarged cycle becomes “closed”. As
previously, the corresponding basin is determined
and the process continues for any remaining non-
labeled states. The time needed for the exact determination of all
successor/predecessor relationships and for the enumer-
ation of the members of a basin grows linearly with the
number of selected states. This fact is instrumental in
rendering the algorithm fast. Another crucial element is
that the size of the search space is reduced each time an
attractor basin is determined. Figure 5 Dependence of the duration of fixed state searches
on the number of network nodes. Explanation in the main text. networks up to certain size). lnet and GenYsis share a
common input format; for GINsim a compatible version
was generated for all relevant runs. Only the lnet test runs, which involved hundreds of
networks, were automated The GenYsis runs, as seen
below, were limited to smaller-size networks while the
GINsim ones were difficult to automate. Therefore, we
ran just a few of them that still generate sufficiently rep-
resentative results. Stable cycle finding The fixed point search described above is performed in
states that satisfy all the system equations. It cannot be
used to detect a cycle since each member state of a cycle
has to be at least 1-discontent. It follows naturally that
relaxing the above restriction can be used for cycle find-
ing. Namely, a search in the enlarged subspace that in-
cludes all the states up to and including k-discontent
states will result in the detection of all j-cycles, where
j ≤k). Unavoidably, the enumeration of more states re-
sults in a reduction of the size of resolvable networks. The value of k can vary from k = 1 (1-cycle search) to
n (search for all cycles). The number of selected states
grows very fast as k increases. Cycle finding is feasible
only when the number of selected states is manageable
because of memory restrictions. In addition, execution
running times increase fast because the algorithm, in Berntenis and Ebeling BMC Bioinformatics 2013, 14:361
http://www.biomedcentral.com/1471-2105/14/361 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361
http://www.biomedcentral.com/1471-2105/14/361 Page 6 of 10 30
40
50
60
70
80
90
100
110
120
−1
0
1
2
3
Network nodes
log10( Time in seconds )
Figure 5 Dependence of the duration of fixed state searches
on the number of network nodes. Explanation in the main text. states are counted and any non-previously labeled
ones are now labeled. (Note that a state can be-
long to multiple attractors.) This step is repeated
until all registered states have been considered. c. Detected cycles, whose members do not flow
towards identified basins, can still remain. Each
one of them may be part of a larger mixed cycle. An iteration process is called which, starting from
an appropriately chosen “seed” cycle, keeps
enlarging it by adding any new states from cycles
with states flowing into it. The iterations continue
until this enlarged cycle becomes “closed”. As
previously, the corresponding basin is determined
and the process continues for any remaining non-
labeled states. states are counted and any non-previously labeled
ones are now labeled. (Note that a state can be-
long to multiple attractors.) This step is repeated
until all registered states have been considered. until all registered states have been considered. c. Detected cycles, whose members do not flow
towards identified basins, can still remain. Each
one of them may be part of a larger mixed cycle. Benchmarking
F
b
h Colored regions indicate attractors that can be analyzed using any
of the three tools GenYsis, GINsim and lnet. For the region in blue,
attractors having at least one discontent node and more than 30
nodes overall, only lnet are applicable. Discontent nodes
0
1
8
120
80
30
0
Network nodes
Grey:
lnet + GINsim + GenYsis
Green:
lnet+ GINsim
Burgundy: lnet + GenYsis
Blue:
lnet
Red:
GenYsis
Figure 4 Schematic comparison of the three algorithms. Colored regions indicate attractors that can be analyzed using any
of the three tools GenYsis, GINsim and lnet. For the region in blue,
attractors having at least one discontent node and more than 30
nodes overall, only lnet are applicable. Discontent nodes
0
1
8
120
80
30
0
Network nodes
Grey:
lnet + GINsim + GenYsis
Green:
lnet+ GINsim
Burgundy: lnet + GenYsis
Blue:
lnet
Red:
GenYsis Figure 4 Schematic comparison of the three algorithms. Colored regions indicate attractors that can be analyzed using any
of the three tools GenYsis, GINsim and lnet. For the region in blue,
attractors having at least one discontent node and more than 30
nodes overall, only lnet are applicable. Figure 4 Schematic comparison of the three algorithms. Colored regions indicate attractors that can be analyzed using any
of the three tools GenYsis, GINsim and lnet. For the region in blue,
attractors having at least one discontent node and more than 30
nodes overall, only lnet are applicable. Network nodes (Log2( all states )) Figure 6 Enumerated states versus the total number of states. Figure 6 Enumerated states versus the total number of states. This log2-log2 plot clearly demonstrates the significant reduction in the
number of analyzed states computed to all possible network states. Berntenis and Ebeling BMC Bioinformatics 2013, 14:361
http://www.biomedcentral.com/1471-2105/14/361 Page 7 of 10 Page 7 of 10 0 1 2 3 4 5 6 7 8
−1
0
1
2
3
4
30−node net
log10( Time in seconds)
0
1
2
3
4
−1
0
1
2
3
4
40−node net
Discontent nodes
0
1
2
3
−1
0
1
2
50−node net
Figure 7 The number of accessible discontent nodes decreases as the number of nodes grows. Computation time for analyses of
networks of networks of different size, depending on the maximum allowed number of discontent nodes. Benchmarking
F
b
h For larger networks, there is a gradual
reduction of the maximum number of discontent nodes the software can handle. This is also mirrored in an increased use of memory (not
shown). For example, for 40-node networks cycles with up to 4 discontent nodes were accessible. Such cycles contain tens or hundreds of states. Discontent nodes Figure 7 The number of accessible discontent nodes decreases as the number of nodes grows. Computation time for analyses of
networks of networks of different size, depending on the maximum allowed number of discontent nodes. For larger networks, there is a gradual
reduction of the maximum number of discontent nodes the software can handle. This is also mirrored in an increased use of memory (not
shown). For example, for 40-node networks cycles with up to 4 discontent nodes were accessible. Such cycles contain tens or hundreds of states. GenYsis was found to be superior in detecting all fixed
points and all cycles for networks up to approximately 30
nodes. The execution time for the larger ones can reach 1
hour and the memory requirements are minimal. For even
larger networks the execution times were higher than our
testing time (1 hour) and memory utilization was becom-
ing important. These results are in overall agreement with
those of Ferhat et al. [17]. It should be emphasized that
GenYsis by design detects all existing cycles, also covering
cycles with thousands of member states. lnet detects fixed states for networks up to 150 nodes,
detects all cycles for networks up to 20 or so nodes and
detects a subset of the cycles for networks with up to 70
nodes. The limiting factor is available computer memory
because the size of the space state regions enumerated
by lnet grows, inevitably, too large. For cycle detection, up to 20-node networks, lnet’s
performance is equivalent to that of GenYsis. From 20 to
30 nodes it is still the same provided, however, that the
lnet search is restricted to k-discontent states with low k
(the actual value of k depends on the network size). Otherwise, it can be significantly slower, for similar rea-
sons as GINsim. The slowdown can be important in the
absence of attractors with very low k (k = 0 or 1) because
these are always detected first and have their basin states
immediately removed from any downstream consider-
ation. Benchmarking
F
b
h Elucidating further the different performances of
the two algorithms, a 30-node network analysis may take
several hours in GenYsis irrespective of the numbers of
fixed states or cycles (no matter how large). On the
other hand, while lnet may fail to detect a very large
cycle, it will find fixed states and small cycles in sub-
second time. GINsim detects fixed states for networks up to ap-
proximately 120 nodes fast and in a memory-efficient
manner as previously reported by Hinkelman [18]. Being
multithreaded, GINsim took also advantage of our multi-
core testing environment. The execution time for larger
networks was, however, higher than our testing time and
sometimes seemed not to terminate at all. Its perform-
ance when searching for stable cycles was rather poor. It
needs already few minutes to analyze 20-node networks
and fails for even moderately larger networks. The rea-
son is that it attempts exhaustive enumeration of all net-
work states. Here it should be noted that GINsim can
handle multiple-valued networks too. Berntenis and Ebeling BMC Bioinformatics 2013, 14:361
http://www.biomedcentral.com/1471-2105/14/361 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361
http://www.biomedcentral.com/1471-2105/14/361 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361
http://www.biomedcentral.com/1471-2105/14/361 Page 8 of 10 2
4
6
8
10
2
3
4
5
6
7
8
Discontent nodes (30−node network)
log10(States)
Figure 8 Even for cycle searches the numbers of enumerated
states remain low. For a 30-node network with approximately
109.03 states, the number of states with no more than 10 discontent
nodes still remains a small fraction of the state space. 2
4
6
8
10
2
3
4
5
6
7
8
Di
d
(30
d
k)
log10(States) A schematic representation of the comparison of the
three algorithms is shown in Figure 4. The remaining
figures focus exclusively on lnet results. The first series of tests targeted the fixed states of net-
works having nodes ranging from 30 up to 150. For each
one node number analyzed, a set of 100 networks was
generated, subject to the rules mentioned earlier. Then
each one of these networks was analyzed by lnet either
searching exclusively for fixed states or for stable cycles
as well. The computer execution times were recorded
and the results were used to generate the plots shown in
Figures 5, 6, 7, 8 9 and 10. Benchmarking
F
b
h In Figure 5 a semi-log plot of the number of nodes
versus the computer execution time in seconds is shown. Data are not shown for the 130, 140 and 150 cases be-
cause insufficient memory caused termination for 20%,
68% and 68% of them respectively. (For those networks
that were successfully analyzed the time duration trends
were in agreement with the trend shown in the plot.) Discontent nodes (30−node network) Figure 8 Even for cycle searches the numbers of enumerated
states remain low. For a 30-node network with approximately
109.03 states, the number of states with no more than 10 discontent
nodes still remains a small fraction of the state space. Next, as has been already explained, the lnet algorithm
relies on a drastic reduction of the number of enume-
rated states. This becomes evident in Figure 6, where
the enumerated states are plotted versus the total
number of system space states (for fixed state searches
only). Above 30 or so nodes lnet is alone in detecting cycles. For increasing numbers of nodes, we have to decrease
the allowed number of discontent nodes for any enu-
merated state because of limited computer memory. This results in fewer detectable cycles. Recall, however,
that k-cycles with low k values are likely to be the most
relevant ones in Boolean networks representing bio-
logical systems. In Figure 7, the focus is on cycle searches. The 3 plots
show computation times for cycle searches in networks
of 30, 40 and 50 nodes, respectively. Data from higher
number of nodes (up to 70) are not shown but follow
similar trends. 0
1
2
3
4
5
6
7
8
0
1
2
3
4
5
6
Discontent nodes
log10(millisecs)
Network nodes
Figure 9 Overall lnet performance. lnet execution time dependence on network size and number of discontent nodes. Figure 9 Overall lnet performance. lnet execution time dependence on network size and number of discontent nodes. Berntenis and Ebeling BMC Bioinformatics 2013, 14:361
http://www.biomedcentral.com/1471-2105/14/361 Page 9 of 10 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361
http://www.biomedcentral.com/1471-2105/14/361 100 120 140 160 180 200 220 240 260 280 300 320
−1.5
−1.0
−0.5
0.0
0.5
Network edges (50−node networks)
log10( Time in seconds )
Figure 10 Effect of the number of edges on fixed state
searches in 50-node networks. Availability and requirements Figure 8, also concerning cycle searches, shows counts
of system states having equal or less numbers of discon-
tent nodes, for increasing numbers of discontent nodes. For a single 30-node network, states with up to 10 dis-
content nodes still cover less than 6% of the entire state
space. Enumerating this fraction is sufficient to detect
existing cycles with up to a few thousand member states. The lnet executable, a file with instructions on how
to use it and examples of input files are provided in
Additional file 1. Operating system(s): The source code can be com-
piled on Windows, Linux and Apple computers. Programming language: ANSI C Figure 9 summarizes all the lnet results concerning
both fixed states and cycles. Restrictions of use by non-academic users: None Finally, Figure 10 shows the effect of increasing the
number of edges for networks with a fixed number of
nodes (50 nodes were chosen). Benchmarking
F
b
h As the number of edges increases
the median lnet execution time increases only slightly but more
network instances take substantially longer to analyze (outlying points). available memory. With growing network size, even fo-
cusing on small sub-spaces of the entire network state
space becomes prohibitively complex at some point. Programming-inspired optimizations will only lead to
marginal improvements because the state space of a Bool-
ean network doubles in size each time a single node is
added. A promising alternative is to apply first one of the
existing network reduction methodologies and then sub-
mit the resulting network to lnet. Such a scenario should
be able to handle networks having (prior to reduction) up
to few hundred nodes. During the preparation of the manuscript we became
aware of a publication by SQ Zhang et al. [19], where a
fixed state detection algorithm, similar to the one pre-
sented here, is described. The authors also provide a
conceptual outline of a cycle-finding version of their algo-
rithm based on a simple periodicity check which, however,
could detect reliably only simple cycles. We did not test
this algorithm because no sufficient information is avail-
able. Finally, there is no discussion of the role of the num-
ber of discontent nodes in cycle detection. Network edges (50−node networks) Figure 10 Effect of the number of edges on fixed state
searches in 50-node networks. As the number of edges increases
the median lnet execution time increases only slightly but more
network instances take substantially longer to analyze (outlying points). Additional file Additional file 1: Includes the lnet executable (LINUX), a help file
(text format) with detailed instructions on how to run lnet and 4
input network files that are used to illustrate the software in the
help file. Authors’ contributions NB wrote the lnet code and performed the comparison tests. NB and ME
conceived the algorithm and wrote the manuscript. Both authors read and
approved the final manuscript. Key characteristics of the approach are the exact enu-
meration of well-defined regions of the network state
spaces and the faithful reproduction of the correspond-
ing state transition graphs. The result is the detection of
all the attractors residing in these regions. Depending on
the overall fraction of the enumerated subspaces, it may
be possible to deduce information about the size of each
attractor basin. Competing interests
k l
d Competing interests
Nikolaos Berntenis and Martin Ebeling are full-time employees of
F. Hoffmann–La Roche Ltd. Both authors declare that they have no competing interests. p
g
Nikolaos Berntenis and Martin Ebeling are full-time employees of
F. Hoffmann–La Roche Ltd. h
h
d
l
h
h
h Acknowledgements The authors would like to thank Abhishek Garg, Julien Dorier and Ioannis
Xenarios for valuable discussions and Laurent Essioux for his support. Received: 20 March 2013 Accepted: 6 December 2013
Published: 13 December 2013 Received: 20 March 2013 Accepted: 6 December 2013
Published: 13 December 2013 Conclusion We present an algorithm that, based on a build-up ap-
proach, greatly reduces the search space for fixed states
and fixed cycles in Boolean networks. It allows for the
fast and reliable detection of all fixed states in networks
of up to 150 nodes, for an edges-to-nodes ratio of up to
3. Highly ordered cycle attractors, which correspond to
the biologically most relevant cycles, are detected for
networks of approximately half the number of nodes. Berntenis and Ebeling BMC Bioinformatics 2013, 14:361
http://www.biomedcentral.com/1471-2105/14/361 Berntenis and Ebeling BMC Bioinformatics 2013, 14:361
http://www.biomedcentral.com/1471-2105/14/361 3. Thomas R: Boolean formalization of genetic control circuits. J Theor Biol
1973, 42:563–585. 4. Thomas R, Thieffry D, Kaufman M: Dynamical behaviour of biological
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1995, 57:277–295. 6. Thomas R, Richard D’A: Biological Feedback. Boca Raton: CRC Press; 1990. 7. Garg A, Xenarios I, Mendoza L, DeMicheli G: Efficient methods for dyna
analysis of genetic networks and in silico gene perturbation
experiments. Lect Notes Comput Sci 2007, 4453:62–76. 8. Di Cara A, Garg A, De Micheli G, Xenarios I, Mendoza L: Dynamic
simulation of regulatory networks using SQUAD. BMC Bioinformatics 2007,
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(10):1378–1380. 13. Naldi A, Remy E, Thieffry D, Chaouiya C: Dynamically consistent reduction
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biochemical control networks. J Theor Biol 1973, 103:103–129. 1. Kauffman S: Metabolic stability and epigenesis in randomly constructed
genetic nets. J Theor Biol 1969, 22:437–467. Execution times of lnet are comparable to or faster
than other approaches. Its limitation is clearly the 2. Glass L, Kauffman SA: The logical analysis of continuous non-linear
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http://www.biomedcentral.com/1471-2105/14/361 Hinkelmann F: Algebraic theory for discrete models in systems biology,
PhD thesis. Virginia Polytechnic Institute, Mathematics Department;
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Systems Biology 2007, 2007:20180. doi:10.1186/1471-2105-14-361
Cite this article as: Berntenis and Ebeling: Detection of attractors of
large Boolean networks via exhaustive enumeration of appropriate
subspaces of the state space. BMC Bioinformatics 2013 14:361. Submit your next manuscript to BioMed Central
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PENGARUH KEMUDAHAN DAN KUALITAS INFORMASI TERHADAP MINAT MAHASISWA DALAM PENGGUNAAN LAYANAN M-BANKING PADA BANK SYARIAH
|
Jurnal Al-Fatih Global Mulia
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57 Jurnal Al-Fatih Global Mulia PENGARUH KEMUDAHAN DAN KUALITAS INFORMASI
TERHADAP MINAT MAHASISWA DALAM PENGGUNAAN
LAYANAN M-BANKING PADA BANK SYARIAH Nurajizah1, Rika Mustika Sari2
Sekolah Tinggi Ekonomi dan Bisnis Islam (STEBI) Global Mulia Cikarang1,2
ajizahstebi@gmail.com1, rikamustika54@gmail.com2 Volume 5 Issue 1 (2023), Pages 57-70
Jurnal Al-Fatih Global Mulia
pISSN 2580-8036, eISSN 2746-7058
https://jurnalglobalmulia.or.id/index.php/alfatih Volume 5 Issue 1 (2023), Pages 57-70
Jurnal Al-Fatih Global Mulia
pISSN 2580-8036, eISSN 2746-7058
https://jurnalglobalmulia.or.id/index.php/alfatih 1 https://www.bankbsi.co.id/news-update/berita/laba-naik-3318-bsi-siap-berlari-raih-pertumbuhan-berkelanjutan.
Diakses tanggal 25 agustus 2022 Abstrak Perkembangan teknologi pada era globalisasi yang berkembang pesat ini dapat menarik
minat para nasabah yang digunakan sebagai fasilitas pendukung pada aktivitas seluruh lapisan
masyarakat. Salah satu teknologi informasi pada perbankan syariah yaitu layanan m-banking
yang merupakan pengembangan dari dua bentuk inovasi bank syariah sebelumnya yaitu sms
banking dan internet banking. Apabila minat telah muncul pada diri calon nasabah, maka
diharapkan calon nasabah dapat memutuskan untuk menggunakan layanan m-banking. Tujuan
dari penelitian ini adalah untuk mengetahui tingkat pengaruh kemudahan dan kualitas informasi
terhadap minat dalam penggunaan layanan mobile banking pada bank syariah secara partial
maupun simultan. Penelitian ini menggunakan metode kuantitatif, data yang digunakan adalah
data primer dan sekunder. Teknik pengumpulan data menggunakan teknik sampling rumus
slovin dengan 93 responden. Analisis data menggunakan analisis regresi linier berganda dan
menggunakan uji validitas, uji reliabilitas, uji asumsi klasik dan uji hipotesis. Pengolahan data
menggunakan program SPSS Versi 25.0 for windows. Hasil penelitian ini dapat disimpulkan
bahwa secara partial variabel kemudahan berpengaruh secara signifikan terhadap minat
mahasiswa dalam penggunaan layanan m-banking pada bank syariah dengan nilai t hitung 4,058
> 1,986 t tabel (df=90) dan nilai sig 0,000 < 0,05. Dan variabel kualitas informasi berpengaruh
secara signifikan terhadap minat mahasiswa dalam penggunaan layanan m-banking pada bank
syariah dengan nilai t hitung 4,570 > 1986 dan nilai sig 0,000 < 0,05. Selanjutnya pengaruh
secara simultan dengan uji F menunjukan bahwa kemudahan dan kualitas informasi
berpengaruh bersama-sama secara signifikan terhadap minat mahasiswa dalam penggunaan
layanan m-banking pada bank syariah dengan nilai F hitung sebesar 122,177 > 3,10. Berdasarkan uji koefisien determinasi menunjukan bahwa pengaruh minat terhadap kemudahan
dan kualitas informasi sebesar 73,1%, sisanya sebesar 26,9% dijelaskan oleh variabel diluar
variabel yang diteliti. Kata Kunci: Kemudahan, Kualitas Informasi, Minat Penggunaan Kata Kunci: Kemudahan, Kualitas Informasi, Minat Penggunaan Abstract Technological developments in this rapidly growing era of globalization can attract the
interest of customers who are used as supporting facilities for activities of all levels of society. One of the information technology in Islamic banking is m-banking service which is the
development of the two previous forms of innovation in Islamic banking, namely SMS banking
and internet banking. If interest has arisen in the prospective customer, it is hoped that the
prospective customer can decide to use m-banking services. The purpose of this study was to
determine the level of influence of the ease and quality of information on interest in the use of
mobile banking services at Islamic banks partially or simultaneously. This study uses
quantitative methods, the data used are primary and secondary data. The data collection
technique used the Slovin formula sampling technique with 93 respondents. Data analysis used
multiple linear regression analysis and used validity test, reliability test, classical assumption
test and hypothesis test. Data processing using SPSS Version 25.0 for windows. The results of
this study can be concluded that partially the convenience variable has a significant effect on
student interest in using m-banking services at Islamic banks with a t-count value of 4.058 >
1.986 t table (df=90) and a sig value of 0.000 <0.05. And the information quality variable has
a significant effect on student interest in using m-banking services at Islamic banks with a t-
count value of 4.570 > 1986 and a sig value of 0.000 <0.05. Furthermore, the simultaneous
effect with the F test shows that the ease and quality of information have a significant effect on
student interest in using m-banking services at Islamic banks with a calculated F value of
122.177 > 3.10. Based on the coefficient of determination test, it shows that the influence of
interest on the ease and quality of information is 73.1%, the remaining 26.9% is explained by
variables outside the variables studied. Keywords: Ease, Information Quality, Interest in Use 57 Jurnal Al-Fatih Global Mulia Pengaruh Kemudahan dan Kualitas Informasi... PENDAHULUAN Perkembangan teknologi pada era globalisasi yang berkembang pesat ini dapat
digunakan sebagai fasilitas pendukung yang digunakan pada aktifitas seluruh lapisan
masyarakat. Saat ini teknologi tidak hanya hadir dalam kehidupan masyarakat, bahkan dapat
dikatakan sebagai kebutuhan primer sebagian besar kelas sosial. Kemajuan dan perkembangan
teknologi informasi yang pesat telah mempengaruhi industri perbankan, seperti halnya m-
banking merupakan salah satu wujud dari perkembangan teknologi tersebut. Alat komunikasi
merupakan salah satu contoh perkembangan teknologi yang saat ini digunakan dalam industri
perbankan. Kehadiran Bank Syariah Indonesia (BSI) telah diresmikan sejak 01 Februari 2021
lalu. BSI melaporkan bahwa pengguna mobile banking-nya sekarang telah menembus 2,5 juta
orang. Kemudian, hingga juni 2021 nilai transaksi kanal digital BSI mencapai Rp. 95,13 triliun,
dengan kontribusi terbesar berasal dari layanan BSI Mobile yang naik 83,56% dari tahun ke
tahun.1 Berdasarkan data - data yang didapat dari Bank Syariah Indoneisa terkait pengguna m-
banking pada tahun 2021 tentunya bank syariah bersaing dalam hal peningkatan penggunaan
layanan m-banking, namun pada kenyataan yang terjadi pada mahasiswa program studi
Perbankan Syariah STEBI Global Mulia Cikarang hal ini berbanding terbalik. Terbukti dengan
tidak banyaknya mahasiswa perbankan syariah STEBI Global Mulia Cikarang yang
mempunyai rekening Bank Syariah sehingga jarang di temui mahasiswa perbankan syariah 58 Jurnal Al-Fatih Global Mulia Nurajizah1, Rika Mustika Sari2 yang memanfaatkan layanan m-banking bank syariah. Pertanggungjawaban keilmuan yang
sudah sepantasnya dilakukan oleh mahasiswa perbankan syariah yang mempraktekan semua
teori yang telah dipelajari salah satunya yaitu mempunyai rekening perbankan syariah. Atas
bekal ilmu-ilmu yang di dapat semasa kuliah seperti pengetahuan pada dunia perbankan syariah
mengenai terbebasnya dari riba, tentu mahasiswa perbankan syariah mempunyai kesadaran
untuk ikut andil pada dunia perbankan syariah. Namun fakta yang terjadi, mahasiswa perbankan
syariah telah mempunyai rekening perbankan syariah atas dasar keterpaksaan dari mata kuliah
yang menganjurkan untuk membuka rekening di bank syariah. karena dirasa mahasiswa
perbankan syariah ini mempunyai banyak pengetahuan dalam dunia perbankan syariah. Dengan
banyaknya bekal ilmu serta kesadaran yang dimiliki bahwa menggunakan bank syariah
dianggap lebih baik, maka penerapannya salah satunya yaitu sudah seharusnya mahasiswa
perbankan syariah mempunyai rekening bank syariah. 2 Jogiyanto. (2007). Sistem Teknologi Keperilakuan, (Yogyakarta: Andi), hal. 111 PENDAHULUAN Dengan mempunyai rekening perbankan
syariah, tentu hal ini akan memicu mahasiswa untuk menggunakan layanan m-banking
sehingga transaksi yang dilakukan akan lebih efektif dan efisien Sebagai lembaga intermediasi, pihak perbankan memang sudah seharusnya memberikan
kualitas layanan yang lebih baik kepada nasabahnya, seperti dalam jasa layanan mobile
banking, sehingga perhatian bank tidak hanya terbatas dari pelayanan secara tatap muka pada
nasabah, tetapi juga pada aspek pelayanan dari jarak jauh melalui m-banking. Dimana hal ini
selain untuk memberikan kemudahan bagi nasabah, juga menghemat waktu bagi mahasiswa
yang ingin melakukan pembayaran SPP melalui m-banking. Oleh karena itu jika kedua m-
banking tersebut memiliki kualitas layanan yang baik, tentu akan mendorong terciptanya
publisitas public kepada calon-calon nasabah potensial, dan pada akhirnya akan menciptakan
nasabah-nasabah yang setia bagi kedua bank. Kemudahan didefinisikan sebagai sejauh mana seseorang percaya bahwa menggunakan
suatu teknologi akan bebas dari usaha. Yang mana dapat memudahakan suatu pekerjaan dari
setiap penggunanya. Dirasa dapat menghemat tenaga, biaya dan waktu atas penggunaan objek
tertentu. Meskipun pendapat seseorang berbeda-beda mengenai kemudahan, salah satunya
kemudahan dalam penggunaan layanan m-banking, dengan adanya aplikasi ini dapat
memudahkan setiap transaski perbankan. Kemudahan penggunaan (easeof use) adalah sebagai
tingkatan di mana seseorang percaya bahwa Teknologi Informasi (TI) dapat dengan mudah
dipahami. Kualitas informasi adalah fitur perlengkapan untuk interaktivitas nasabah, fitur
merupakan kriteria penting yang menarik perhatian nasabah di dalam penyampaian jasa mobile
banking. Informasi merupakan hasil pengolahan data yang memberikan arti dan manfaat. Kualitas informasi adalah tingkat dimana informasi memiliki karakteristik isi, bentuk, dan
waktu, yang memberikannya nilai bagi para pemakai akhir tertentu. Sehingga dapat dikatakan
bahwa baik buruknya suatu informasi dapat dinilai melalui kualitas informasi yang didapat,
semakin baik kualitas informasi yang didapat akan memberikan kesan positif bagi para
pengguna teknologi informasi Teori TAM (Technology Acceptance Model) Technology Acceptance Model (TAM) merupakan perilaku yang pada umumnya
digunakan untuk mejelaskan penerimaan individual terhadap penggunaan sistem teknologi. 4
dimana Teori Technology Acceptance Model (TAM) yang dikemukakan oleh (Davis, 1989)5,
TAM berpendapat bahwa suatu penerimaan individual terhadap sistem teknologi informasi
telah ditentukan oleh dua konsep yang telah diamati. Dua konsep utama ini adalah kegunaan
persepsian (perceived usefulness) dan kemudahan penggunaan persepsian (perceived ease of
use). )
Menurut Jogiyanto teori ini memiliki beberapa kelebihan, yaitu:6 g y
p
y
a. TAM merupakan model perilaku (behavior) yang bermanfaat untuk menjawab pertanyaan
mengapa banyak sistem teknologi informasi yang gagal diterapkan karena pemakaiannya
tidak mempunyai minat (intention) untuk menggunakannya. Tidak banyak model-model
penerapan sistem teknologi informasi yang memasukkan faktor psikologis atau perilaku
(behavior) di dalam modelnya dan TAM adalah salah satu model untuk
mempertimbangkannya. b. TAM dibangun dengan dasar teori yang kuat. c. TAM telah diuji dengan banyak penelitian dan hasilnya sebagian besar mendukung dan
menyimpulkan bahwasannya TAM merupakan model penelitian yang baik. c. TAM telah diuji dengan banyak penelitian dan hasilnya sebagian besar mendukung dan
menyimpulkan bahwasannya TAM merupakan model penelitian yang baik. y
p
y
p
p
y
g
d. TAM merupakan model persimoni (persimonious) yaitu model sederhana tetapi valid. Kualitas Informasi d. TAM merupakan model persimoni (persimonious) yaitu model sederhana tetapi valid. Kualitas Informasi
M d l T
i D L
& M L Model Teori DeLone & McLean Model Teori DeLone & McLean Model DeLone dan McLean digunakan untuk mengukur keberhasilan sistem informasi,
model ini disebut Sederhana namun dianggap sangat efektif oleh para peneliti (DeLone.W.H &
McLean. E.R., 1992).7 D&M Model membuat teori Penelitian teoritis dan empiris berdasarkan
sistem informasi yang dibuat oleh para peneliti di sekitarnya 1970-an dan 1980-an. Bahwa Informasi merupakan hasil pengolahan data yang memberikan arti dan manfaat. Kualitas informasi dalah sejauh mana informasi memiliki isi, bentuk, dan karakteristik temporal
yang memberikan nilai kepada pengguna akhir tertentu. Sehingga dapat dikatakan bahwa baik
buruknya suatu informasi dapat dinilai melalui kualitas informasi yang didapat, semakin baik
kualitas informasi yang didapat akan memberikan kesan positif bagi para pengguna teknologi
informasi.8 Menurut D&M Model Kualitas dari suatu informasi mencangkup indikator seb
9 Menurut D&M Model Kualitas dari suatu informasi mencangkup indikator sebagai
berikut:9 Menurut D&M Model Kualitas dari suatu informasi mencangkup indikator
berikut:9 a. Akurat Akurat artinya informasi harus bebas dari kesalahan dan tidak lazim atau
menyesatkan. Akurat juga berarti bahwa informasi harus secara jelas mencerminkan
maksud dan tujuan. 3 Sauca Ananda Pranidana, “Analisis Faktor-Faktor yang Mempengaruhi Nasabah Bank BCA untuk Menggunakan
Klik-BC”
4 Hamzah, 2009, “Penerimaan Teknologi Pengguna Terhadap Sistem Informasi Dengan Metode Technology
Acceptance Model”. Jurnal Pilar Nusa Mandiri Vol. IX No.2.Hal 172
5 Davis, F.D. 1989. Perceived usefulness, perceived ease of use, and user acceptance of information technology.
MIS Quarterly. 13(3): 319-339
6 Jogiyanto. (2007). Sistem Teknologi Keperilakuan, (Yogyakarta: Andi),
7 DeLone.W.H & McLean. E.R., 1992. Informatiom System Success: The Quest For The Dependent Variable.
Pp.60-95.
8 Azhar Susanto, Sistem Informasi Manajemen:Konsep dan Pengembangannya, (Bandung: Linggar Jaya, 2004),
hal. 40.
9 Azhar Susanto, Sistem Informasi Manajemen:Konsep dan Pengembangannya, (Bandung: Linggar Jaya, 2004),
hal. 43. Kemudahan Kemudahan diartikan sampai mana seseorang merasa yakin dan percaya jika
menggunakan suatu teknologi akan bebas dari usaha.2 Yang mana dapat memudahakan suatu
pekerjaan dari setiap penggunanya. Dianggap dapat menghemat tenaga, biaya dan waktu atas
penggunaan objek tertentu. Meskipun pendapat seseorang berbeda-beda mengenai kemudahan,
salah satunya kemudahan dalam penggunaan layanan mbanking, dengan adanya aplikasi ini
dapat memudahkan setiap transaksi perbankan. Pranidana berpendapat bahwa “Kemudahan 59 Jurnal Al-Fatih Global Mulia Pengaruh Kemudahan dan Kualitas Informasi... penggunaan (ease of use) adalah sebagai tingkatan dimana seseorang percaya bahwa teknologi
informasi (TI) dapat dengan mudah dipahami”.3 penggunaan (ease of use) adalah sebagai tingkatan dimana seseorang percaya bahwa teknologi
informasi (TI) dapat dengan mudah dipahami”.3 3 Sauca Ananda Pranidana, “Analisis Faktor-Faktor yang Mempengaruhi Nasabah Bank BCA untuk Menggunakan
Klik-BC” 10 Slameto, 2010, “Belajar & Faktor-Faktor yang mempengaruhi”, (Jakarta:PT Rineka Cipta)
11 Jogiyanto. (2007). Sistem Teknologi Keperilakuan, (Yogyakarta: Andi)
12 Undang-Undang Nomor 21 tahun 2008 tentang perbankan syariah ayat 1 Teori TAM (Technology Acceptance Model) Informasi harus akurat karena dapat terjadi banyak gangguan (noise) a. Akurat Akurat artinya informasi harus bebas dari kesalahan dan tidak lazim atau
menyesatkan. Akurat juga berarti bahwa informasi harus secara jelas mencerminkan
maksud dan tujuan. Informasi harus akurat karena dapat terjadi banyak gangguan (noise) 60 Jurnal Al-Fatih Global Mulia Nurajizah1, Rika Mustika Sari2 yang dapat mengubah atau merusak informasi dari sumber informasi ke penerima
informasi. yang dapat mengubah atau merusak informasi dari sumber informasi ke penerima
informasi. b. Tepat pada waktunya Tepat waktu artinya informasi yang sampai pada penerima tidak
boleh terlambat. Informasi yang terlupakan tidak akan lagi memiliki nilai karena
informasi merupakan dasar pengambilan keputusan. Keputusan untuk datang terlambat
dapat berakibat fatal bagi organisasi atau asosiasi. Cepatnya teknologi yang harus didapat
itu disebabkan tingginya nilai informasi, sehingga dibutuhkan teknologi-teknologi yang
modern untuk mendapatkan, mengolah dan mengirimkannya. Sehingga ketepatan waktu
pada sistem teknologi informasi sangat diperlukan. c. Relevan Relevan berarti informasi tersebut memiliki manfaat untuk penggunanya. Relevansi informasi untuk tiap-tiap orang dari satu dengan yang lainnya berbeda-beda. Misalnya informasi mengenai sebab kerusakan mesin produksi kepada akuntan
perusahaan adalah kurang relevan dan akan lebih relevan apabila ditunjukkan kepada ahli
teknik perusahaan. Sehingga informasi tepat sasarannya. d. Lengkap Lengkap artinya informasi yang diberikan harus diterima sepenuhnya oleh
penerima. Informasi yang diterima tidak boleh setengahsetengah. sehingga penerima
informasi dapat menggunakan informasi tersebut semaksimal mungkin. Informasi yang
lengkap akan memberikan dampak positif bagi penggunanya. Ketika informasi dapat
memuat semua indikator di atas, maka kualitas informasi dapat dikatakan baik dan layak
untuk digunakan. Dalam lingkup ini, kualitas informasi yang dimaksud adalah semua
informasi yang dimuat dalam layanan m-banking khususnya pada Bank Syariah. Minat Minat merupakan sutau keinginan perasaan yang dimiliki setiap individu tentu berbeda-
beda, hal ini dijadikan pengaruh dalam terbentuknya minat terhadap sesuatu. Hal ini tentu akan
menentukan seseorang tersebut aktif tidaknya dalam melakukan suatu pekerjaan dan dalam
situasi kondisi apapun. Perasaan senang maupun sedih akan mempengaruhi minat dari individu
itu sendiri. Selain itu, minat juga akan mempengaruhi sesuatu yang dilihat atau diamati. Hal ini
tentu ada hubungannya dengan keinginan atau kebutuhan dari individu itu sendiri. Ada
beberapa pendapat minat menurut ahli a. Minat adalah suatu rasa lebih suka dan rasa karakteristik pada suatu hal atau aktivitas,
tanpa ada yang menyuruh. Minat pada dasarnya adalah menerima hubungan antara diri
sendiri dengan hal-hal eksternal. Semakin kuat atau dekat hubungannya, semakin besar
minatnya.10 y
b. Minat perilaku (behavioral intention) merupakan kecenderungan untuk mendorong
seseorang malakukan suatu tindakan. 11 b. Minat perilaku (behavioral intention) merupakan kecenderungan untuk mendorong
seseorang malakukan suatu tindakan. 11 c. Menurut Salahudin Minat dapat diartikan suatu keadaan dimana apabila seseorang melihat
ciri-ciri atau arti sementara situasi yang dihubungkan dengan keinginan atau kebutuhan
sendiri. minat sebagai perhatian yang mengandung unsur perasaan. B
k S
i h c. Menurut Salahudin Minat dapat diartikan suatu keadaan dimana apabila seseorang melihat
ciri-ciri atau arti sementara situasi yang dihubungkan dengan keinginan atau kebutuhan
sendiri. minat sebagai perhatian yang mengandung unsur perasaan. g y
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12 Undang-Undang Nomor 21 tahun 2008 tentang perbankan syariah ayat 1 11 Jogiyanto. (2007). Sistem Teknologi Keperilakuan, (Yogyakarta: Andi) 10 Slameto, 2010, “Belajar & Faktor-Faktor yang mempengaruhi”, (Jakarta:PT Rineka Cipta) Jogiyanto. (2007). Sistem Teknologi Keperilakuan, (Yogyakarta: Andi)
12 Undang-Undang Nomor 21 tahun 2008 tentang perbankan syariah ayat 1 Bank Syariah Menurut Pasal 1 UU Nomor 21 Tahun 200812, bank adalah badan usaha yang
menghimpun dana masyarakat dalam bentuk simpanan dan menyalurkannya kepada
masyarakat dalam bentuk kredit atau lainnya untuk menaikkan standar kehidupan masyarakat
luas. Bank memiliki dua jenis, yaitu bank konvensional dan bank syariah. Bank Konvensional
adalah bank yang melakukan bisnis dengan cara Konvensional. Bank Konvensional meliputi
Bank Umum Konvensional (BUK) dan Bank Perkreditan Rakyat (BPR). Bank syariah adalah
bank yang menjalankan usaha sesuai dengan prinsip syariah. Bank syariah antara lain Bank 61 Jurnal Al-Fatih Global Mulia Pengaruh Kemudahan dan Kualitas Informasi... Umum Syariah (BUS) dan Bank Pembiayaan Rakyat Syariah (BPRS). Prinsip dasar syariah
adalah dasar hukum islam. Berlandaskan penetapan fatwa dibidang syariah, lembaga memiliki
kewenangan dan kebijakan dalam kegiatan perbankan.13 BUS merupakan bank syariah yang kegiatannya menyediakan jasa pembayaran. BPRS
adalah bank syariah yang menjalankan kegiatan usahanya dan tidak memberikan jasa lalu lintas
pembayaran. Unit Usaha Syariah (UUS) adalah unit kerja kantor pusat Bank Umum
Konvensional, sebagai kantor utama dari kantor atau unit yang melakukan kegiatan usaha sesuai
dengan prinsip syariah, atau sebagai unit kerja kantor cabang. Bank yang melakukan kegiatan
usaha di luar negeri, biasanya sebagai kantor pusat cabang unit usaha syariah. g
y
g
p
g
y
Di dalam Pasal 2 UU Nomor 21 Tahun 2008, kegiatan usaha pada perbankan syariah
diatur berdasarkan prinsip syariah, demokrasi ekonomi, dan prinsip kehati-hatian. Selanjutnya
mengenai tujuan Perbankan Syariah, Pasal 3 Undang-Undang No 21 Tahun 2008, menyatakan
bahwa Perbankan Syariah bertujuan untuk mendukung terselenggaranya pembangunan
nasional dalam rangka meningkatkan keadilan, kebersaman dan pemerataan masyarakat. Sejarah Berdirinya Bank Syariah di Indonesia Lahirnya Bank Syariah pertama di Indonesia yaitu terbentuknya PT Bank Muamalat
Indonesia (BMI) yang merupakan hasil kerja tim perbankan MUI yang berdirinya
ditandatangani pada tanggal 1 November 1991. Untuk saat ini BMI sudah memiliki beberapa
cabang di kota besar seperti Jakarta, Surabaya, Bandung, Makassar, dan beberapa kota-kota
besar lainnya.14 Di Indonesia, Bank Syariah yang pertama kali didirikan pada tahun 1992 adalah bank
Muamalah Indonesia. Walaupun terbilang lambat dalam perkembangannya tetapi jika
dibandingkan dengan negara-negara muslim yang ada di dunia, perbankan syariah di Indonesia
akan terus berkembang dan bersaing dalam dunia perbankan negara lainnya. Bank Syariah
hanya memiliki satu unit bank pada periode tahun 1992-1998, kemudian pada tahun 2004 telah
bertambah menjadi 20 unit bank syariah di Indonesia, ditahun 2005 bertambah 88 unit bank
syariah, terakhir menurut data OJK pada tahun 2016 jumlah Bank Syariah di Indonesia
mencapai 166 unit. p
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14 Heri Sudarsono, Bank dan Lembaga Keuangan Syariah (Yogyakarta: Ekonisia, 2008), h.30 g
j
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)
16 Ikatan Bankir Indonesia (IBI) dan Lembaga Sertifikasi Profesi Perbankan (LSPP), Mengenal Operasional
Perbankan 1, (Jakarta: PT Gramedia Pustaka Utama, 2014), hal. 59. 13 Rizal Yaya, Aji Erlangga Martawireja, Ahim Abdurahim, Akuntansi Perbankan Syariah Teori Dan Praktik
Kontemporer (Jakarta: Salemba Empat, 2016), hal.48. g
g
y
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15 Otoritas Jasa Keuangan, 2015, Bijak Ber-Electric Banking. (Jakarta:OJK) 14 Heri Sudarsono, Bank dan Lembaga Keuangan Syariah (Yogyakarta: Eko Kelebihan m-banking g
1. Mobile banking menawarkan beberapa jasa-jasa yang cukup menarik untuk diminat 2. Selain bisa menghemat waktu, m-banking juga bisa menghemat biaya. 3. Akses nasabah dengan bank bisa dilakukan dengan jasa-jasanya kapanpun dan
dimanapun. 4. Nasabah bisa mengetahui jika telah terjadi penarikan uang bukan dari nasabah itu
sendiri dan bisa langsung melakukan pemblokiran ATM. 1. Strategi kompetitif dari pihak bank pada layanan m-banking bisa memberikan nilai
tambah kepada nasabahnya. 2. Biaya pengurusan nasabah dapat berkurang. 18 Kekurangan M-banking18
a. Jenis OS handphone Kekurangan M-banking18 a. Jenis OS handphone c. Jenis kartu yang digunakan d. Kecepatan data/jaringan suatu wilayah e. batasan limit transaksi dalam sehari f. Aktifasi setiap bulan g. gangguan pada sistem layanan m-banking Bank Syariah Ini menunjukkan bahwa Bank Indonesia mampu bersaing dengan bank-
bank konvensional, dan semakin tahun mengalami kemajuan yang lumayan pesat.15
Tujuan Berdirinya Bank Syariah Setelah mengetahui tentang suatu bank konvensional yang dirasa mengalami kegagalan
menjalani suatu peran utamanya yang menghubungkan pemilik modal dengan pihak yang
memerlukan dana, maka Bank Syariah dibentuk untuk tujuan sebagai berikut.16 a. Untuk mengarahkan suatu kegiatan ekonomi dalam bermuamalah secara syariat islam,
seperti terhindarnya dari praktek riba dalam dunia perbankan. b. Kegiatan Investasi dibuat untuk meratakan pendapatan dan menciptakan suatu kead
dibidang ekonomi. b. Kegiatan Investasi dibuat untuk meratakan pendapatan dan menciptakan suatu keadilan
dibidang ekonomi. d
Untuk peningkatan taraf hidup umat muslim peluang usaha yang besar akan dibuka untuk d. Untuk peningkatan taraf hidup umat muslim, peluang usaha yang besar akan dibuka u
mereka kelompok miskin agar diarakahkan ke berwirausaha. e. Untuk mengurangi tingkat kemiskinan, merupakan program utama dari negara-ne
muslim yang sedang berkembang. f. Untuk menjaga keadaan yang stabil pada ekonomi/moneter pemerintah. Diharap
mampu menghindari inflasi akibat penerapan sistem bunga pada aktivitas bank syari p
g
p
p
g p
y
g. Untuk mengurangi minat umat muslim menjadi nasabah pada Bank Konvensional. 62 Jurnal Al-Fatih Global Mulia Nurajizah1, Rika Mustika Sari2 17 Syamsul Hadi Novi, “Faktor-Faktor Yang Mempengaruhi Layanan Mobile Banking”, Universitas Islam
Indonesia, h. 58. M-Banking Mobile banking atau yang sering disebut m-banking ialah suatu layanan perbankan yang
dapat diakses melalui handphone GSM (Global for Mobile Communication). m-banking adalah
suatu layanan perbankan yang menerapkan suatu teknologi informasi.17 Layanan perbankan ini
akan menjadikan peluang bagi bank untuk menawarkan nilai tambah kepada pengguna atau
nasabah. M-banking juga memberikan suatu layanan untuk mendukung kelancaran dan
kemudahan dalam aktivitas perbankan. Keefektifan dan keefisienan nasabah yang akan
melakukan berbagai transasksi di perbankan jika tidak memiliki fasilitas pendukung seperti
handphone dan internet akan menjadi kesulitan bagi nasabah. Jika setiap pengguna memiliki
handphone dan internet maka mereka bisa bertansaksi dimanapun dan kapanpun. Ada beberapa
jenis transaksi Mobile banking, sebagai berikut: 1. Mengetahui informasi saldo, Transfer uang, mutasi rekening, informasi nilai tukar. 2. Pembayaran (kartu kredit, PLN, telepon, handphone,listrik, asuransi) 3. Pembelian (pulsa isi ulang, saham). Kegiatan transaksi mobile banking bisa dilakukan dimana saja dan kapan saja, salah
satu keuntungan diperolehnya nasabah selain menghemat waktu, yaitu nasabah juga bisa
mengontrol rekening dan melakukan transaksi perbankan lainnya dengan cara menggunakan
ponsel. Habsari, wawancara dengan penulis, Bank Syariah Mandiri KC Bandar Jaya, Lampung, 31 Mei 2018. 18 Santi Habsari, wawancara dengan penulis, Bank Syariah Mandiri KC Bandar Jaya, Lampung, 31 Mei 2018. 19 Sarwono Jonathan, Rumus-Rumus Populer dalam SPSS 22 untuk Riset Skripsi, Yogyakarta: C.V Andi
Offset, 2015. hal. 101. METODE PENELITIAN Penelitan ini menggunakan metode kuantitatif. Jenis dan sumber data menggunakan
data primer dan data sekunder. Populasi yang diambil adalah mahasiswa angkatan 2018-2021
program studi perbankan syariah STEBI Global Mulia Cikarang. Dengan teknik pengambilan
sampling menggunakan rumus slovin yang di dapat yaitu 93 responden dari 120 mahasiswa. Teknik pengumpulan data pada penelitian ini menggunakan metode perpustakaan dan
kuesioner dengan instrumen pengukuran menggunakan skala likert. 63 Jurnal Al-Fatih Global Mulia Pengaruh Kemudahan dan Kualitas Informasi... Peneliti ini menggunakan metode analisis regresi linier berganda dengan menggunakan
program komputer yaitu Software Statistical Package For The Social Science (SPSS) versi 25.0. Penerapan metode ini akan menghasilkan tingkat hubungan antara variabel-variabel bebas
(independen) terhadap variabel terkaitnya (dependen) serta arah hubungan yang terjadi
(hubungan negative atau positive). Yaitu dengan menggunakan Uji Validitas, Uji Reliabilitas
Uji Asumsi klasik meliputi uji normalitas, uji multikolinearitas dan uji heteroskedastisitas, lalu
juga menggunakan uji hipotesis meliputi uji t (partial), uji F (simultan),uji koefisien determinasi
dan menggunakan uji anaisis regresi linier berganda. adapun persamaan regresinya adalah
sebagai berikut : Y= a + b₁X₁ + b₂X₂ + e
Keterangan :
Y
= Variabel terikat (minat penggunaan layanan m-banking)
a, b₁, b₂ = Bilangan Konstanta
X₁
= Kemudahan
X₂
= Kualitas Informasi
e
= Residual Eror dari masing-masing variabel HASIL DAN PEMBAHASAN 20 Paturohman, I. (2019). SPSS 25 Main Subject: Linear Regression Analysis. Bandung. HASIL DAN PEMBAHASAN 1. Hasil Uji Validitas 1. Hasil Uji Validitas Korelasi product moment pearson merupakan pengukuran parametik, akan
menghasilkan koefisien yang berfungsi untuk mengukur kekuatan hubungan linier antara
dua variabel, jika hubungan dua variabel tidak linier, maka koefisien korelasi
pearson tersebut tidak mencerminkan kekuatan hubungan dua variabel yang sedang
diteliti, meski dua variabel mempunyai hubungan kuat.19 Tabel 1
Hasil Koefisien Pearson Product Moment
No Pertanyaan
Pearson Correlation
Sig. Keterangan
Kemudahan (X₁)
1
0,842
0,000
Valid
2
0,850
0,000
Valid
3
0,841
0,000
Valid
4
0,789
0,000
Valid
5
0,774
0,000
Valid
6
0,762
0,000
Valid
7
0,877
0,000
Valid
8
0,729
0,000
Valid
Kualitas Informasi (X₂)
1
0,790
0,000
Valid
2
0,706
0.000
Valid
3
0,714
0,000
Valid
4
0,831
0,000
Valid
5
0,824
0,000
Valid
6
0,859
0,000
Valid
7
0,749
0,000
Valid
8
0,732
0,000
Valid 64 Jurnal Al-Fatih Global Mulia Nurajizah1, Rika Mustika Sari2 Minat Penggunaan (Y)
1
0,811
0,000
Valid
2
0,581
0,000
Valid
3
0,729
0,000
Valid
4
0,858
0,000
Valid
5
0,819
0,000
Valid
6
0,852
0,000
Valid
7
0,865
0,000
Valid
8
0,861
0,000
Valid
9
0,793
0,000
Valid
Sumber : Hasil Pengelolahan SPSS 2022 Berdasarkan Tabel 1 menunjukan bahwa seluruh variabel mempunyai 25 item
pernyataan yang diberikan kepada 93 responden mempunyai nilai r hitung yang lebih besar
dari r tabel yaitu 0.2039 dan nilai signifikansinya lebih kecil dari 0,05, sehingga dapat
dikatakan bahwa semua indikator pertanyaan pada variabel penelitian adalah valid. 2. 2. Hasil Uji Reliablitas Hasil Uji Reliablitas
Tabel 2
Hasil Uji Reliabilitas
Variabel
Alpha Cronbanch’s
Kesimpulan
Kemudahan
0,924
Reliabel
Kualitas Infromasi
0,905
Reliabel
Minat Penggunaan
0,925
Reliabel
Sumber : Hasil Pengelolahan SPSS 2022 Tabel 2 menunjukan bahwa nilai cronbach’s alpha atas variabel kemudahan sebesar
0,924, kualitas informasi sebesar 0,905 dan minat penggunaan sebesar 0,925. Dengan
demikian, dapat disimpulkan bahwa pernyataan dalam kuesioner ini reliabel karena
mempunyai nilai cronbach’s alpha > 0,60. Hal ini menunjukan bahwa setiap item pernyataan
yang digunakan akan mampu memperoleh data yang konsisten berarti bila pernyataan itu
diajukan kembali akan diperoleh jawaban yang relatif sama dengan jawaban yang
sebelumnya. 65 Jurnal Al-Fatih Global Mulia 3. Uji Asumsi Klasik a. Uji Normalitas
Uji normalitas digunakan untuk menguji apakah dalam model regresi, data variabel
dependen dan independen yang dipakai terdistribusi normal atau tidak. Sebuah data
penelitian yang baik adalah yang datanya berdistribusi normal (Ghozali, 2016). Pada
penelitian ini uji normalitas menggunakan metode analisa grafik. Dasar pengambilan keputusan untuk mendeteksi kenormalan adalah jika data menyebar
di sekitar garis diagonal dan mengikuti arah diagonal, maka model regresi memenuhi
asumsi normalitas dan begitu juga dengan sebaliknya.20
1) Metode P-P Plot of Regression Standardized Residual. Berikut ini output
perhit ngan SPSS ersi 25 0 a. Uji Normalitas
Uji normalitas digunakan untuk menguji apakah dalam model regresi, data variabel
dependen dan independen yang dipakai terdistribusi normal atau tidak. Sebuah data
penelitian yang baik adalah yang datanya berdistribusi normal (Ghozali, 2016). Pada
penelitian ini uji normalitas menggunakan metode analisa grafik. p
j
gg
g
Dasar pengambilan keputusan untuk mendeteksi kenormalan adalah jika data menyebar
di sekitar garis diagonal dan mengikuti arah diagonal, maka model regresi memenuhi
asumsi normalitas dan begitu juga dengan sebaliknya.20 p
j
gg
g
Dasar pengambilan keputusan untuk mendeteksi kenormalan adalah jika data menyebar
di sekitar garis diagonal dan mengikuti arah diagonal, maka model regresi memenuhi
asumsi normalitas dan begitu juga dengan sebaliknya.20 g
j g
g
y
1) Metode P-P Plot of Regression Standardized Residual. Berikut ini output
perhitungan SPSS versi 25.0. 1) Metode P-P Plot of Regression Standardized Residual. Berikut ini output
perhitungan SPSS versi 25.0. 65 Jurnal Al-Fatih Global Mulia Pengaruh Kemudahan dan Kualitas Informasi... Berdasarkan gambar 4.2 di atas dapat disimpulkan bahwa grafik normal P-
Plot terlihat titik-titik menyebar disekitar garis diagonal dan penyebarannya
mengikuti arah diagonal, maka grafik menunjukan bahwa model regresi layak
dipakai karena memenuhi asumsi normalitas. 2) Metode One Sample Kolmogorov Smirnov. Berikut output perhitungan SPSS Versi
25.0. Berdasarkan gambar 4.2 di atas dapat disimpulkan bahwa grafik normal P-
Plot terlihat titik-titik menyebar disekitar garis diagonal dan penyebarannya
mengikuti arah diagonal, maka grafik menunjukan bahwa model regresi layak
dipakai karena memenuhi asumsi normalitas. p
2) Metode One Sample Kolmogorov Smirnov. Berikut output perhitungan SPSS Versi
25.0. One-Sample Kolmogorov-Smirnov Test
Unstandardized
Residual
N
93
Normal Parametersa,b
Mean
.0000000
Std. Deviation
2.72222596
Most
Extreme
Differences
Absolute
.088
Positive
.082
Negative
-.088
Test Statistic
.088
Asymp. Sig. (2-tailed)
.073c
a. Test distribution is Normal. b. Calculated from data. c. Lilliefors Significance Correction. 66 Jurnal Al-Fatih Global Mulia b. Uji Multikolinearitas p
p
j
p
g
2) Metode Uji Glejser, berikut output hasil SPSS Versi 25.0
hasil uji Heteroskedastisitas metode Glejser, diketahui nilai signifikansi
variabel X₁ adalah 0,969, dan nilai signifikansi variabel X₂ adalah 0,730. Nilai
signifikansi kedua variabel ini > 0,05, maka disimpulkan bahwa data tidak
mengalami Heteroskedastisitas, dengan kata lain tidak terjadi keragaman varian
dari nilai residual terhadap semua pengamatan yang ada pada model regresi. 2) Metode Uji Glejser, berikut output hasil SPSS Versi 25.0
hasil uji Heteroskedastisitas metode Glejser, diketahui nilai signifikansi
variabel X₁ adalah 0,969, dan nilai signifikansi variabel X₂ adalah 0,730. Nilai
signifikansi kedua variabel ini > 0,05, maka disimpulkan bahwa data tidak
mengalami Heteroskedastisitas, dengan kata lain tidak terjadi keragaman varian
dari nilai residual terhadap semua pengamatan yang ada pada model regresi. b. Uji Multikolinearitas b. Uji Multikolinearitas
Multikolinearitas adalah ditemukan adanya korelasi yang sempurna atau
mendekati sempurna antar variabel independen dan model regresi (Ghozali I. , 2016). Jika nilai tolerance > 0,05 maka artinya tidak terjadi multikolinearitas. Dan jika nilai VIF
< 5,00 maka tidak terjadi multikolinearitas. Hal ini bisa dikatakan dengan model regresi
yang baik karena tidak terjadi multikolinearitas. j
Multikolinearitas adalah ditemukan adanya korelasi yang sempurna atau
mendekati sempurna antar variabel independen dan model regresi (Ghozali I. , 2016). Jika nilai tolerance > 0,05 maka artinya tidak terjadi multikolinearitas. Dan jika nilai VIF
< 5,00 maka tidak terjadi multikolinearitas. Hal ini bisa dikatakan dengan model regresi
yang baik karena tidak terjadi multikolinearitas. Dari data yang telah di uji, nilai tolerence tiap variabel lebih besar dari 0,05 dan
nilai VIF lebih kecil dari nilai 5,00. pada analisi data output uji multikolinearitas bisa
disimpulkan : 1) Nilai Tolerence variabel X₁ dan X₂ = 0,282 > 0,05
Artinya : tidak terjadi multikolinearitas pada variabel bebas 2) Nilai VIF Variabel X₁ dan X₂ = 3,552 < 5,00
Artinya : tidak terjadi multikolinearitas pada variabel bebas c. Uji Heteroskedastisitas
Uji Heteroskedastisitas adalah keadaan yang mana dalam model regresi terjadi
ketidaksamaan variansi dan residual pada satu pengamatan ke pengamatan yang lain. Model regresi yang baik adalah tidak terjadi heteroskedastisitas (Ghozali I. , 2016 edisi
8). c. Uji Heteroskedastisitas
Uji Heteroskedastisitas adalah keadaan yang mana dalam model regresi terjadi
ketidaksamaan variansi dan residual pada satu pengamatan ke pengamatan yang lain. Model regresi yang baik adalah tidak terjadi heteroskedastisitas (Ghozali I. , 2016 edisi
8). 1) Metode Scatterplot, berikut output hasil SPSS Versi 25.0
Terlihat bahwa distribusi data tidak teratur dan tidak membentuk pola
tertentu, serta tersebar diatas maupun dibawah angka nol pada sumbu Y. Hal ni
dapat disimpulkan tidak terjadi heteroskedastisitas pada model regresi. 2) Metode Uji Glejser, berikut output hasil SPSS Versi 25.0
hasil uji Heteroskedastisitas metode Glejser, diketahui nilai signifikansi
variabel X₁ adalah 0,969, dan nilai signifikansi variabel X₂ adalah 0,730. Nilai
signifikansi kedua variabel ini > 0,05, maka disimpulkan bahwa data tidak
mengalami Heteroskedastisitas, dengan kata lain tidak terjadi keragaman varian
d i il i
id
l t h d
t
d
d
d l
i 1) Metode Scatterplot, berikut output hasil SPSS Versi 25.0
Terlihat bahwa distribusi data tidak teratur dan tidak membentuk pola
tertentu, serta tersebar diatas maupun dibawah angka nol pada sumbu Y. Hal ni
dapat disimpulkan tidak terjadi heteroskedastisitas pada model regresi. 3. Uji Asumsi Klasik Berdasarkan Tabel 4.13 output SPSS di atas, diketahui bahwa nilai signifikasi
Asymp Sig sebesar 0,073 > 0,05, maka sesuai dengan dasar pengambilan keputusan
dalam uji normalitas one sample kolmogorov smirnov di atas, dapat disimpulkan
bahwa data berdistribusi normal. Dengan demikian asumsi atau persyaratan
normalitas dalam model regresi sudah terpenuhi. 66 Jurnal Al-Fatih Global Mulia Nurajizah1, Rika Mustika Sari2 b. Uji Multikolinearitas
Multikolinearitas adalah ditemukan adanya korelasi yang sempurna atau
mendekati sempurna antar variabel independen dan model regresi (Ghozali I. , 2016). Jika nilai tolerance > 0,05 maka artinya tidak terjadi multikolinearitas. Dan jika nilai VIF
< 5,00 maka tidak terjadi multikolinearitas. Hal ini bisa dikatakan dengan model regresi
yang baik karena tidak terjadi multikolinearitas. Dari data yang telah di uji, nilai tolerence tiap variabel lebih besar dari 0,05 dan
nilai VIF lebih kecil dari nilai 5,00. pada analisi data output uji multikolinearitas bisa
disimpulkan :
1) Nilai Tolerence variabel X₁ dan X₂ = 0,282 > 0,05
Artinya : tidak terjadi multikolinearitas pada variabel bebas
2) Nilai VIF Variabel X₁ dan X₂ = 3,552 < 5,00
Artinya : tidak terjadi multikolinearitas pada variabel bebas 4. Uji Hipotesis 4. Uji Hipotesis
a. Uji Partial (Uji-t)
Uji t dilakukan untuk menunjukan seberapa jauh pengaruh setiap variabel bebas
Kemudahan (X₁) dan Kualitas Informasi (X₂) terhadap Minat Penggunaan (Y) (Ghozali I. , 2016). Berdasarkan hasil uji partial, diketahui bahwa nilai t hitung variabel kemudahan (X₁)
sebesar 4,058 > nilai t tabel sebesar 1,986 (df = n - k) atau df = 93 – 3 = 90). Selain itu,
nilai signifikasi t bernilai 0,000 < 0,05, sehingga H0 ditolak dan H₁ diterima. Hal ini
menunjukan bahwa variabel kemudahan (X₁) berpengaruh secara signifikan terhadap minat
penggunaan (Y). Kemudian bahwa nilai t hitung variabel kualitas informasi (X₂) sebesar 4,570 > nilai t
tabel sebesar 1,986 (df=90). Selain itu. Nilai signifikansi t bernilai 0,000 < 0.05, sehingga
H0 ditolak dan H₂ diterima. Hal ini menunjukan bahwa variabel Kualitas Informasi (X₂)
berpengaruh secara signifikan terhadap minat penggunaan (Y). 4. Uji Hipotesis
a. Uji Partial (Uji-t)
Uji t dilakukan untuk menunjukan seberapa jauh pengaruh setiap variabel bebas
Kemudahan (X₁) dan Kualitas Informasi (X₂) terhadap Minat Penggunaan (Y) (Ghozali I. , 2016). Berdasarkan hasil uji partial, diketahui bahwa nilai t hitung variabel kemudahan (X₁)
sebesar 4,058 > nilai t tabel sebesar 1,986 (df = n - k) atau df = 93 – 3 = 90). Selain itu,
nilai signifikasi t bernilai 0,000 < 0,05, sehingga H0 ditolak dan H₁ diterima. Hal ini
menunjukan bahwa variabel kemudahan (X₁) berpengaruh secara signifikan terhadap minat
penggunaan (Y). Kemudian bahwa nilai t hitung variabel kualitas informasi (X₂) sebesar 4,570 > nilai t
tabel sebesar 1,986 (df=90). Selain itu. Nilai signifikansi t bernilai 0,000 < 0.05, sehingga
H0 ditolak dan H₂ diterima. Hal ini menunjukan bahwa variabel Kualitas Informasi (X₂)
berpengaruh secara signifikan terhadap minat penggunaan (Y). p
g
g
p
p
gg
b. Uji Simultan (Uji F)
Uji F dilakukan untuk menunjukan apakah variabel bebas Kemudahan (X₁) dan
Kualitas Informasi (X₂) yang dimasukan dalam model regresi secara bersama-sama
berpengaruh terhadap variabel terikat Minat Penggunaan (Y) (Ghozali I. , 2016) b. Uji Simultan (Uji F)
Uji F dilakukan untuk menunjukan apakah variabel bebas Kemudahan (X₁) dan
Kualitas Informasi (X₂) yang dimasukan dalam model regresi secara bersama-sama
berpengaruh terhadap variabel terikat Minat Penggunaan (Y) (Ghozali I. , 2016) b. Uji Simultan (Uji F)
Uji F dilakukan untuk menunjukan apakah variabel bebas Kemudahan (X₁) dan
Kualitas Informasi (X₂) yang dimasukan dalam model regresi secara bersama-sama
berpengaruh terhadap variabel terikat Minat Penggunaan (Y) (Ghozali I. 4. Uji Hipotesis , 2016) Berdasarkan hasil Uji ANOVA atas Ftest pada tabel didapatkan f hitung sebesar
122,177 dengan tingkat signifikan 0,05, karena F hitung > F tabel (122,177 > 3,10) dan
tingkat signifikasi 0,000 < 0,05, maka model regresi dapat digunakan untuk memprediksi
minat penggunaan (Y) atau dikatakan bahwa variabel kemudahan (X₁) dan kualitas 67 Jurnal Al-Fatih Global Mulia Pengaruh Kemudahan dan Kualitas Informasi... informasi (X₂) secara bersama-sama berpengaruh secara nyata terhadap variabel minat
penggunaan (Y). c. Uji Koefisien Determinasi (R Square)
Koefisien determinasi menunjukkan sejauh mana tingkat hubungan antara variabel
dependen dengan independen atau sebaliknya sejauh mana konstribusi variabel independen
mempengaruhi variabel dependen. c. Uji Koefisien Determinasi (R Square)
Koefisien determinasi menunjukkan sejauh mana tingkat hubungan antara variabel
dependen dengan independen atau sebaliknya sejauh mana konstribusi variabel independen
mempengaruhi variabel dependen. p
g
p
Dari hasil uji koefisien determinasi menjelaskan besarnya nilai korelasi/hubungan (R)
yaitu sebesar 0,855 (85,5%). Dari output tersebut diperoleh koefisien determinasi (R
Square) sebesar 0,731 (73,1%). Angka ini mengandung pengertian bahwa 73,1% Minat
Penggunaan (Y) dapat dijelaskan melalui variabel Kemudahan (X₁) dan Kualitas Informasi
(X₂). Sisanya sebesar 26,9% dijelaskan oleh variabel lain diluar variabel yang diteliti. d Uji Analisis Regresi Linier Berganda Dari hasil uji koefisien determinasi menjelaskan besarnya nilai korelasi/hubungan (R)
yaitu sebesar 0,855 (85,5%). Dari output tersebut diperoleh koefisien determinasi (R
Square) sebesar 0,731 (73,1%). Angka ini mengandung pengertian bahwa 73,1% Minat
Penggunaan (Y) dapat dijelaskan melalui variabel Kemudahan (X₁) dan Kualitas Informasi
(X₂). Sisanya sebesar 26,9% dijelaskan oleh variabel lain diluar variabel yang diteliti. d. Uji Analisis Regresi Linier Berganda Uji regresi linier berganda adalah analisis untuk mengukur besarnya pengaruh antara
dua atau lebih variabel independen terhadap variabel dependen. Berikut adalah hasil uji
analisis regresi liniar berganda dengan perhitungan SPSS Versi 25.0. Berdasarkan Uji Analisis Regresi Linear Berganda, maka pengaruh variabel
kemudahan (X₁) dan variabel Kualitas Informasi (X₂) terhadap minat penggunaan (Y)
memiliki persamaan regresi sebagai berikut : p
g
g
Y= 0,533 + 0,503X₁ + 0,585X₂ + e Y= 0,533 + 0,503X₁ + 0,585X₂ + e
Penjelasan persamaan regresi di atas adalah sebagai berikut : Penjelasan persamaan regresi di atas adalah sebagai berikut : 1) Nilai konstanta sebesar 0,533 yang berarti nilai minat penggunaan adalah 0,533,
saat X₁ dan X₂ adalah nol (0). 4. Uji Hipotesis 2) Nilai koefisien variabel kemudahan mengalami peningkatan satu satuan sedangkan
variabel kualitas informasi konstanta atau tidak ada atau sebesar 0,503 maka Y
(minat penggunaan) akan mengalami peningkatan sebesar 0,503 dengan asumsi
variabel kemudahan (X₁) bernilai tetap. Nilai koefisien variabel kualitas informasi mengalami peningkatan satu satuan
sedangkan variabel kemudahan konstan atau tidak ada atau sebesar 0,585 maka Y (minat
penggunaan) akan mengalami peningkatan sebesar 0,585 dengan asumsi variabel kualitas
informasi (X₂) bernilai tetap. 68 Jurnal Al-Fatih Global Mulia KESIMPULAN Berdasarkan hasil penelitian yang telah dilakukan dan telah melalui tahap pengumpulan
data, pengolahan data, analisis data, dan interpretasi hasil analisis mengenai pengaruh
kemudahan dan kualitas informasi terhadap minat mahasiswa dalam penggunaan layanan m-
banking pada Bank Syariah, dengan menggunakan data yang terdistribusi normal dan tidak
terdapat multikolinearitas, serta tidak adanya masalah heteroskedastisitas maka dihasilkan
kesimpulan sebagai berikut: 1. Variabel Kemudahan
Hasil uji t nilai t hitung variabel kemudahan (X₁) sebesar 4,058 > nilai t tabel sebesar 1,986
(df=90). Selain itu, nilai signifikasi bernilai 0,000 < 0,05. sehingga H0 ditolak dan H₁
diterima. Hal ini menunjukan bahwa kemudahan (X₁) berpengaruh secara signifikan
terhadap minat penggunaan (Y). 2. Variabel Kualitas Informasi
Hasil uji t nilai t hitung variabel kualitas Informasi (X₂) sebesar 4,570 > nilai t tabel sebesar
1,986 (df=90). Selain itu, nilai signifikasi bernilai 0,000 < 0,05, sehingga H0 ditolak dan H₂
diterima. Hal ini menunjukan bahwa Kualitas Informasi (X₂) berpengaruh secara signifikan
terhadap minat penggunaan (Y). 3. Hasil Uji F hitung sebesar 122,177 dengan tingkat signifikan 0,000, karena F hitung > F tabel
(122,177 > 3,10) dan tingkat signifikasi 0,000 < 0,05, sehingga H0 ditolak dan H₃ diterima. 68 Jurnal Al-Fatih Global Mulia Nurajizah1, Rika Mustika Sari2 Hal ini menunjukan bahwa variabel kemudahan (X₁) dan kualitas informasi (X₂) secara
bersama-sama berpengaruh secara signifikan terhadap variabel minat penggunaan (Y). Berdasarkan uji koefisien determinasi menunjukan kemudahan (X₁) dan kualitas informasi
(X₂) secara bersama-sama berpengaruh terhadap minat dalam penggunaan layanan m-
banking pada bank syariah (Y) sebesar 73,1%. Hal ini menunjukan bahwa variabel kemudahan (X₁) dan kualitas informasi (X₂) secara
bersama-sama berpengaruh secara signifikan terhadap variabel minat penggunaan (Y). Berdasarkan uji koefisien determinasi menunjukan kemudahan (X₁) dan kualitas informasi
(X₂) secara bersama-sama berpengaruh terhadap minat dalam penggunaan layanan m-
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Low-head pumped hydro storage: A review of applicable technologies for design, grid integration, control and modelling
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Citation for the original published paper (version of record):
Hoffstaedt, J., Truijen, D., Fahlbeck, J. et al (2022). Low-head pumped hydro storage: A review of
applicable technologies for design, grid
integration, control and modelling. Renewable and Sustainable Energy Reviews, 158. http://dx.doi.org/10.1016/j.rser.2022.112119 N.B. When citing this work, cite the original published paper. research.chalmers.se offers the possibility of retrieving research publications produced at Chalmers University of Technology. It
covers all kind of research output: articles, dissertations, conference papers, reports etc. since 2004. research.chalmers.se is
administrated and maintained by Chalmers Library research.chalmers.se offers the possibility of retrieving research publications produced at Chalmers University of Technology. It
covers all kind of research output: articles, dissertations, conference papers, reports etc. since 2004. research.chalmers.se is
administrated and maintained by Chalmers Library A R T I C L E
I N F O Keywords:
Low-head pumped hydro storage
Energy storage
Grid stability
Renewables integration
Energy transition
Reversible pump-turbine To counteract a potential reduction in grid stability caused by a rapidly growing share of intermittent
renewable energy sources within our electrical grids, large scale deployment of energy storage will become
indispensable. Pumped hydro storage is widely regarded as the most cost-effective option for this. However,
its application is traditionally limited to certain topographic features. Expanding its operating range to low-
head scenarios could unlock the potential of widespread deployment in regions where so far it has not yet
been feasible. This review aims at giving a multi-disciplinary insight on technologies that are applicable
for low-head (2-30 m) pumped hydro storage, in terms of design, grid integration, control, and modelling. A general overview and the historical development of pumped hydro storage are presented and trends for
further innovation and a shift towards application in low-head scenarios are identified. Key drivers for
future deployment and the technological and economic challenges to do so are discussed. Based on these
challenges, technologies in the field of pumped hydro storage are reviewed and specifically analysed regarding
their fitness for low-head application. This is done for pump and turbine design and configuration, electric
machines and control, as well as modelling. Further aspects regarding grid integration are discussed. Among
conventional machines, it is found that, for high-flow low-head application, axial flow pump-turbines with
variable speed drives are the most suitable. Machines such as Archimedes screws, counter-rotating and rotary
positive displacement reversible pump-turbines have potential to emerge as innovative solutions. Coupled axial
flux permanent magnet synchronous motor-generators are the most promising electric machines. To ensure grid
stability, grid-forming control alongside bulk energy storage with capabilities of providing synthetic inertia next
to other ancillary services are required. Low-head pumped hydro storage: A review of applicable technologies for
design, grid integration, control and modelling Low-head pumped hydro storage: A review of applicable technologies for
design, grid integration, control and modelling J.P. Hoffstaedt a,∗, D.P.K. Truijen b, J. Fahlbeck c, L.H.A. Gans d, M. Qudaih e, A.J. Laguna a,
J.D.M. De Kooning b, K. Stockman b, H. Nilsson c, P.-T. Storli d, B. Engel e, M. Marence f,
J.D. Bricker g,h a Faculty of Mechanical, Maritime and Materials Engineering, Department of Maritime and Transport Technology, Delft University of Technology, Mekelweg
2 2628 CD Delft The Netherlands a Faculty of Mechanical, Maritime and Materials Engineering, Department of Maritime and Transport Technology, Delft University of Technology, Mekelweg
2, 2628 CD Delft, The Netherlands f Electromechanical Systems & Metal Engineering, Ghent University & FlandersMake@UGent - Corelab EEDT-MP, Sint-Martens-Latemlaan
rijk, Belgium b Department of Electromechanical Systems & Metal Engineering, Ghent University & FlandersMake@UGent - Corelab EEDT-MP, Sint-Martens-Latemlaan
2B, 8500 Kortrijk, Belgium c Department of Mechanics and Maritime Sciences, Division of Fluid Dynamics, Chalmers University of Technology, 412 96 Gothenburg, Sweden
d Department of Energy and Process Engineering, Waterpower Laboratory, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway
e Elenia Institute for High Voltage Technology and Power Systems, Technische Universität Braunschweig, Schleinitzstraße 23, 38106 Braunschweig, Germany
f IHE Delft Institute for Water Education, Westvest 7, 2611 AX Delft, The Netherlands g Department of Hydraulic Engineering, Hydraulic Structures and Flood Risk, Delft University of Technology, The Netherlands
h Department of Civil and Environmental Engineering, University of Michigan, 2350 Hayward, Ann Arbor, MI 48109-2125, USA Contents lists available at ScienceDirect Contents lists available at ScienceDirect 1364-0321/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
p //
g/
/j
Received 8 September 2021; Received in revised form 17 December 2021; Accepted 9 January 2022 E-mail address: J.P.Hoffstaedt@tudelft.nl (J.P. Hoffstaedt). (article starts on next page) Renewable and Sustainable Energy Reviews 158 (2022) 112119 Renewable and Sustainable Energy Reviews 158 (2022) 112119 1364-0321/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http:/
Received 8 September 2021; Received in revised form 17 December 2021; Accepted 9 January 2022 A R T I C L E
I N F O Abbreviations: ADRC, Active Distribution Rejection Control; AF-PMSM, Axial Flux Permanent Magnet Synchronous Machine; ANN, Artificial Neural
Network; AS, Ancillary Services; CAES, Compressed Air Energy Storage; CRPT, Counter-Rotating Pump-Turbine; DSO, Distribution System Operator; DSSR,
Double-Stator Single-Rotor; DTC, Direct Torque Control; EIA, Energy Information Administration; ESHA, European Small Hydropower Association; ESOEI,
Energy Storage On Energy Invested; ESS, Energy Storage System; FOC, Field Oriented Control; IRES, Intermittent Renewable Energy Source; LCOS, Levelised
Cost Of Storage; MEPT, Maximum Efficiency Point Tracking; MMD, Modular Machine Drive; MPC, Model Predictive Control; MPPT, Maximum Power Point
Tracking; MTPA, Maximum Torque Per Ampere; PAT, Pump As Turbine; PHS, Pumped Hydro Storage; PLL, Phase Locked Loop; PM, Permanent Magnet; PMSM,
Permanent Magnet Synchronous Machine; PTO, Power Take-Off; PWM, Pulse-Width Modulation; RPT, Reversible Pump-Turbine; SSDR, Single-Stator
Double-Rotor; SSSR, Single-Stator Single-Rotor; SVM, Space Vector Modulation; TSO, Transmission System Operator
∗Corresponding author. Received 8 September 2021; Received in revised form 17 December 2021; Accepted 9 January 2022 1. Introduction and analyse technologies based on their applicability for low-head
utilisation. First, an overview of PHS and its historical development is
given. Based on this, recent trends leading to further innovation in the
field are identified, and the potential and necessity of storage technolo-
gies are discussed. Finally, technological and economic challenges are
explored, and the key advancements that could contribute to economic
and technical viability are isolated. Building on this, in the major tech-
nological fields – pump-turbine design and configuration, control and
electric machinery, as well as modelling – the most promising technolo-
gies are compared and their fitness for low-head application is assessed. Additionally, implications of grid integration are discussed, including
further elaboration on the significance of integrating large-scale energy
storage, such as low-head PHS into world-wide grids. In a global effort to reduce greenhouse gas emissions, renewables
are now the second biggest contributor to the world-wide electricity
mix, claiming a total share of 29% in 2020 [1]. Although hydropower
takes the largest share within that mix of renewables, solar photo-
voltaics and wind generation experience steep average annual growth
rates of 36.5% and 23%, respectively, since 1990 [2]. Both of these
technologies, however, significantly differ in their generation char-
acteristics when compared to traditional thermal power plants. This
trend towards an increase in intermittent generation, coupled with
a reduction in spinning reserves, could undermine grid stability. To
counteract these effects, grid-scale deployment of energy storage is
indispensable. There are complementary approaches to balance demand and sup-
ply in an electricity grid, such as an increase in flexible generation,
demand management, or exporting and importing electricity. Nonethe-
less, at certain penetration levels of renewables, to reduce the risk of
grid instability, a heterogeneous pool of storage solutions is needed. A wide variety of such storage technologies – including capacitors,
flywheels, electro-chemical batteries, compressed air energy storage
(CAES), molten-salt or hydrogen storage – is available to balance the
grid in the timescale from seconds up to seasonal variations. Crucial
factors for large-scale balancing include energy and power capacity as
well as fast response times while maintaining high efficiencies. Aside
from fulfilling these criteria, the major driver towards commercial
deployment is the levelised cost of storage (LCOS); leading in this are
pumped hydro storage (PHS) and CAES [3]. 2. Overview and historical development of pumped hydro storage Pumped hydro storage is an amended concept to conventional
hydropower as it cannot only extract, but also store energy. This is
achieved by converting electrical to potential energy and vice versa in
the form of pumping and releasing water between a lower and a higher
reservoir. The energy conversion occurs by using pumps and turbines
either combined in a reversible (binary set) or separate configuration
(ternary and quaternary sets). The power of such a system, as well as
the amount of energy that can be extracted or stored, is proportional
to the product of head and water flow or volume, respectively. Hence,
a higher head results in a reduced flow for a given desired power and
smaller reservoirs for a given storage capacity. It does not, therefore
just correlate with scaled-down reservoirs but also smaller remaining
civil structures and machinery, historically leading to reduced cost and
a significant economic advantage of utilising high-head differences [7]. Of today’s bulk energy storage integrated into the world-wide grids,
over 90% is comprised of PHS of which the vast majority are high-head
applications. According to the International Hydropower Association,
in 2019, the global installed capacity reached 158 GW with the biggest
contributors being China making up 30.3 GW of the share, Japan
27.6 GW, and the United States 22.9 GW [8]. Fig. 2 shows the distribu-
tion of global storage capacity that is operational, under construction,
planned, and announced as of 2021. Pumped hydro storage is a mature and well-known technology that
has been used since the beginning of the 20th century. In 2020, it
contributed with 90.3% of the world’s energy storage capacity [5]. However, while some regions reach the limits of economically viable
PHS that can be implemented, others lack entirely the necessary to-
pographic features. Traditional PHS relies on high heads to realise the
expected power and storage capacity. Most of the plants produce in the
order of 1000–1500 MW of power, with round-trip efficiencies which
are commonly in the range of 70%–85% [6]. In comparison, the next largest contributors to bulk energy storage
are electro-chemical battery storage – rapidly growing with a total
capacity of 14.2 GW – and thermal storage with 2.9 GW in 2020 [5]. To explain the historic market dominance of PHS and understand
recent trends, several factors have to be taken into account. 2. Overview and historical development of pumped hydro storage Pumped
hydro storage utilising reversible pump-turbines has been available as
a mature and cost-effective solution for the better part of a century
with an estimated energy based capital cost of 5–100 $/kWh [10]. To-
day, compressed air energy storage is considered mature and reliable,
offering similarly low capital cost between 2–50 $/kWh, and electro-
chemical batteries offer high energy density with higher costs, and
experience drastic growth while the impact of hydrogen-based storage
in the energy transition is largely expected to be substantial [10]. Aside from its use to store energy, hydropower is regarded as the
foremost renewable generation method when it comes to flexibility
and improving grid stability. Due to the proven advantages of hydro-
electric power generation, wide-ranging research efforts have focused
on conceptual adaptations and technological advancements utilising
low- and ultra-low-head scenarios. Some of these technologies, such as
wastewater, run-of-river hydropower, or tidal barrages have seen pro-
totyping and commercial deployment. However, theoretical attention
and practical implementation towards low-head PHS has been limited. Fig. 1 shows a conceptual drawing of what such a system may consist
of when deploying a reversible pump-turbine coupled to a motor-
generator that is connected to the grid via an AC-DC-AC converter for
variable speed operation. However, PHS‘s dominance is not only due to its historic lead but
can also be attributed to its technical, economic, and sustainability
advantages. These include high efficiencies, large achievable capacities,
and long lifetimes. Compared to rapidly expanding battery storage that
can be used wherever it is most needed, one clear advantage is this
durability. It is currently assumed that a battery system will last around
15–20 years, but on the other hand, the oldest hydropower plant in
Norway has been operating for over 120 years [11,12]. Prolonged
lifetimes are one factor improving the sustainability of PHS compared
to other storage solutions. Others include maturity, low capital and
operating cost, as well as low energy and carbon dioxide density. Based on these and other factors regarding economic, performance,
technological, and environmental considerations, Ren et al. ranked PHS
as the most sustainable storage technology [13]. The lack of attention on low-head PHS can be partly explained
through high levelised cost of storage (LCOS) caused by extensive civil
structures, enlarged machinery, lower round-trip efficiencies, and lim-
ited flexibility to provide ancillary services (AS). https://doi.org/10.1016/j.rser.2022.112119 J.P. Hoffstaedt et al. J.P. Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 1. Introduction An alternative approach is
based on the so-called energy stored on energy invested (ESOEI), which
gives an estimate of the relation between the stored energy during the
lifetime of a system and the energy required to construct the system. Also for this metric, PHS and CAES are, by far, in the lead [4]. 2. Overview and historical development of pumped hydro storage The predicted increase
in demand for energy balancing and AS in the upcoming decades will
likely justify increased LCOS. Additionally, technological advancements
could significantly contribute to a reduction in LCOS. Addressing the
technological challenges and overcoming economic barriers of low-
head PHS could unlock the potential of integrating large-scale energy
storage into the grids of regions where it has not been feasible so far. For the given reasons, research and development towards shifting
the operating range of PHS to low heads is scarce. Using a multi-
disciplinary approach, the main goal of this research is to review 2 J.P. Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 Fig. 1. Schematic showing a low-Head PHS concept and its main components. Fig. 1. Schematic showing a low-Head PHS concept and its main components. Fig. 2. Global PHS Capacity [9]. ther advantages of PHS include suitability for long term storage
become the norm since the middle of the 20th century [6] Experie Fig. 1. Schematic showing a low-Head PHS concept and its main components. Fig. 1. Schematic showing a low-Head PHS concept and its main components. Fig. 1. Schematic showing a low-Head PHS concept and its main components. Fig. 2. Global PHS Capacity [9]. Fig. 2. Global PHS Capacity [9]. Further advantages of PHS include suitability for long-term storage
– since hardly any storage losses occur other than seepage and evap-
oration – and quick availability due to short switch-on and switch-off
times. With these factors ensuring a significant share within a heteroge-
neous pool of storage technologies, one major disadvantage of PHS has
historically been its topographic constraints. A switch from river-based
to closed-loop off-river systems could overcome some of the constraints
and increase the potential for deployment [14,15]. Nonetheless, regions
with flat topographies still do not offer viable sites. become the norm since the middle of the 20th century [6]. Experiencing
a major boom in Europe, parts of Asia, and North America, a large
portion of today‘s installed PHS capacity was constructed in the 1960s,
1970s, and 1980s; in most countries, this occurred alongside rapidly in-
creasing nuclear power. The gained flexibility that PHS plants provided
allowed them to match a varying demand with the baseload generation
of nuclear power plants. 2. Overview and historical development of pumped hydro storage An example where this was particularly rele-
vant is Japan due to its lack of interconnections to other countries as
well as their strong strategy towards nuclear energy. In the United States, another reason for the growing capacity of
PHS during that period was the energy crisis in the 1970s, leading
to an increased cost of fossil fuels allowing PHS to grow as a substi-
tute for peak balancing [16]. After that period, development slowed
down in most regions with the major exception of China. Its rapidly
growing economy and correlated energy demand largely satisfied by
non-flexible coal plants required major energy storage. 2.3. Potential of deployment and scalability in Europe Resulting from the rapid transition that grids are experiencing
worldwide, the need for energy storage is evident. However, there are
a variety of factors influencing the actual storage demand and its ex-
pected progression during the coming decades. First and foremost, this
is the growth in intermittent and converter coupled renewables. While
a direct correlation between renewable penetration levels and storage
demand can be assumed, mitigating factors such as improved genera-
tion forecasting and the continuous development of renewables able to
provide AS will allow for later deployment of energy storage. Further
factors to consider include the flexibility of remaining generators in
the grids and the emerging need for additional operating reserves,
improved demand management, as well as further grid expansion and
interconnection. The second major trend is expanding the operating range and
application. One of the most limiting factors in the potential use of
large-scale PHS has been the fact that not many locations could offer
economically viable deployment. These were traditionally mountainous
regions accessing water with enough space to construct extensive civil
structures. There is a large potential in Europe to deploy further mini
and small hydropower plants to counteract the effects of higher renew-
able penetration levels. However, this does not apply to countries with
flat topographies, such as Denmark, the Netherlands, or Belgium [21]. Additionally, to achieve the balancing capabilities of pumped storage
systems, larger plants typically provide better economies of scale. Suitable locations for such are rare in Europe and some countries like
Japan are considered to have used nearly all available sites [22]. This
limited availability of appropriate locations drives the development of
new approaches. Examples for a promising change of approach are
underwater PHS or gravity energy storage. Bearing these factors in mind, it becomes clear that storage demand
will heavily depend on individual grid characteristics and may vary
in different regions. Based on Germany as an example, additional
short-term storage can be expected at renewable shares between 40%
and 60% and long-term storage between 60% and 80%. Above 80%
and towards a fully renewable generation, bulk energy storage on all
timescales is not only required in order to avoid extensive renewable
energy curtailing, ensure grid stability and power quality, but will be a
cost-effective solution in the GW range [29]. J.P. Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 to low and ultra low-head applications, including the potential use of
seawater, while improving its capability to provide AS. If technological
advancements allow for economic viability, large-scale low-head PHS
could be integrated in regions where PHS so far was not a feasible
solution. Up until this point, pump-turbines were coupled to fixed speed
motor-generators. The next significant development occurred in the
1990s when variable speed operation was introduced in Japan. The
ability to adjust the angular velocity of the runners allowed for higher
efficiencies under changing conditions, reduced the switching time
between pump and turbine mode, and facilitated higher ramp rates and
quicker response times [17]. Later trends for PHS show the usage of ternary units. With ternary
units, a separate pump and turbine are connected on a single shaft to an
electric machine that can work either as a motor or a generator [26]. This configuration presents a very flexible and fast response range,
shows higher efficiencies than reversible machines, and can utilise
hydraulic short circuits for optimal power in- or outtake [27,28]. The major drawback with ternary units is that they requires higher
investment and maintenance costs compared to a reversible unit [28]. In a low-head scenario, the increase in investment cost would be even
greater since a high-power, low-head machine needs to be large in
order to handle a high flow rate. Thus, it is suggested as a less attractive
alternative for low-head PHS. 2.2. Recent trends After an initial reduction of PHS deployment around the turn of
the millennium, the rapidly growing share of intermittent renewable
energy sources (IRES) in the last couple of decades sparked new interest
in sustainable flexible generation as well as large-scale energy storage
solutions. This caused increasing attention towards the rehabilitation
of old hydropower plants and an expansion of PHS [18]. While PHS
experienced a much longer development process than competitive tech-
nologies and could hence be considered mature, two major trends can
be identified pushing further innovation in the field. The first one is derived from the change in grid characteristics
caused by a reduction in spinning reserves. A growing number of
converter coupled renewables raise the necessity for external provision
of AS. To provide these using PHS, research efforts focus on devel-
oping improved control and machinery but also novel concepts, such
as hybrid storage solutions. Examples of such concepts could be the
coupling of conventional PHS with flywheels for frequency control or
supercapacitors providing virtual inertia [19]. Hybrid storage solutions
incorporating PHS, such as hybrid pumped and battery storage, are also
particularly suited for off-grid applications [20]. 2.1. Early deployment and progression in the 𝟐𝟎th century Not long after hydropower began to generate electricity, the first
small-scale PHS plants were constructed in the mountainous regions
of central Europe in the beginning of the 20th century. Initially using
separate pumps and turbines, combined reversible pump-turbines have 3 J.P. Hoffstaedt et al. 2.4. Technological and economic challenges Radial-
or mixed-flow machines are preferable for pumps with a specific speed
of 𝛺s < 2.7, and as the specific speed increases (2.6 < 𝛺s < 11.6), an
axial configuration is more suitable [45]. Here, 𝛺is the runner rotational speed in rad/s, 𝑄is the volumetric
flow rate in m3/s, and 𝐻is the head in metre. Stepanoff [42] explained
in 1948 that a higher specific speed results in a smaller, and thus
cheaper, machine. With a higher flow rate, the blade design differs
significantly, as illustrated in Fig. 3. On the other hand, machines with
low rotational speeds and small shear forces (e.g. Archimedes screw and
positive displacement machines) are more fish-friendly [43,44]. Radial-
or mixed-flow machines are preferable for pumps with a specific speed
of 𝛺s < 2.7, and as the specific speed increases (2.6 < 𝛺s < 11.6), an
axial configuration is more suitable [45]. Based on these challenges, three main areas can be identified where
significant progress could contribute to making low-head PHS techni-
cally and economically competitive. These are pump-turbine design and
configuration, grid integration, and electrical machines and control. Research in these fields will be discussed in the chapters following. Additionally, modelling approaches that may aid the development are
compared. Carravetta et al. [46] postulate that axial-flow pumps can be used
as PAT for heads between 1–5 m and flow rates up to 1000 l/s. They
also claim that mixed-flow PATs can be used for heads in the region
of 5–15 m and flow rates of 50–150 l/s. Bogenrieder [47] stated that
radial pump-turbines are suitable to use for heads that are above 60 me-
tre, with a power exceeding 50 MW. Typically, radial- or mixed-flow
machines work best for high heads and low flow rates. For exam-
ple, regular Francis-like pump-turbines (mixed-flow) are the common
choice when it comes to mid- to high-head applications, but the head
variations at low-head operation would greatly affect efficiency [48]. Mixed-flow machines can be used as low-head PHS if the flow rate is
low. However, according to Eq. (1), this implies that the power will also
be low. Multiple machines could be used in parallel to increase the total
power. Breeze [48] suggests that a Deriaz, mixed-flow machine can be
used for heads between 20–100 m. 2.4. Technological and economic challenges The definition of low-head is not unanimous among different coun-
tries and researchers. For example, the U.S. Energy Information Admin-
istration (EIA) considers low-head when the head is less than 30 metre
and Okot [36] classifies it as when 5 < 𝐻< 15 metre. In this work, the
European Small Hydropower Association (ESHA) classification defines
low-head, and states that low-head hydropower plants have a head of
2-30 metre [37]. Low-head PHS has not yet seen deployment on a significant scale
within our grids. This is largely due to the increased upfront cost
required. While highly dependent on the individual project and site,
the major contributor to the initial CAPEX of PHS projects in general
are civil structures, including the reservoir, penstock, and lining [31]. Due to larger masses of water being stored in the reservoir and flowing
through the penstock when it comes to low-head applications, their
contribution to the overall economic viability can only be assumed to
be significant. All aspects regarding civil components as well as detailed
economic analyses of low-head PHS systems are, however, outside of
the scope of this review. The overall efficiency of a low-head power plant is more sensitive
to head losses than a high-head alternative, and low-head PHS requires
that the pipelines are short to be economically feasible [38]. This is
because head losses are proportional to the pipeline length and the
flow velocity squared, which is a further incentive for not using ternary
units in a low-head case since they require more pipelines and would
thus decrease the plant‘s overall efficiency. With the higher flow rate
of high-power low-head PHS, larger reservoirs are required to store the
same amount of energy as a corresponding high-head application [34]. This is because the energy storage capacity is a function of the water
mass and head. Apart from that, other conditions such as the type of
machine (radial-, mixed-, or axial-flow), operation (variable or fixed
speed), and reservoir configuration may apply when choosing the best
reversible pump-turbine configuration [39]. Chapallaz et al. [40] stated
that, in practice, almost any hydro pump can also be used as a turbine. The reverse is, however, not the case. As an example, impulse turbines
(Pelton or Turgo) cannot be used as pumps. Low-head PHS would be most competitive utilising a storage ca-
pacity able to provide balancing in the timescale of hours to days. 2.4. Technological and economic challenges This places it in the middle between lithium-ion batteries appropri-
ate for shorter and hydrogen storage appropriate for longer periods. The LCOS of new high-head PHS systems ranges from 50 e/MWh to
80 e/MWh [32]. Initially low-head plants may not be able to compete
with this. However, changing demand characteristics of the electricity
markets, as well as further development and improvement of techno-
logical aspects, significantly influence economic viability and thereby
the potential of large-scale deployment. The increase in renewables in
world-wide grids will lead to rising demands, not just for short- and
long-term balancing but also the provision of AS increasing the value
of both. Additional revenue from the provision AS could hence increase
economic viability. The design and characteristics of any hydro pump and turbine
are determined by its conditions of operation. In turbomachinery, the
specific speed is one key parameter to select the most appropriate
reversible pump-turbine or using a pump as turbine (PAT). In this
paper, it is defined in accordance with Dixon and Hall [41] as Eq. (2),
and Table 1 shows ranges of specific speeds for various machines. Further drivers making PHS economically more attractive are grow-
ing interconnections to other grids opening up additional markets
as well as technical advances such as higher efficiencies across a
broader operating range [22]. Technological progression can help fa-
cilitate these drivers. A reduction of switching times between pump
and turbine mode together with higher power ramp rates will allow
for enhanced capabilities to provide AS as well as maximise balancing. Aside from regulatory changes, such as carbon taxation, electricity
price margins have been identified as one of the major drivers towards
PHS utilisation. Improved round-trip efficiencies directly correlate with
higher revenues for the operator and therefore result in increased
utilisation [33]. 𝛺s = 𝛺𝑄1∕2
(𝑔𝐻)3∕4
(2) 𝛺s = 𝛺𝑄1∕2
(𝑔𝐻)3∕4 (2) Here, 𝛺is the runner rotational speed in rad/s, 𝑄is the volumetric
flow rate in m3/s, and 𝐻is the head in metre. Stepanoff [42] explained
in 1948 that a higher specific speed results in a smaller, and thus
cheaper, machine. With a higher flow rate, the blade design differs
significantly, as illustrated in Fig. 3. On the other hand, machines with
low rotational speeds and small shear forces (e.g. Archimedes screw and
positive displacement machines) are more fish-friendly [43,44]. 2.4. Technological and economic challenges This is because its design is closer
to an axial machine compared to a conventional Francis-like pump-
turbine and the Deriaz design also presents adjustable blades [48,49]. Breeze further expresses the necessity of variable speed drives to extend
the operational region at high efficiency. 3. Pump-turbine design and configuration for low-head pumped
hydro storage The choices when selecting the type of reversible pump-turbine
(RPT) unit, or evaluating using a pump as turbine (PAT), are governed
by a number of factors. The first thing to evaluate is the power of the
hydropower plant, which is a function of head and flow rate and the
general formula is given by Eq. (1). (1) 𝑃= 𝜌𝑔𝐻𝑄𝜂
(1) 2.3. Potential of deployment and scalability in Europe Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 high volumetric flow rate are also regarded as having great potential
to unleash new opportunities for pumped hydro storage [35]. 2.3. Potential of deployment and scalability in Europe In less flexible grids, for
example utilising large-scale nuclear power to cover the base load, the
need for extensive storage will be reached at much lower renewable
levels. The former is a recently developed and tested concept based on
submerging a hollow sphere offshore and using the static pressure
difference for energy storage. The surrounding sea acts as the upper
reservoir and the sphere as the lower which can be filled to generate
electricity or emptied to store it. Initial model-scale tests have been
successful; it is a freely scalable technology without issues regarding
land use and considered cost competitive with PHS and compressed
air storage [23]. Using seawater in general for PHS is so far an un-
common practice, but has been investigated as a solution for isolated
grids [24]. If technical, environmental, and economic challenges are
overcome, utilising seawater could be another promising expansion of
PHS’s potential deployment. An update to the European Green Deal has raised the ambition to
reduce greenhouse gas emission by 55% until 2030 compared to the
standard of 1990 paving the way for a carbon neutral energy supply by
2050. Consequently, the share of intermittent renewables will need to
increase faster. Recent estimates see a growth towards 70% renewable
power generation in 2030 [30]. Depending on the flexibility of this share of renewables in different
regions, a rapid increase in demand for storage and the provision of
AS can be expected with PHS being a promising candidate to fill the
gap. For countries with a flat topography, economically viable low-
head PHS could bear a huge potential to cover the growing demand. This is especially relevant if a large coastline is available, opening up
the possibility for seawater application. Coastal applications also come
with the additional benefit of proximity to large-scale IRES, such as
offshore wind farms. The latter similarly decouples the fundamental principle of PHS
from its topographic restrictions. Storage is done via gravitational
potential energy. However, energy is stored or extracted respectively
by moving a piston of large mass up and down using water powered by
a pump-turbine for conversion. While still under development, initial
economic evaluations show it to have a potentially attractive LCOS
compared to other storage technologies [25]. An alternative that is not less promising and will potentially suit
both these trends is to extend the operating range of conventional PHS 4 J.P. Hoffstaedt et al. J.P. Table 1 stated that centrifugal machines have preferable efficiency as a function
of flow rate; however, axial machines have a flatter efficiency curve
as a function of head. This further suggests that an axial machine
is preferable in a low-head scenario due to the fact that part-load
operations will be less influenced by the large variation in head of a
low-head PHS application. Ranges of specific speed for various machines [45]. Lately, axial-flow pump-turbines with two runners, rotating in op-
posite direction from one another, have been proposed as an alternative
for low-head PHS. They are usually referred to as counter-rotating
pump-turbines (CRPT) due to the rotation of the individual runners,
as illustrated in Fig. 4. According to Furukawa [55], the advantages of
those machines are that they can be of smaller size, have a more stable
head-flow rate characteristic curve, and have a wider range of high
efficiency with individual speed control of the runners when compared
to a single runner axial machine. Several numerical studies predict that
a well designed low-head counter-rotating pump-turbine may achieve
efficiencies of up to 80%–90% in both pump and turbine mode [56–58]. Fahlbeck et al. [59] showed numerical results for a prototype counter-
rotating pump-turbine in pump mode with a peak efficiency of 91%,
heads up to 12 metre, flow rates between 60–160 m3/s, and a maximum
power of almost 14 MW. bValues should be regarded only as a reference number, since it is not common to
indicate a specific speed for these machines. for a machine with high power at low heads. An axial machine also
allows for cheaper civil structures. In turbine mode, the runner rotates
due to the torque that is generated by the flow-induced runner blade
pressure and suction sides. The electric generator extracts power by
a balancing counteracting torque at the particular rotational speed. In pump mode, an electric motor adds power to the runner in the
form of torque at the particular rotational speed. A flow is developed
due to the rotating runner blade pressure and suction sides, causing
a balancing counteracting torque. The pressure change is in an axial
machine primarily due to the change of relative flow velocity [45]. This is because the tangential velocity of the runner and the cross-
sectional area are constant along a streamline in an axial machine. Table 1 In
a centrifugal machine, the flow must change direction from axial to
radial (pump mode), or radial to axial (turbine mode), as shown in
Fig. 3. The principles for the head rise in a centrifugal machine are
here described in pump mode for brevity. As the flow goes through
the machine, the cross-sectional area and the tangential velocity of
the runner increase with the radius through the machine. The absolute
flow velocity will decrease as the cross-sectional area increases, due to
continuity. According to Bernoulli’s principle, the static pressure will
increase with the change of absolute velocity squared [50]. The main
cause of the static pressure rise is, however, due to centrifugal effects
caused by the increase of the runner’s tangential velocity, and passage
diffusion, due to a reduction in the relative flow velocity, through the
machine [45]. The result is that the exit blade velocity needs to be
small in order to limit the pressure rise in a low-head application. This
means that the machine needs to be small and that the entrance-to-
exit diameters decrease with the decreasing head [51]. The smaller size
further limits the flow rate and thus the power. Additional non-conventional machines have also been studied as
low-head PHS. The Archimedes screw, depicted in Fig. 4, is a viable
option for heads between 2–10 m, discharge ranges up to 15 m3/s,
and power output of up to 355 kW [36,43,60]. The Archimedes screw
enables lower installation and maintenance costs compared to other
conventional pump-turbines and can reach efficiencies of up to 90%
in turbine mode [61,62]. An additional benefit is that the Archimedes
screw presents better conditions for fish-friendliness when compared to
conventional bladed pump-turbines [43,61,63]. Positive displacement (PD) pumps are usually chosen when the
system requires low specific speeds [45]. Some PD pumps can also
represent a good alternative when reversible flows must be taken into
account, thus resembling a PAT [64]. Positive displacement pumps are
self-priming, typically produce low flow rates, can handle big variations
in head without significantly changing their efficiency, and are often
regarded as a good choice for viscous fluids or fluids with the presence
of solids or precipitates that need to be handled [45,50]. Rotary positive
displacement machines have already been studied as micro hydro
turbines in water supply pipelines with pressures up to 5 bar (hydraulic
head equivalent to 51 metre) and presented efficiencies between 60%–
80% [65–68]. 𝑃= 𝜌𝑔𝐻𝑄𝜂 Here, 𝜌is the density of water, 𝑔is the gravity acceleration, 𝐻is the
head, 𝑄is the volumetric flow rate, and 𝜂is the overall efficiency of
the power plant. The gravity acceleration and water density can be
regarded as constant. The equation shows that if the head is low, the
flow rate must be large in order to produce high power [34]. With
a large flow rate, the diameter of pipelines and the runner need to
be large as well to limit the flow velocity, and thus hydraulic losses
in the system. High-head conditions are usually preferable to build
pump storage hydropower plants. However, low-head solutions with The reason why an axial machine is preferable in a low-head
application is that it allows for a higher flow rate, which is necessary 5 J.P. Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 Renewable and Sustainable Energy Reviews 158 (2022) 112119
J.P. Hoffstaedt et al. Fig. 3. Principle view of bladed pump-turbine configurations, note that the term ‘‘centrifugal’’ refers to both the radial- and mixed-flow. The drawing is based on principles shown
in [40–42,46]. Fig. 3. Principle view of bladed pump-turbine configurations, note that the term ‘‘centrifugal’’ refers to both the radial- and mixed-flow. The drawing is based on principles shown
in [40 42 46] Fig. 3. Principle view of bladed pump-turbine configurations, note that the term ‘‘centrifugal’’ refers to both the radial- and mixed-flow. The drawing is based on principles shown
in [40–42,46]. iew of bladed pump-turbine configurations, note that the term ‘‘centrifugal’’ refers to both the radial- and mixed-flow. The drawing is bas mp-turbine configurations, note that the term ‘‘centrifugal’’ refers to both the radial- and mixed-flow. The drawing is based on principles sh Table 1
Ranges of specific speed for various machines [45]. Technology
Specific speed 𝛺𝑠
Axiala
2.6–11.6
Mixed
0.6–2.7
Radial
0.1–0.8
Archimedes screwb
0.03–0.39
Positive displacementb
0.01–0.13
aCRPT is classified as an axial machine. bValues should be regarded only as a reference number, since it is not common to
indicate a specific speed for these machines. 4. Grid integration of energy storage systems A reliable electrical power grid is a balanced system. As generation
and demand fluctuate perpetually, transmission system operators (TSO)
and distribution system operators (DSO) have to keep the system
balance everywhere in the electrical grid. This balance ensures that the
grid operates at its nominal frequency (50 or 60 Hz) and that voltage
and power load remain within a certain limit at all times. However, the
higher the penetration of intermittent renewable energy sources, the
more insecure this balance. Thus, the increasing penetration of IRES is
a challenge that TSO and DSO have to handle [71,72]. At the present time, power systems rely on conventional power
plants utilising synchronous generators contributing significantly to
the stabilisation of the electrical power system, using the rotating
masses in their generators (rotors). The synchronous generators keep
the frequency steady at its nominal value due to their large flywheel
masses and thereby assure system stability. In the case of generation
or load fluctuation leading to sudden grid frequency deviations, the
rotor’s combined inertia keeps the generators rotating and consequently
supports the grid stability [73]. On the contrary, not all IRES have large
rotating masses and most are integrated into the grid via converters,
subsequently decoupling the rotating masses from the grid frequency. Therefore, they do not have any natural inertia (spinning reserve) and
thus operate in an entirely different way than synchronous generators. As of today, the grid-connected converters for IRES follow the grid
frequency by using a phase locked loop (PLL). This tracks the grid
frequency in order to keep the IRES converters synchronised to the grid. The PLL control concept is known as grid-following control [73,74]. 𝑃m −𝑃e = 𝐽𝜔0
d𝜔
d𝑡
(3)
𝜔= d𝜗
d𝑡
(4) 𝑃m −𝑃e = 𝐽𝜔0
d𝜔
d𝑡
(3)
𝜔= d𝜗
d𝑡
(4) 𝑃m −𝑃e = 𝐽𝜔0
d𝜔
d𝑡 (3) 𝜔= d𝜗
d𝑡 𝜔= d𝜗
d𝑡 (4) Here, 𝑃m is the mechanical power, 𝑃e is the electrical power, 𝜔0 is
the nominal angular frequency, 𝜔is the output angular frequency, 𝜗
is the rotation angle, and J is the total moment of inertia of the rotor
mass. Fig. 5 shows a simplified block diagram for a synthetic electrical
inertia control system. As illustrated, ESS are needed along the grid-
forming control to provide the necessary synthetic electrical inertia. Table 1 From all the available PD alternatives, the lobe and gear
pump configurations – illustrated in Fig. 4 – are the most suitable
options to handle reversible flow. Given the low specific speed, PD
pumps could most likely be regarded as a fish-friendly technology [44]. On the other hand, only the lobe design seems to handle fish and solid
without extra mitigation measures. A few small-scale projects have
tested PD RPTs [64]. However, further investigations and real-scale In general, pump-turbines are worse at part-load conditions com-
pared to a pure pump or turbine since the pump-turbine design is
often a trade-off to reach acceptable performance at design condi-
tions [52,53]. Delgado et al. [54] reported that it is hard to predict
part-load performance for PATs, especially in turbine mode since pump
manufacturers usually do not supply any data of this. Stepanoff [42] 6 J.P. Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 Renewable and Sustainable Energy Reviews 158 (2022
Hoffstaedt et al. Fig. 4. Principle view of non-conventional pump-turbine configurations. The drawing is based on principles showed in [45,55,56]. tions. The drawing is based on principles showed in [45,55,56]. Fig. 5. Simplified block diagram for synthetic electrical inertia control [80,81]. Here,
𝛥f is the deviation of system frequency, ROCOF is the rate of change of frequency, J is
the virtual inertia control constant, 𝛥𝑃imitate is the active power of the converter, and
𝛥𝑃inertia is the emulated virtual inertia power that could be imitated into the system. Fig. 4. Principle view of non-conventional pump-turbine configurations. The drawing is based on principles showed in [45,55,56]. Fig. 5. Simplified block diagram for synthetic electrical inertia control [80,81]. Here,
𝛥f is the deviation of system frequency, ROCOF is the rate of change of frequency, J is
the virtual inertia control constant, 𝛥𝑃imitate is the active power of the converter, and
𝛥𝑃inertia is the emulated virtual inertia power that could be imitated into the system. prototypes are still needed to validate the use of PD RPTs in low-head
pumped storage application. Pump-turbines in PHS applications can operate at fixed or vari-
able rotational speed. Variable speed machines take advantage of a
wider operating range at high efficiency and can thus produce power
in a wider spectrum [38,69]. Moreover, variable speed units ensure
greater penetration and bring more flexibility to PHS operations, es-
pecially for smaller machines [70]. Table 1 Despite the technical advantages,
this technology is about 30% more expensive than fixed speed units. Thus, the choice between the two speed control options relies on both
techno-economic and demand aspects [38,70]. Fig. 5. Simplified block diagram for synthetic electrical inertia control [80,81]. Here,
𝛥f is the deviation of system frequency, ROCOF is the rate of change of frequency, J is
the virtual inertia control constant, 𝛥𝑃imitate is the active power of the converter, and
𝛥𝑃inertia is the emulated virtual inertia power that could be imitated into the system. controlled converter acts as an AC voltage source with stated voltage,
phase, and frequency. By controlling the voltage magnitude and fre-
quency, the converter behaves very similar to a synchronous generator. The fundamental difference between grid-following and grid-forming
is the way of synchronisation. By applying the swing Eqs. (3) and
(4) [77], the grid-forming control strategy calculates the voltage an-
gle and amplitude deviation, using current power transfer. It is thus
self-synchronising. Therefore, a converter using grid-forming control
coupled with an ESS is currently being discussed as a viable alternative
to imitate the synchronous generator‘s behaviour regarding frequency
control, especially its ability to provide synthetic electrical inertia [73,
74,78]. In power plants with rotating mass and consequent inertia that
are decoupled from the grid frequency, an additional control loop is
required that gives a power reference proportional to the derivative
of grid frequency. To provide the additional power requested by the
synthetic inertia, the plant may still rely on the physical inertia present
but due to its decoupled nature depends on said synthetic inertia
control. 4. Grid integration of energy storage systems This shows that ESS are an important factor in the energy transi-
tion and will play a key role in the future. Energy storage systems
will provide inertia for local grid stability as well as other necessary
AS, such as steady state voltage control, fast reactive current injec-
tions, short-circuit current, black start capability, and island operation
capability [79]. To tackle the challenge of increasing IRES and decreasing natural
system inertia without affecting the system stability, two approaches
are feasible. The first is to maintain a minimum number of rotat-
ing machines. Among other purposes, the contribution of short-circuit
power and voltage support can provide the necessary inertia to the
transmission system in a case of disturbances in the grid [75,76]. The second solution is through IRES itself. This occurs by using the
capabilities of the power electronics, or energy storage systems (ESS),
to provide and ensure a stable grid frequency without any synchronous
rotating machines. For this purpose, a grid-forming control mode is cur-
rently being developed and tested in many research projects. Here, the Moreover, ESS will also need to compensate for weather and sea-
sonal fluctuations in the power supply from IRES, specially from wind
and solar power. For all the previous reasons, ESS are becoming in-
creasingly important. New possibilities for medium and long-term ESS
with sufficient storage capacity and flexibility, in accordance with 7 J.P. Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 the respective requirements, are needed to meet the growing demand
from IRES. Low-head PHS system is a power generation system and
serves at the same time as an ESS. This makes the integration of
PHS (low-head or high-head) via grid-forming controlled converter a
vitally important milestone of the energy transition in order to provide
the necessary storage capacity needed for grid stability and flexibility. The grid integration of low-head PHS via a grid-forming, controlled
converter will not only be of great significance for countries with flat
topographies such as Denmark, Belgium, and the Netherlands. It will
also be essential for countries with a high share of offshore wind energy
as these could enable the concept of energy islands. of a stator yoke and the corresponding iron losses can be averted. This
can be achieved by using a north–south PM arrangement of the rotors. Then, the flux path is completely axial, obviating the magnetic function
of the yoke. 5.1. Electric machines In traditional high-head, high-power PHS, synchronous machines
with excitation winding and direct grid connection are used. However,
doubly-fed induction machines have been adopted in Europe since
2006 for lower power applications. Doubly-fed induction machines
are coupled to a partially rated converter with rotor winding to in-
crease the operating range, which increases turbine efficiency at lower
speeds [82]. As can be seen from Eq. (2), RPT operation at low head
and high power reduces the nominal rotational speed for a fixed specific
speed. Therefore, the power take-off (PTO) in low-head PHS needs to
be designed to operate at high efficiency for low rotational speeds. Furthermore, variable speed RPTs require a highly efficient PTO over
a wide operating range. Doubly-fed induction machines with a gearbox
were the classical choice for such low-speed applications. However,
with the recent decrease in cost of power electronics, permanent mag-
net synchronous machines (PMSM) with a fully rated converter are
opted for instead [83–90]. Advantages include a high power density,
high efficiency, and controllability over a wide operating range [91,
92]. Furthermore, PMSMs with a large pole number avert the use of
reduction gearing, which reduces energy losses and increases reliabil-
ity [93,94]. However, the increased cost of permanent magnets (PM)
and converter losses limits its application for high-power installations. It can be concluded that the PMSM is currently the most sensible
electric machine technology for modern low-head PHS due to its high
efficiency and direct-drive capability, although the use of rare earth
materials is a drawback. The principles of axial flux design, modularity
for fault tolerance and data-driven condition monitoring are likely to
play a role in the further improvement of the PMSM. A more recent development in PMSMs is the axial flux PMSM
(AF-PMSM), which has a magnetic flux direction parallel to the axis
of rotation, in contrast to their radial counterparts. These disc-type
machines have a high diameter-to-length ratio, can accommodate high
pole numbers, and are suitable for high-torque low-speed applica-
tions [95–97]. They have a higher power density and use less core
iron, leading to a lower weight [96,98,99], which in turn results in a
higher torque-to-weight ratio. The possible topologies are single-stator
single-rotor (SSSR), double-stator single-rotor (DSSR), or single-stator
double-rotor (SSDR). 5.1. Electric machines Furthermore, different concepts can be differ-
entiated on the use of surface mounted or interior PMs, slotted or
slotless armature, presence or absence of stator core, concentrated or
distributed windings, etc. [100]. 4. Grid integration of energy storage systems The single-stator double-rotor topology has already been
adopted in wind and tidal turbine applications [104,105]. An SSDR
with coreless stator maximises efficiency, while averting cogging torque
and torque ripple [106–108]. Since the flux path is axial, grain-oriented
material – which has greater magnetic permeability in one direction –
can be used in the stator slots. This results in significantly lower iron
losses compared to non-oriented material [109], while reducing PM use
compared to coreless alternatives [100]. Especially in high-voltage electric machines, the vast majority of oc-
curring faults are stator faults, followed by rotor and bearing
faults [110]. Therefore, AF-PMSMs with concentrated windings can
offer a significant advantage by adopting a Modular Machine Drive
(MMD) design. If a fault arises in one of the stator windings, the MMD
can compensate this with the other modules and remain functional
albeit the maximum power is reduced [111,112]. This fault-tolerant
design improves the reliability of the electric machine, which is a
considerable advantage for a low-head PHS system providing grid
support. The reliability can be further increased by means of condition
monitoring techniques and fault or anomaly detection methods [113,
114]. Thanks to the drastic increase in computational power in the past
years (both local and in the cloud), these techniques have become more
data-driven, relying on, e.g., machine learning [115,116], including
artificial neural networks [117], support vector machines [118], and
deep learning [119,120]. The use of digital twins for predictive main-
tenance of mechanical components [121] or the full drivetrain [122]
shows promising results and offer a perspective for the future of
condition monitoring [123]. These techniques can be applied on the
electric machine, and in extension on the whole drivetrain. Current,
voltage, magnetic flux, speed, temperature, and vibration signals can be
captured on the electric machine and serve as inputs for the condition
monitoring system. 5.3.1. Maximum power point tracking algorithms Maximum power point tracking (MPPT)-based algorithms are used
to find the optimal speed setpoint for a certain power setpoint in tur-
bine mode. In a low-head turbine, this power setpoint is the maximum
available power, hence the name MPPT. In grid-supporting PHS, this
may not be the case, as the power setpoints depend on grid frequency. Therefore, adjustments need to be made to the existing algorithms. The MPPT algorithms can be divided into direct and indirect meth-
ods. Direct MPPT algorithms are based on iterative extremum-seeking
control algorithms. These algorithms require limited knowledge of the
system, but are inherently slow due to their iterative behaviour, making
them less suitable for grid-supporting PHS. However, they can still be
used in storage systems with lower dynamic requirements because of
their simplicity. Indirect methods are based on a model of the system,
making them more dynamic but less flexible. Most of the existing
MPPT control methods rely on flow rate measurement. However, a flow
rate sensor is costly and has a certain error. In low-head RPTs, the
accuracy can further decrease due to a non-uniform flow, a short intake,
and high turbulence [133]. To evade these drawbacks, Borkowski and
Dariusz [90] presented a flow rate estimator. The estimator is based on
an artificial neural network (ANN), which is trained by experimental
data. Active distribution rejection control (ADRC) is used in torque and
speed control to account for known and unknown electrical, hydrauli-
cal, or mechanical disturbances in the system, increasing performance
and robustness. Guo et al. [84] applies a first order ADRC for a
PMSM in a hydropower application, where the known disturbances are
mechanical friction and hydraulic torque. A second order state observer
is used to estimate the rotational speed and hydraulic torque. ADCR is
especially useful in low-head high-power PHS, since any change in the
system tubes has a significant influence on the head losses, because of
the high flow rate at low head. Direct torque control (DTC) is an alternative control method to FOC. In DTC, the electromagnetic torque and stator flux are controlled by
switching between a discrete number of stator voltage vectors, which
in turn form the stator flux vector interacting with the rotor flux. Based on the torque and flux linkage reference and the current flux
vector position, a lookup table is consulted to select the optimal voltage
vector. If e.g. 5.2. Torque and speed control 7. Control schematic of DTC. Fig. 7. Control schematic of DTC. Fig. 6. Decoupled field oriented control of a PMSM with estimated back-EMF
feedforward. estimator based on phase voltages and currents is used (bottom). These
estimated values are compared to torque and flux setpoints. Hysteresis
controllers then determine the proper voltage vector from a lookup
table, resulting in the switching signals. Direct torque control has a
slightly better torque response compared to FOC and does not require a
position sensor [128,129]. However, DTC relies on an accurate estima-
tor. Especially at low speeds, an estimator based on phase voltages and
currents cannot accurately estimate the stator flux [130] which makes
it less suitable for low-head PHS. Although some improved estimator
algorithms have been studied [130], this drawback is best averted by
using a position sensor in the estimator. Disadvantages of DTC include
variable switching frequency, high harmonic current distortion, and
torque ripple [128,129,131]. To achieve a smoother dynamic response
and thus less torque and flux ripple, space vector modulation (SVM) is
used instead of the lookup table [128,129,132]. Here, 𝑝is the pole pair number and 𝛹PM is the constant flux of the
permanent magnets. Fig. 6 shows the control scheme of a field oriented
controlled PMSM. On the bottom right, the stator currents are measured
and transformed to the rotating 𝑞, 𝑑reference frame. These signals
are compared with the setpoints on the left and controlled by two PI
controllers. These controllers determine the duty ratios resulting in the
Pulse-Width Modulated (PWM) signals for the converter. In FOC, ̂𝑖d is
set to 0. Eq. (7) shows that the machine torque is directly proportional
to 𝑖q, resulting in a highly dynamic control. To achieve decoupled
control of both currents, the coupling terms in Eq. (7) are used as a
feedforward. Furthermore, a back-EMF estimator can be implemented
in the 𝑞current control. Although FOC is highly dynamic and easy to implement, setting
𝑖d = 0 is not the most efficient way to reach a desired torque setpoint
for a PMSM with saliency, like an interior magnet PMSM. Therefore,
maximum torque per ampere (MTPA) control can reduce copper losses
and increase overall efficiency in low-head hydropower applications. The MTPA accomplishes this by minimising 𝑖s =
√
𝑖2
q + 𝑖2
d for every
torque setpoint [124]. 5.2. Torque and speed control Applications with interior magnet PMSMs in
wind turbines found a reduction in Joule losses (up to 4.2%), while
maintaining a dynamic response to changing torque setpoints [124,
125]. 5.3. Power control In the power control of low-head PHS, the goal is to reach a
power setpoint as fast and efficiently as possible. Three main control
parameters are determined: the pump/turbine rotational speed 𝛺; the
inlet vane angle 𝛼; and the blade pitch 𝛽. An RPT with only 𝛺as
control parameter is defined as a non-regulated RPT. A single- and
double-regulated RPT further include, respectively inlet vane control,
and inlet vane and blade pitch control. In low-head PHS, a regulated
RPT is recommended, because it allows the RPT to be operated at high
efficiency in a large operating range of heads, flow rate, and power
setpoint. The position sensor plays a critical role in FOC. However, a po-
sition sensor is costly and its signal can contain noise. Therefore,
saliency-based sensorless rotor position estimators [88,89,126,127] are
proposed for low-power systems, since they can increase reliability and
reduce cost [89]. For low rotational speed runners, as in low-head PHS,
the saliency-based approach is the most suitable [89]. Here, a high
pulse frequency is injected, while the current response, which depends
on the rotor magnetic flux position, is observed. 5.2. Torque and speed control In variable speed PHS, the machine speed is altered to reach a
power setpoint as fast and precise as possible, both in pump and turbine
mode. Therefore, the machine torque must be precisely controlled. Field oriented control (FOC) is a vector control method that has been
widely used in low-head micro-hydropower installations [83–89]. The
main advantage is an independent control of the machine torque, and
thus, highly dynamic performance. This is necessary in PHS to quickly
react to changes to the rapidly fluctuating grid frequency. The general
principle of FOC is to regulate the 𝑖d and 𝑖q currents in the rotating
reference frame. The electrical dynamics of a PMSM can be modelled
by Eqs. (5) and (6). Here, 𝛺𝑒𝛹PM is the back-EMF of the permanent
magnets. 𝑅is the stator resistance. 𝐿q and 𝐿d are the 𝑞and 𝑑axis
inductances, respectively. 𝛺𝑒𝐿𝑖is the armature reaction EMF, through
which the 𝑞and 𝑑schemes are coupled. Single-stator single-rotor topologies [101] are simple in design and
compact. However, there is a strong imbalanced axial force between the
stator and rotor. Therefore, the rotor disc width needs to be increased
to avoid twisting [96]. Double-stator single-rotor topologies [102]
are a valuable alternative to SSDR topologies. Double-stator single-
rotor uses fewer PMs, but experiences more copper losses due to poor
winding utilisation [96]. A DSSR machine with integrated permanent
magnets has a high power-to-inertia ratio, since the rotor disc serves
no magnetic purpose and is eliminated [100]. The reduced inertia is
a significant benefit in a grid-supporting low-head PHS. In a slotted
stator AF-PMSM, cogging torque results from the interaction between
the PMs and the stator slots. This undesired torque can be significantly
reduced by changing the angle between both stators [103]. However,
this also reduces power output. Finally, SSDR is deemed the most
favourable AF-PMSM topology. Next to decreased copper losses, the use 𝑣d = 𝑅𝑖d + 𝐿d
d𝑖d
d𝑡−𝛺e𝐿q𝑖q
(5)
𝑣q = 𝑅𝑖q + 𝐿q
d𝑖q
d𝑡+ 𝛺e(𝐿d𝑖d + 𝛹PM)
(6) (5) (6) By regulating the 𝑑and 𝑞axis currents, the torque can be regulated as
shown in the general torque Eq. (7) of the PMSM. 𝑇= 𝑝3
2
[𝛹PM𝑖q + (𝐿d −𝐿q)𝑖d𝑖q
]
(7) 𝑇= 𝑝3
2
[𝛹PM𝑖q + (𝐿d −𝐿q)𝑖d𝑖q
] (7) 8 Renewable and Sustainable Energy Reviews 158 (2022) 112119 J.P. Hoffstaedt et al. Fig. 6. Decoupled field oriented control of a PMSM with estimated back-EMF
feedforward. Fig. 7. Control schematic of DTC. Fig. J.P. Hoffstaedt et al. Especially in a low-head high-power system,
where the flow rate is high, these losses have a significant influence on
the efficiency. Gradient descent control is a direct maximum efficiency point track-
ing (MEPT) algorithm that allows multiple control variables, opposed
to the perturb and observe algorithm. However, to derive the efficiency,
an accurate flow sensor is necessary, which was discussed to be a
challenge in low-head PHS systems [133]. On every operating point,
the control variables are incremented with the direction of their partial
derivatives of efficiency at the current operating point, multiplied by a
step size 𝑘[138]. In Eq. (10), 𝛼is the vane opening and 𝛽is the blade
pitch. 𝛺11 = 𝛺𝐷
√
𝐻
,
𝑄11 =
𝑄
𝐷2√
𝐻
(12) (12) 𝑄11 and 𝛺11 can also be compensated when the Reynolds number of the
real system differs from the design [145]. Fraile-Ardanuy et al. [146]
applied a hill chart to a control system in order to find the optimal
efficiency speed for given 𝛼and measured 𝑄. For a reduced-scale
RPT [147], a lookup table is trained based on measurements. The
lookup table is used to find ̂𝛺for given measured 𝐻and ̂𝑃. However,
the speed is controlled by 𝛼, while it was shown in the paper that
𝑄11 = 𝑓(𝛼, 𝛺). However, the proposed control system is promising for
low-head hydropower if both 𝛺and 𝛼are controlled separately. Then,
the RPT could be controlled to reach ̂𝑃at the highest efficiency for a
certain measured 𝐻. 𝑄11 and 𝛺11 can also be compensated when the Reynolds number of the
real system differs from the design [145]. Fraile-Ardanuy et al. [146]
applied a hill chart to a control system in order to find the optimal
efficiency speed for given 𝛼and measured 𝑄. For a reduced-scale
RPT [147], a lookup table is trained based on measurements. The
lookup table is used to find ̂𝛺for given measured 𝐻and ̂𝑃. However,
the speed is controlled by 𝛼, while it was shown in the paper that
𝑄11 = 𝑓(𝛼, 𝛺). However, the proposed control system is promising for
low-head hydropower if both 𝛺and 𝛼are controlled separately. Then,
the RPT could be controlled to reach ̂𝑃at the highest efficiency for a
certain measured 𝐻. 𝛥𝛼= 𝑘𝜕𝜂
𝜕𝛼
𝛥𝛽= 𝑘𝜕𝜂
𝜕𝛽
𝛥𝛺= 𝑘𝜕𝜂
𝜕𝛺
(10) (10) If the time constants of the control parameters are known, 𝑘can
be chosen differently for each control parameter. J.P. Hoffstaedt et al. If 𝐾is too
low after oscillations and the MPP shifts, the dynamic response is
poor. To allow both dynamic response and minimal power fluctuation
around the MPP, 𝐾can be taken proportional to the power gradient
𝛥𝑃
𝛥𝛺[134]. Due to the parabolic nature of the turbine characteristics,
𝐾is high when far away from the MPP and gradually decreases when
the MPP is neared. Note that using this gradient-based step size cannot
be used to reach a lower power setpoint ̂𝑃. However, the step size
here can be proportional to | ̂𝑃−𝑃|. In wind turbine applications, fuzzy
logic is recently used to find the value of 𝐾, where the perturbed
variable is the generator voltage, which is proportional to the generator
speed [135–137]. Inherent to this method is that the system will still perturb 𝛺when the
MPP is reached, resulting in an oscillation around the MPP. Step size
𝐾is an adaptive value that increases when the power is continuously
rising and decreases when the power is fluctuating [83]. However,
the dynamic behaviour of this method is not optimal. If 𝐾is too
low after oscillations and the MPP shifts, the dynamic response is
poor. To allow both dynamic response and minimal power fluctuation
around the MPP, 𝐾can be taken proportional to the power gradient
𝛥𝑃
𝛥𝛺[134]. Due to the parabolic nature of the turbine characteristics,
𝐾is high when far away from the MPP and gradually decreases when
the MPP is neared. Note that using this gradient-based step size cannot
be used to reach a lower power setpoint ̂𝑃. However, the step size
here can be proportional to | ̂𝑃−𝑃|. In wind turbine applications, fuzzy
logic is recently used to find the value of 𝐾, where the perturbed
variable is the generator voltage, which is proportional to the generator
speed [135–137]. Hill charts define the relation between flow rate 𝑄, rotational speed
𝛺, inlet vane angle 𝛼, and efficiency 𝜂for a constant head 𝐻. Therefore,
if 𝛺and 𝛼are known, 𝑄and 𝜂can be read from the graph. Furthermore,
𝛼and 𝜂are plotted versus the unitary rotational speed 𝛺11 and unitary
flow rate 𝑄11 in Eq. (12), making hill charts scalable for different heads. However, in a real system, the losses 𝐻L(𝑄) have to be taken into
account, making it difficult to estimate the net head across the turbine
without a flow rate sensor. J.P. Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 Although this model can be used in low-head control models, it is
important to note that flow rate Q is a function of head and rotational
speed 𝑄= 𝑓(𝐻, 𝛺) for a non-regulated RPT. Therefore, changing 𝛺will
affect flow rate 𝑄as well. Zhang et al. [142] proposes a polynomial
empirical equation for the efficiency of a turbine 𝜂ℎ(𝛺, 𝑄), where the
coefficients can be derived from experimental data. Borkowski and
Dariusz [143] used an ANN to compose and validate an efficiency
equation 𝜂ℎ(𝛺, 𝑄) together with a flow rate characteristic 𝑄(𝛺, 𝛼) for
a regulated turbine. The control system based on these characteristics
requires no flow rate sensing. However, the 𝑄characterisation is for a
constant head and the flow rate is approximated by a linear function of
speed. This limits its application under varying head at low rotational
speeds. Among direct MPPT control methods, the perturb and observe
algorithm has been investigated for non-regulated low-head turbines. The principle operation consists of altering or perturbing the rotational
speed 𝛺, i.e. accelerating or decelerating, and analysing the change in
output power 𝑃, measured at the electric machine or converter. If the
power has increased, the sign of d𝛺is maintained and the procedure
continues. Otherwise, d𝛺is reversed [83]. Eq. (8) shows how the speed
setpoint ̂𝛺is altered after each iteration. Note that 𝛿(𝑡) implies the sign
of d𝛺. ̂𝛺= ∫
𝑡𝑘−1+𝑇𝑠
𝑡𝑘−1
𝐾𝛿(𝑡) d𝑡
(8)
𝛿(𝑡) = sgn(𝑃𝑘−𝑃𝑘−1) sgn(𝛺𝑘−𝛺𝑘−1)
(9) (8) (9) Similarly, ANN has also been used to form lookup tables [144]. Lookup tables can be constructed over a large operating range during
on-site measurements or by using an existing dataset. Pérez-Diaz and
Fraile-Ardanuy [144] use two ANNs to train the head and efficiency
for input parameters 𝑄, 𝛺, and 𝛼. A possible application of the result-
ing lookup tables is to find reference
̂𝛺and ̂𝛼to reach the optimal
efficiency for a given head. In this control system, no flow sensor is
necessary, thus reducing cost and increasing reliability, especially for
low-head systems. Inherent to this method is that the system will still perturb 𝛺when the
MPP is reached, resulting in an oscillation around the MPP. Step size
𝐾is an adaptive value that increases when the power is continuously
rising and decreases when the power is fluctuating [83]. However,
the dynamic behaviour of this method is not optimal. J.P. Hoffstaedt et al. Furthermore, 𝑘can
be adaptive and defined by a line search algorithm at every itera-
tion [139]. Although this control algorithm shows great potential, any
disturbances on the gradient estimation due to measurement error
or mutual influence between control parameters has a great impact
on the convergence [138]. Therefore, a moving average filter and a
Kalman filter can, respectively, be used to increase robustness [90,140]. Furthermore, Borkowski [90] accounts for the time delay of flow rate
settlement after a change in turbine control parameter further reduce
risk of a non-converging control. One drawback of using turbine characteristics is that the electrical
machine and converter losses are not included. Therefore, the overall
MPP may differ from the turbine MPP [87]. De Kooning et al. [148]
found that the MPP displacement in wind turbines was greater for low
wind and thus lower rotational speeds. In direct MPPT methods, these
losses are included if the power is measured on the converter side. An-
other drawback of indirect MPPT methods is that they do not account
for system performance deterioration over a long time period. However,
reinforcement learning, as proposed for wind turbines [149,150], can
solve this problem at the cost of a higher real computational intensity. Indirect MPPT algorithms rely on prior knowledge of the system in
order to determine the optimal torque or speed reference to achieve a
power setpoint. Recently, this system knowledge is mostly captured in
the form of empirical equations, hill charts, or lookup tables, which are
derived from measurements or numerical analysis like computational
fluid dynamics. Márquez et al. [141] derived an empirical formula for
a propeller turbine, which is a modified Kaplan turbine, designed for
low-heads and low flow rates. Eq. (11) relates the non-regulated turbine
efficiency to flow rate and turbine speed. 5.3.1. Maximum power point tracking algorithms the torque must be increased, a voltage vector is selected
so that the angle between stator and rotor flux is increased. Fig. 7
visualises the control schematic. To find the torque and stator flux, an 9 6. Modelling of low-head systems The overall objective of developing a model of a given system is
to have a representation of the real world. Since such a model will
always be a simplified depiction, it is crucial to weigh which aspects
are essential and what should be left out or simplified. In the case of
numerical models, this also helps to improve performance and reduce
the computational resources necessary. At the end, a well formulated
model allows to predict the behaviour of a system to a greater extent
and wider scenarios than experiments and interpolating empirical data. These predictions are crucial in developing such systems to understand
performance and dynamics, aid optimisation, and can be required for
accurate control during operation. The mathematical models are typi-
cally derived from first principles, such as balance equations of mass,
energy, or momentum, but can also be based on phenomenological or
empirical observations or a mixture of both. 𝜕𝐻
𝜕𝑡= −𝑈𝜕𝐻
𝜕𝑥−𝑎2
𝑔
𝜕𝑈
𝜕𝑥
(14)
𝜕𝑈
𝜕𝑡= −𝑈𝜕𝑈
𝜕𝑥−𝑓𝑈|𝑈|
2𝐷
−𝑔𝜕𝐻
𝜕𝑥
(15) (14) (15) Here we have H as the pressure head, U as water velocity, a as the
pressure wave velocity, g as gravitational acceleration, D as the conduit
diameter, and f as friction factor. If appropriate, simplifications can
be made neglecting velocity head or friction losses. The set of partial
differential equations can be solved either directly or by transforming
it first into a series of ordinary differential equations. Models for high-head PHS are comprehensive and well researched
while attention to low-head PHS applications has been limited. Fun-
damentally, the same approaches can be used. There are, however,
differences in the relevance of individual model components. From a
hydrodynamic point of view, the major difference is a shift towards
higher flow and reduced head for a given power. The increase in the
mass flow rate of water may cause the system to be more prone to water
hammer effects. Cavitation is a further effect to consider when choosing
model components for a low-head scenario. Reaction turbines, such as
Francis or Kaplan turbines that are suitable for medium- and low-head
applications, are considered more susceptible to the effect [157]. Comparisons of similar approaches have shown that treating the
conduit as rigid results in a reduction in computational resources neces-
sary and hence, decreased simulation time. However, if the underlying
scenario requires, higher accuracy can be achieved when considering
elasticity and compressibility effects [163,164]. 5.3.2. Model predictive control However, the benefits
of adapting MPC for low-head PHS could be substantial, because of the
potentially increased influence of transient effects, as described in Sec-
tion 6.1. For a dynamic system providing frequency support, the MPC’s
computational intensity increases even further, which might slow down
the optimisation algorithm. However, with the recent advancements
made in parallel computing with, e.g., multi-core processors (CPUs) and
many-core processors such as graphical processing units (GPUs), the
MPC process can be accelerated [156], making it suitable for complex
dynamic systems like low-head PHS. of variable speed control which may cause even lower speeds at off-
design operation points, this may, for example, not just affect the choice
of motor-generator architecture but could also affect the drivetrain
losses. When choosing modelling approaches for individual system
components, considering these characteristics specific for low-head PHS
helps to cover the relevant aspects while also optimising performance. 5.3.2. Model predictive control An important factor in RPT operation and control that is often
overlooked in traditional MPPT strategies is the transient effect of the
water supply system caused by a control action. The transient flow
equations are described in Section 6.1. Fang et al. [151] stated that
increasing the control action magnitude actually decreased the output
power, while the settling and maximum turbine pressure deviation
increased. Therefore, it can be seen how using an MPPT control that
does not account for these effects can have poor dynamic behaviour 𝜂ℎ(𝛺, 𝑄) = 3.33 𝑄
[
1
2
(
90
𝜆𝑖
+ 𝑄+ 0.78
)
𝑒
−50
𝜆𝑖
]
(11)
𝜆𝑖=
[
1
(𝜆+ 0.089) −0.0035
]−1
,
𝜆= 𝑅𝐴𝛺
𝑄 (11) 10 J.P. Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 when applied to a real system. In some studies on MPPT control, the
water inertia time 𝑇𝑤is incorporated as a time delay on the control
action [90] or as an extra mechanical inertia on the RPT [152]. How-
ever, this does not fully capture the transient effects. Therefore, model
predictive control (MPC) is applied to PHS systems [153–155]. In MPC,
a detailed model of the full PHS system, including hydraulic transients,
losses, and an RPT model, are used. Based on a certain operating state
setpoint, an internal optimisation algorithm simulates control actions
and observes the predicted outcomes of the model. The outcomes are
then given a cost value based on the power response. These predictions
are made for multiple future time samples. Each time sample, this
process is repeated. Therefore, MPC is an accurate control method
that can work on complex systems, at the cost of a significantly high
computational intensity. Furthermore, MPC can also incorporate system
constraints, such as maximum pressure deviation and mechanical rate
limits. Chaoshun et al. [153] proposed using a nonlinear MPC, which
includes the elastic water hammer effect in a high-head PHS plant. Liang et al. [154] used MPC to define the optimal switching time
between pump and turbine mode for a multi-RPT PHS plant. However,
these studies did not include pressure constraints, which are especially
important in systems with long pipelines. The MPC for a 40 metre
PHS plant by Mennemann et al. [155] included this effect. MPC is
currently mostly investigated for high-head PHS. 6.1. Hydrodynamics One of the most common approaches in PHS is to model the
penstock as a rigid conduit in addition to the consideration of water
being an incompressible fluid. A widespread approach making these
assumptions is based on the net force on the body of water which can be
given through both the rate of change of momentum and the differences
in pressure head to obtain the change in time of the volumetric flow
rate as shown in Eq. (13) [158–160]. 𝐿d𝑄
d𝑡= (𝐻−𝐻T −𝐻L
) 𝐴𝑔 𝐿d𝑄
d𝑡= (𝐻−𝐻T −𝐻L
) 𝐴𝑔
(13) (13) Here, Q is the volumetric flow rate through the conduit and turbine,
H is the head over the body of water, 𝐻T is the turbine head, 𝐻L
represents the head losses within the conduit, g is the gravitational
acceleration, A is the conduit cross-sectional area, and L is the conduit
length. The turbine head can be obtained either from empirical data
or physical models as a function of both its rotational speed and flow
rate, while the major and minor head losses are typically represented
through well known hydraulic models, such as the Darcy–Weisbach
formulation in combination with the Colebrook equation [50]. To
complete the conduit component of the system model, these equations
are typically coupled with equations relating to a governor, such as a
gate or guide vanes and mechanical power of the turbine as given in
Eq. (1). Using the above mentioned combination of ordinary differential and
algebraic equations to model the water column as a rigid body is one
the simplest methods to cover the system dynamics. However, to accu-
rately depict transients in the system, such as travelling pressure waves,
an approach using coupled partial differential equations considering
compressibility and elasticity is required [161]. This could be partic-
ularly relevant in applications where enlarged mass flow rates of water
are subject to sudden changes of flow rates and heads, such as low-head
PHS. A commonly used 1-D approach considering these effects uses
the so-called, water hammer equations. These relate pressure head and
water velocity as a function of position and time as shown in Eqs. (14)
and (15) [162,163]. 6.2. Power take-off However, it is important to include
the whole system with its losses to have precise control. Model
predictive control is a computationally intense control method
that can account for transient effects in complex systems, making
it a valuable option for low-head PHS. 𝐽d𝜔
d𝑡= 𝜏h −𝜏g −𝐷f𝜔
(17) 𝐽d𝜔
d𝑡= 𝜏h −𝜏g −𝐷f𝜔 (17) This ordinary differential equation relates the change in angular ve-
locity 𝜔and rotational mass moment of inertia of the system J to
the balance of hydraulic torque 𝜏h, generator torque 𝜏g, as well as
friction typically represented as a viscous damping torque 𝐷f𝜔. Typical
implementations modelling the electrical dynamics of motor-generators
use space-vector representation with the d-q frame of reference. An
example for such an approach for a PMSM can be found in Eqs. (5)
and (6) described in Section 5.2. This ordinary differential equation relates the change in angular ve-
locity 𝜔and rotational mass moment of inertia of the system J to
the balance of hydraulic torque 𝜏h, generator torque 𝜏g, as well as
friction typically represented as a viscous damping torque 𝐷f𝜔. Typical
implementations modelling the electrical dynamics of motor-generators
use space-vector representation with the d-q frame of reference. An
example for such an approach for a PMSM can be found in Eqs. (5)
and (6) described in Section 5.2. • When modelling low-head PHS, the same fundamental approaches
of traditional PHS can be used. However, the change in system
characteristics being a shift to larger masses of water and reduced
head requires more attention on certain model components. Mod-
elling hydrodynamics in more detail allows to cover transients
such as water hammer and estimate the risk of cavitation. Paying
further attention to changing motor-generator dynamics can help
to accurately predict performance and improve control. • When modelling low-head PHS, the same fundamental approaches
of traditional PHS can be used. However, the change in system
characteristics being a shift to larger masses of water and reduced
head requires more attention on certain model components. Mod-
elling hydrodynamics in more detail allows to cover transients
such as water hammer and estimate the risk of cavitation. Paying
further attention to changing motor-generator dynamics can help
to accurately predict performance and improve control. A further point of interest to modelling the PTO of low-head PHS
in combination with variable speed operation is a potential shift to
higher Joule and reduced iron losses. 6.2. Power take-off Variable speed operation of PHS does not just enable to work at
improved efficiency under varying conditions but also improves the ca-
pability to provide AS. When modelling newly developed low-head PHS
systems, this should be considered. For variable speed systems in low-
head PHS as well as related and comparable wind power generation, a
variety of power take-off architectures and controls are available. Com-
monly used are models representing doubly-fed induction or permanent
magnet synchronous motor-generators, respectively, in combination
with drivetrain models of the change in angular velocity based on
torque balance and inertia [161,170,171]. To model the drivetrain, the
simplest model consider a lumped rigid body approach with a rigid
shaft such that all rotating masses and hence rotational inertias can
be added together, leading to Eq. (17). • For the power take-off, axial flux PMSMs are the most promising
electric machines for low-head PHS due to their high efficiency,
high power density, and suitability for high-torque-low-speed
operation. The machine torque can be controlled to achieve a
speed setpoint by either field oriented control or direct torque
control, with the latter having a slightly better torque response if
a position sensor is used, but increasing torque ripple. Active dis-
tribution rejection control can be used to complement the torque
and speed control, increasing performance and robustness. To
derive the speed setpoint, MPPT algorithms based on RPT models
are suitable for low-head PHS because of their short response time
and steady power output. However, it is important to include
the whole system with its losses to have precise control. Model
predictive control is a computationally intense control method
that can account for transient effects in complex systems, making
it a valuable option for low-head PHS. • For the power take-off, axial flux PMSMs are the most promising
electric machines for low-head PHS due to their high efficiency,
high power density, and suitability for high-torque-low-speed
operation. The machine torque can be controlled to achieve a
speed setpoint by either field oriented control or direct torque
control, with the latter having a slightly better torque response if
a position sensor is used, but increasing torque ripple. Active dis-
tribution rejection control can be used to complement the torque
and speed control, increasing performance and robustness. To
derive the speed setpoint, MPPT algorithms based on RPT models
are suitable for low-head PHS because of their short response time
and steady power output. (16) Such cavitation numbers characterise how close the local reference
pressure is to the vapour pressure of the fluid. It is calculated from
the reference pressure 𝑝r, the vapour pressure at the given temperature
𝑝v(𝑇), and the characteristic system pressure difference 𝛥𝑝. Approaches
using various forms of the cavitation number have shown widespread
use in a variety of hydrodynamic applications. It has been found,
however, that studies giving too much emphasis to it, result in poor
repeatability and inconsistency between them. This is due to a wide
variety of definitions of the cavitation number as well as the neglect
of other factors, such as the influence of geometry, flow velocity, or
fluid temperature [167]. More accurate approaches typically include
representations of the dynamics of the vapour bubble cluster. While
computationally more intensive, these allow to model growth and
collapse of the nuclei based on the Rayleigh–Plesset equation derived
from the conservation of mass allowing to solve for the time-dependent
vapour bubble radius [168,169]. • In a low-head context, the choice of pump-turbine design is highly
dependent on the flow rate of the system. Axial flow pump-
turbines, with variable speed drives, are the most suitable solution
for high flow rates which leads to higher power outputs. On the
other hand, other designs, such as Archimedes screw or rotary
positive displacement configurations, can be beneficial at lower
flow rates and micro- or small-scale locations. • The discussion on grid integration has shown that, to compensate
for an increase in intermittent generation and a reduction in spin-
ning reserves, a combination of grid-forming control alongside
bulk energy storage is necessary. To ensure grid stability, such
systems will need to provide synthetic inertia next to other an-
cillary services, namely steady state voltage control, fast reactive
current injections, short circuit currents, black start, and island
operation capability. 6. Modelling of low-head systems Aside from travelling pressure waves, another effect that may be
of higher relevance in a low-head high-flow system is cavitation. The
inception of cavitation is dependent on the amount and type of particles
in the water allowing for nuclei to sustain [165]. Low-head PHS is
a promising candidate for seawater applications in coastal regions. In
such, breaking waves mixing particles from the seabed with the salty
water could increase the risk for cavitation to occur. Its likelihood is The different relation between flow and head also affects turbo-
machinery and power take-off. The shift to a higher flow and lower
pressure at the machine side closer to the upper reservoir typically
results in lower angular velocity and higher torque at the runner and
motor-generator. This is due to an increased runner tip speed as a
consequence of enlarged machines. In combination with the likely use 11 Renewable and Sustainable Energy Reviews 158 (2022) 112119 J.P. Hoffstaedt et al. Renewable and Sustainable Energy Reviews 158 (2022) 112119 also increased when pump-turbines work at off-design conditions as a
consequence of variable speed operation. 7. Conclusion Due to the rapid rise of intermittent renewable energy sources,
penetration levels will exceed what can be compensated for by alter-
native stability measures, and large-scale integration of energy storage
will become imperative. This increase in demand for short- and long-
term balancing and the provision of ancillary services will contribute
to novel systems turning into cost-effective solutions. While pumped
hydro storage is a promising candidate to improve grid stability, its
limitations in deployability call for a conceptual adaptation. Shift-
ing the operating range from traditional high-head towards low-head
applications could pave the way to utilise PHS in regions where so
far it had not been feasible. Further technological advancements can
significantly contribute to enhancing its capability to improve grid
stability while also making it cost competitive. Based on the lack of
research on low-head PHS, this review discusses challenges and the
potential of low-head PHS while giving an overview of pumped storage
technologies and their applicability to low-head applications. The main
outcomes are the following: This formation of vapour bubbles occurs when the local refer-
ence pressure in the fluid reaches its vapour pressure. Fundamental
numerical estimates for the likelihood of cavitation occurrence use dif-
ferent forms of a dimensionless cavitation parameter, such as Thoma‘s
cavitation number shown in Eq. (16) [166]. 𝜎= 𝑝r −𝑝v(𝑇)
𝛥𝑝
(16) 𝜎= 𝑝r −𝑝v(𝑇)
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in revenue from energy arbitrage and the provision of AS combined
with improved control strategies optimised for these may serve as a
further proof of concept. Additionally, modelling and simulation efforts
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https://revistas.javeriana.edu.co/index.php/MAGIS/article/download/25682/22072, https://www.redalyc.org/journal/2810/281060621014/281060621014.pdf, https://dialnet.unirioja.es/descarga/articulo/7768746.pdf
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Análisis de publicaciones de los primeros 20 números de magis
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Magis
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Análisis de publicaciones
de los primeros 20
magis
números de magis
Analysis of publications for the first 20 editions of magis
Fecha de recepción: DICIEMBRE 16 DE 2018 / Fecha de aceptación: FEBRERO 20 DE 2019 / Fecha de disponibilidad en línea: MARZO DE 2019
doi: 10.11144/Javeriana.m11-23.appm
Christian Hederich-Martínez
hederich@pedagogica.edu.co
Universidad P edagógica N acional , C olombia
https://orcid.org/0000-0003-1080-9973
C atalina Roa-C asas
roa.a@javeriana.edu.co
Pontificia Universidad Javeriana , C olombia
https://orcid.org/0000-0002-8104-5782
Resumen
Este artículo analiza el comportamiento de magis en sus 10
primeros años. Se analizan las investigaciones que se han publicado en 20 números, de 2008 a 2017, con una metodología
que recoge estadísticas descriptivas y un análisis multivariante
de redes de palabras claves. Los resultados presentan tipos de
artículos publicados, enfoques de investigación, indicadores
de colaboración. El análisis multivariante identifica siete diferentes conglomerados temáticos, definidos por coocurrencias
de palabras clave, que resultan asociados con niveles educativos específicos y con momentos de aparición en la revista.
Palabras claves
Artículo científico; publicación científica;
palabra clave; citación
Abstract
This article analyzes the behavior of magis in the first 10
years. The researches published in these first 20 editions,
from 2008 to 2017, have been analyzed using a methodology
that gathers descriptive statistical data and applies a multivariate analysis of keyword networks. The results indicate the
types of articles published so far, their research approaches,
collaboration indicators, quotes and downloads. Multivariate
analysis identifies seven different clusters (groups) of topics
defined by the concurrence of keywords associated to specific educative level and the number of times they have been
included in the journal.
Keywords
Scientific article, scientific publication, keyword, citation
Para citar este artículo / To cite this article
Hederich-Martínez, C. & Roa-Casas, C. (2019). Análisis de publicaciones de los primeros 20 números de magis. magis, Revista Internacional de
Investigación en Educación, 11 (23), 221-242. doi: 10.11144/Javeriana.m11-23.appm
VOLUMEN 11 / NÚMERO 23 / ENERO-JUNIO DE 2019 / ISSN 2027-1182 / BOGOTÁ-COLOMBIA / Páginas 221-242
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Descripción del artículo |
Article description
Este artículo se deriva de un proceso de investigación que analiza el comportamiento
de magis en sus 10 primeros años.
Las revistas científicas se han convertido en el escenario fundamental para la publicación de resultados de investigaciones en los diferentes
campos del conocimiento. Conforman un espacio de intercambio activo y
continuo, en la medida en que ponen a disposición de los investigadores
un repositorio de los avances en su área. De este modo, posibilitan y promueven un intercambio permanente de información y conocimiento entre
especialistas de diferentes instituciones y países, pues los resultados de
investigaciones realizadas en diversos lugares del mundo coexisten en una
publicación. Así, las revistas científicas ponen a disposición de los lectores
artículos originales para ser leídos, valorados y difundidos entre quienes
investigan un área. En este sentido, estas publicaciones constituyen la base
de nuevos procesos que retoman y cuestionan los resultados publicados,
para avanzar hacia la producción de otros nuevos.
En línea con lo anterior, la valoración de los artículos que se publican en determinada área es un acercamiento a la investigación que una
comunidad científica desarrolla. Así, valorar la producción de un campo
es adentrarse en la dinámica de la investigación misma, con sus aportes,
intereses, tensiones y cuestionamientos, para reconocerla como base de
nuevas indagaciones. La continua publicación de artículos por parte de
las revistas, y su disposición en bases de datos e índices internacionales, si
bien no garantiza la lectura de los resultados de las investigaciones, sí hace
posible su visibilidad en espacios a los cuales acudirán los interesados en
su uso y divulgación.
Desde sus planteamientos de origen, magis nace para ser un espacio
académico que permita cualificar prácticas de investigación en el campo
educativo en Colombia y la región (García, 2008). En su definición etimológica, magis significa más y mejor. En el marco de las definiciones de
la revista, el término se refiere al compromiso, la actitud y el empeño de
superación personal de maestros y alumnos, en busca de la realización
del potencial de bondad que hay en cada ser humano. De este modo, su
acercamiento al campo de conocimiento de la educación se postula desde
su comprensión interdisciplinaria, en el marco de una perspectiva cultural,
en un interés por la profesionalización de los agentes y las instituciones
educativas, y un compromiso ético con la educación.
En ese sentido se formula que magis promueva buenas prácticas
pedagógicas, didácticas y de gestión educativa, en línea con la manera
en que estas son manifestación de la formación integral de quienes las
agencian, orientan y estudian, y cómo son apropiadas por los estudiantes
cuando son acompañados por el maestro. En el marco de lo anterior,
desde sus inicios, magis se dirige a investigadores de diferentes disciplinas que generan conocimiento sobre estas prácticas, y busca, también,
llegar a maestros en formación y en ejercicio de los diferentes niveles de
la educación que pretendan construir un panorama profundo de las prácticas educativas y su investigación, además de funcionarios de instancias
gubernamentales que tienen a su cargo la formulación de políticas y directrices de gestión.
magis cumple sus 10 primeros años y nos interesa ver las formas que
han tomado esas apuestas iniciales que fundaron la revista. Así, este análisis se propone como marco de comprensión de su comportamiento en
este tiempo. En este sentido, da cuenta de las investigaciones que se han
publicado en 20 números, de 2008 a 2017, y hace un balance de los tipos
de artículos, países, idiomas e instituciones presentados, el nivel de colaboración entre diferentes autores e instituciones, las metodologías seguidas,
las descargas y citaciones, y un análisis de las redes y las tendencias temáticas que marcan las palabras clave de los artículos publicados. Se trata de
Metodología
PÁGINA
Procedimiento
Se calcularon las estadísticas descriptivas de la
base total de artículos, comenzando con la distribución de frecuencias, continuando con el cálculo de
medidas de tendencia central y concluyendo con un
análisis multivariante de redes de palabras claves para
determinar la coocurrencia de todas las palabras definidas por el autor e incluidas en el resumen. Estos
datos fueron agrupados en tres grandes temas, que
aparecen bajo estos títulos en el artículo: primero,
aquellos que resultaban ser indicadores de colaboración; segundo, los relacionados con un alto indicador
de uso, descarga y citaciones de los artículos publicados; y, tercero, los que describen asociaciones temáticas y de naturaleza semántica, que cierran el análisis.
Resultados
Descripción general
Los textos incluyen reportes de investigación,
reflexión y revisiones, presentaciones de la revista, artículos del cuerpo editorial (editoriales), ensayos, entrevistas, ponencias y reseñas. La tipología de artículos
con la que se realizó el procesamiento no necesariamente corresponde a la que se indica en la descripción
de cada artículo, dado que esta ha cambiado a lo largo
de los números publicados. Para este análisis, se organizaron de acuerdo con las siguientes definiciones:
Muestra
Para el análisis se trabajó sobre la totalidad de
los artículos publicados por magis : desde el volumen
1, número 1, publicado en 2008 hasta el volumen 10,
número 20, publicado en 2017. En total, se consideraron 251 textos.
La base de datos trabajada identifica títulos de
los artículos, cada uno de sus autores, sus instituciones
de afiliación y los países de su localización, tipología
del artículo, metodología seguida —en el caso de los
reportes de investigación—, número de descargas del
archivo PDF en plataforma y de citaciones en Google
magis
Académico y Scopus, y palabras clave definidas por los
autores y presentes en el abstract de los artículos.
Es importante anotar que, en estos veinte números, magis ha editado tres números monográficos:
el número 8, de 2011, dedicado al tema de violencia
en las escuelas; el 12, publicado en 2013, enfocado en
la enseñanza de las ciencias y la diversidad cultural;
y el número 14, disponible en 2014, con foco en los
dilemas y los desafíos de la investigación cualitativa
en educación. Interesa, de manera especial, examinar
el comportamiento de estos números en el contexto
general de lo publicado, a fin de alimentar decisiones
futuras al respecto con base en la evidencia disponible.
• Presentación: textos elaborados por el editor
o el director de la revista para anunciar los
artículos que componen el número.
• Editorial: textos de reflexión y/o análisis sobre procesos o productos de la revista magis
que permiten analizar asuntos concretos de
su trayectoria. Son elaborados por miembros
del comité editorial y/o del equipo editorial.
• Artículos de investigación: textos inéditos
que presentan procesos y resultados de investigaciones terminadas.
Análisis de publicaciones de los primeros 20 números de magis
una mirada en doble sentido. Interna, en la medida en
que permite ver el comportamiento de magis en sus 10
primeros años y su marco de relación con los planteamientos iniciales que le dan origen. Externa, pues da
cuenta de un balance de la investigación en educación
en esos años.
Desde otro punto de vista, creemos que la experiencia acumulada en magis puede ser útil a otras
revistas científicas en el campo de la educación y, en
general, de las ciencias sociales. Desde sus inicios,
magis se dispuso y se mantuvo en el índice bibliográfico nacional —Publindex—, y fue la primera revista
científica nacional del ámbito de la educación en ser
aceptada en la base de Scopus. En este sentido, su
experiencia puede ser muy útil para otras revistas nacionales que empiezan a seguir sus pasos.
Es importante anotar que no se pretende hacer
un juicio de valor sobre la calidad de lo publicado en
estos años. La revista ha seguido procesos editoriales que, en mucho, incluso han excedido los controles
habitualmente establecidos por las revistas científicas
en relación con el número, la diversidad y el nivel de
formación de los revisores, la cantidad de instancias
y los filtros utilizados. Al respecto, estamos bastante
seguros de la calidad de lo publicado. Sin embargo, las
reglas que rigen el uso, la circulación y la difusión de
un artículo particular o de un número completo aún
están bajo nuestra lupa, y nos interesa comprenderlas mucho mejor. Este análisis es, también, un intento
para avanzar en ese sentido.
A continuación, se analiza el comportamiento de magis en estos 10 años, con una metodología
que recoge estadísticas descriptivas y un análisis multivariante de redes de palabras claves. Los resultados
presentan tipos de artículos publicados, enfoques de
investigación, indicadores de colaboración, citas y descargas, y asuntos temáticos como niveles educativos
objeto de las investigaciones y coocurrencia de palabras clave para visibilizar temáticas preferentes que
definen ciertas tendencias.
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• Artículos de reflexión derivados de investigación: textos de reflexión que presentan elaboraciones teóricas, interpretaciones
críticas, conceptualizaciones relacionadas con aspectos epistemológicos o construcciones metodológicas de una o más investigaciones realizadas por los autores.
• Artículos de revisión: revisiones exhaustivas de los avances y
tendencias en un campo de conocimiento, una temática o una
problemática particular. Se caracterizan por presentar una cuidadosa revisión bibliográfica de mínimo 50 referencias citadas.
• Entrevistas: transcripción de una entrevista oral realizada a un
investigador que se destaca por su trayectoria en el campo de la
educación y la investigación en educación.
• Reseñas: textos expositivos sobre el contenido de un libro en educación, con el interés de promover su lectura.
• Ensayos: documentos de reflexión en educación, no derivados de
procesos de investigación concretos.
• Ponencias: textos que se han presentado en eventos académicos
y que recogen reflexiones relevantes y vigentes sobre educación
e investigación en los campos pertinentes.
En este punto puede ser importante distinguir los textos que son
propiamente artículos de otros tipos de documentos que no lo son. Entre
los primeros están los ensayos, los reportes de investigación, las reflexiones
derivadas de investigación y las revisiones. Estos son 203 artículos, que
representan el 80,9% de los textos publicados por la revista. El porcentaje
restante está representado por 48 textos, que corresponden al 19,1% del
total, y agrupan presentaciones de los números, textos editoriales, entrevistas, ponencias y reseñas. Estos últimos no serán tenidos en cuenta en lo
que sigue. La tabla 1 muestra la distribución de la totalidad de los textos
publicados en los primeros veinte números de magis. Una simple división
nos indica que, en promedio, cada número de magis ha sacado a la luz 12,5
textos, de los cuales 10 son artículos.
Tabla 1
Distribución de textos publicados en magis por tipología
Tipología de textos
No.
(%)
% en
artículos
Ensayo
19
7,6
9,4
Investigación
107
42,6
52,7
Reflexión derivada de investigación
61
24,3
30,0
Revisión
16
6,4
7,9
Subtotal
203
80,9
100,0
Presentación
20
8,0
41,7
Editorial
4
1,6
8,3
Entrevista
4
1,6
8,3
Ponencia
4
1,6
8,3
Reseña
16
6,4
33,3
Subtotal
48
19,1
100,0
251
100,0
Artículos
Otros
documentos
Total
Fuente: elaboración propia
En relación con los artículos que serán objeto de nuestro análisis, la
gran mayoría de los textos corresponde a reportes de investigación (107 artículos, que representan el 52,2% del total), seguido por reflexiones derivadas de investigación (61, 30,5%). Los ensayos y las revisiones son los tipos
de artículo con menor frecuencia de publicación en magis, con porcentajes
menores a 10%. Ver gráfica 1.
Revisión
Reflexión derivado
de investigación
Investigación
Fuente: elaboración propia
De la totalidad de los reportes de investigación, el 53,3% corresponde a investigaciones de enfoque cualitativo (57 artículos), mientras que
solo 35 casos son investigaciones de enfoque cuantitativo (32,7%). En 15
investigaciones (14,0%), el enfoque elegido es de tipo mixto.
Tabla 2
Distribución de investigaciones por enfoque de investigación
Enfoque
No.
(%)
Cualitativo
57
53,3
Cuantitativo
35
32,7
Mixto
15
14,0
Total
107
100,0
Fuente: elaboración propia
La mayoría de los textos publicados está en castellano (86,7%), solo
15 artículos están escritos en idioma inglés (7,4%), 11 en portugués (5,4%)
y solo 1 en francés (0,4%).
Indicadores de colaboración
En general, los textos son elaborados por un número variable de
autores, hasta un máximo de diez. En su mayor parte, los textos poseen un
solo autor (88 artículos, que representan el 43,3% del total) o dos autores
(67, 33,0%). El 92,1% de los artículos es escrito por tres autores o menos.
Ver tabla 3.
PÁGINA
Análisis de publicaciones de los primeros 20 números de magis
Ensayo
magis
Gráfica 1
Distribución de artículos por tipología
225
Tabla 3
Tabla 4
Distribución de artículos por número de autores
Distribución de artículos por país de la institución
magis
de afiliación de los autores
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226
Número de autores
No.
(%)
1
88
43,3
2
67
33,0
3
32
15,8
4
9
5
País
No.
(%)
Alemania
1
0,5
Argentina
13
6,0
Australia
2
0,9
4,4
Brasil
13
6,0
3
1,5
Canadá
1
0,5
6
3
1,5
Chile
25
11,5
10
1
0,5
Colombia
80
36,9
Ecuador
2
0,9
Total
203
100,0
España
40
18,4
Estados Unidos
5
2,3
Francia
1
0,5
Grecia
1
0,5
Inglaterra
1
0,5
Israel
1
0,5
México
23
10,6
Perú
1
0,5
Portugal
3
1,4
Reino Unido
2
0,9
Uruguay
1
0,5
Venezuela
1
0,5
Total
217
100,0
Fuente: elaboración propia
Los autores de los artículos publicados aparecen
afiliados a un total de 155 instituciones. Aquellas de
aparición más frecuente son, en su orden, la institución de casa, la Pontificia Universidad Javeriana con
22 apariciones (14,2%), la Universidad Distrital con 16
apariciones (10,3%) y la Universidad Pedagógica Nacional de Colombia con 14 apariciones (9,0%). Otras
instituciones con menores niveles de aparición son la
Universidad de Antioquia con 9 apariciones (5,8%) y,
en este punto, aparecen dos instituciones extranjeras:
la Universidad de Barcelona y CONICET, cada una con
5 apariciones (3,2%). Las demás instituciones muestran cuatro o menos apariciones.
El número de instituciones diferentes que colaboran para producir un único artículo alcanza un
máximo de siete. La mayoría de los artículos están firmados por uno o varios autores de una misma institución (148, 72,9%). En 45 casos (22,2%) figuran dos
instituciones. En un caso bastante atípico, los diez autores que firman un artículo aparecen afiliados a siete
instituciones diferentes.
En consonancia con lo anterior, la colaboración
internacional no es muy alta. Solo en 14 artículos
(6,9%), los autores del texto trabajan en instituciones
localizadas en países diferentes.
Con respecto al país en el que están ubicadas las
instituciones representadas por los diferentes autores,
en Colombia está el 36,9%. Otros países que muestran
buenos niveles de participación en la revista son España (18,4%), Chile (11,5%), México (10,6%), Argentina
(6,0%) y Brasil (6,0%). Entre estos seis países se ubica
casi el 90% de la participación. Las instituciones ubicadas en otros países muestran, cada una, menos del
2% de participación.
Fuente: elaboración propia
Citas y descargas
magis es una revista de acceso abierto. Los datos
de descarga de los documentos en formato PDF representan una buena aproximación al grado de circulación del conocimiento que difunde, aunque, debe
mencionarse, no es el único ni el más preciso. Una vez
descargado un archivo en formato PDF, este puede
ser reproducido y enviado un número indefinido de
veces, sin que quede registro alguno de ello. Aun así,
el dato de descargas puede darnos una buena idea
de la demanda y del interés generado por el artículo
entre los lectores.
La tabla 5 muestra las medidas de tendencia
central de tres indicadores: el número de descargas de
archivos en formato PDF en la web de la revista, el número de citas registradas en la plataforma de Google
Académico y el número de citas registradas en revistas
indexadas en la base de Scopus.
Tabla 5
Mínimos, máximos, medias y desviaciones típicas de las descargas y citaciones de artículos de magis
Mínimo
Máximo
Media
Desviación estándar
Citas Scopus
0
13
1,0
1,75
Citas Google Académico
0
231
11,3
21,80
Descargas PDF
143
47.524
1.313,8
3.493,48
Fuente: elaboración propia
magis
Los resultados muestran que los artículos son descargados en formato PDF en numerosas ocasiones. En promedio, cada artículo ha sido descargado 1.313,9 veces. Si bien hay enormes diferencias al respecto entre
los artículos (D. E. = 3.493,5).
Acoso escolar. De las causas, origen y manifestaciones a la pregunta
por el sentido que le otorgan los actores es el artículo más descargado
entre los publicados por magis ; esta revisión, publicada en 2011, en el volumen 4, número 8, de Luis Evelio Castillo-Pulido, tiene 47.524 descargas,
desde su publicación hasta diciembre de 2018. Este dato, sin embargo, es
atípico, por lo que será excluido de los siguientes análisis. A buena distancia del anterior, el segundo artículo con mayor frecuencia de descargas es
El informe de investigación con estudio de casos, una reflexión derivada
de investigación, de Froylán Antonio Ceballos-Herrera (2009), con 9.214
descargas. La tabla 6 muestra los 20 artículos más descargados.
Análisis de publicaciones de los primeros 20 números de magis
PÁGINA
Tabla 6
Lista de los 20 artículos de magis más descargados
Volumen
4
1
2
8
1
Número
8
2
3
17
2
Año
2011
2009
2009
2016
2009
Tipo
Referencia
Revisión
Castillo-Pulido, L. (2011). El acoso escolar. De las causas,
origen y manifestaciones a la pregunta por el sentido que
le otorgan los actores. Magis, Revista Internacional de
Investigación en Educación, 4 (8), 415-428. Recuperado a
partir de https://revistas.javeriana.edu.co/index.php/MAGIS/
article/view/3572
Descargas
47.524
Reflexión
Ceballos-Herrera, F. (2009). El informe de investigación
con estudio de casos. Magis, Revista Internacional de
Investigación en Educación, 1 (2), 413-423. Recuperado a
partir de https://revistas.javeriana.edu.co/index.php/MAGIS/
article/view/3394
9.214
Investigación
González-Moreno, C.; Solovieva, Y. & Quintanar-Rojas,
L. (2009). La actividad de juego temático de roles en la
formación del pensamiento reflexivo en preescolares. Magis,
Revista Internacional de Investigación en Educación, 2 (3),
173-190. Recuperado a partir de https://revistas.javeriana.
edu.co/index.php/MAGIS/article/view/3407
6.561
Investigación
Montanares-Vargas, E. & Llancavil-Llancavil, D. (2016). Uso
de fuentes históricas en formación inicial de profesores.
Magis, Revista Internacional de Investigación en Educación,
8 (17), 85-98. https://doi.org/10.11144/Javeriana.m8-17.ufhf
6.537
Ensayo
Hoyos-Vásquez, G. (2009). Educación para un nuevo
humanismo. Magis, Revista Internacional de Investigación
en Educación, 1 (2), 425-433. Recuperado a partir de
https://revistas.javeriana.edu.co/index.php/MAGIS/article/
view/3395
5.821
Continúa
227
Volumen
1
magis
1
VOLUMEN 11 / NÚMERO 23 / ENERO-JUNIO DE 2019 / ISSN 2027-1182 / BOGOTÁ-COLOMBIA / Páginas 221-242
PÁGINA
9
Número
1
1
19
Año
2008
2008
2017
Tipo
Referencia
Reflexión
Touriñan-López, J. (2008). Teoría de la educación:
investigación disciplinar y retos epistemológicos. Magis,
Revista Internacional de Investigación en Educación, 1 (1),
175-194. Recuperado a partir de https://revistas.javeriana.
edu.co/index.php/MAGIS/article/view/3363
5.588
Reflexión
Murillo-Torrecilla, F. J. (2008). Los modelos multinivel como
herramienta para la investigación educativa. Magis, Revista
Internacional de Investigación en Educación, 1 (1), 45-62.
Recuperado a partir de https://revistas.javeriana.edu.co/
index.php/MAGIS/article/view/3355
4.463
Investigación
Duque-Gómez, L. (2017). Conflicto social colombiano:
representación en textos escolares de ciencias sociales.
Magis, Revista Internacional de Investigación en Educación,
9 (19), 49-68. https://doi.org/10.11144/Javeriana.m9-19.cscr
4.237
Investigación
Orellana-García, P. & Melo-Hurtado, C. (2014). Ambiente
letrado y estrategias didácticas en la educación preescolar
chilena. Magis, Revista Internacional de Investigación
en Educación, 6 (13), 113-128. https://doi.org/10.11144/
Javeriana.M6-13.ALED
4.199
Reflexión
Mireles-Vargas, O. (2015). Metodología de la investigación:
operaciones para develar representaciones sociales. Magis,
Revista Internacional de Investigación en Educación, 8 (16),
149-166. https://doi.org/10.11144/Javeriana.m8-16.miop
4.050
Investigación
Torres-Puentes, E. & Arias-Gómez, D. (2011). La pedagogía
crítica en la experiencia carcelaria de presas políticas. Magis,
Revista Internacional de Investigación en Educación, 4 (7),
27-43. Recuperado a partir de https://revistas.javeriana.edu.
co/index.php/MAGIS/article/view/3554
3.871
Reflexión
Herrera-González, J. D. (2010). La formación de docentes
investigadores: el estatuto científico de la investigación
pedagógica. Magis, Revista Internacional de Investigación
en Educación, 3 (5), 53-62. Recuperado a partir de https://
revistas.javeriana.edu.co/index.php/MAGIS/article/
view/3526
3.284
Revisión
Uribe-Álvarez, G. & Camargo-Martínez, Z. (2011). Prácticas de
lectura y escritura académicas en la universidad colombiana.
Magis, Revista Internacional de Investigación en Educación, 3
(6), 317-341. Recuperado a partir de https://revistas.javeriana.
edu.co/index.php/MAGIS/article/view/3543
3.260
Revisión
Palacios-Mena, N. & Herrera-González, J. (2013).
Subjetividad, socialización política y derechos en la escuela.
Magis, Revista Internacional de Investigación en Educación,
5(11), 413-437. Recuperado a partir de https://revistas.
javeriana.edu.co/index.php/MAGIS/article/view/5710
3.129
Revisión
Bolívar-Botía, A. (2010). ¿Cómo un liderazgo pedagógico
y distribuido mejora los logros académicos? Revisión de la
investigación y propuesta. Magis, Revista Internacional de
Investigación en Educación, 3 (5), 79-106. Recuperado a
partir de https://revistas.javeriana.edu.co/index.php/MAGIS/
article/view/3528
3.086
Revisión
Andrade-Lotero, L. (2012). Teoría de la carga cognitiva,
diseño multimedia y aprendizaje: un estado del arte. Magis,
Revista Internacional de Investigación en Educación, 5 (10),
75-92. Recuperado a partir de https://revistas.javeriana.edu.
co/index.php/MAGIS/article/view/4166
2.685
Investigación
Estévez-Nenninger, E.; Valdés-Cuervo, Á.; Arreola-Olivarría,
C. & Zavala-Escalante, M. (2014). Creencias sobre enseñanza
y aprendizaje en docentes universitarios. Magis, Revista
Internacional de Investigación en Educación, 6 (13), 49-64.
https://doi.org/10.11144/Javeriana.M6-13.CSEA
2.670
228
6
8
4
3
3
5
3
5
6
13
16
7
5
6
11
5
10
13
2014
2015
2011
2010
2011
2013
2010
2012
2014
Descargas
Continúa
7
2
2
14
3
Año
2009
2014
2009
Tipo
Referencia
Reflexión
Bozu, Z. (2009). El profesorado universitario novel y
su proceso de inducción profesional. Magis, Revista
Internacional de Investigación en Educación, 1 (2), 317-328.
Recuperado a partir de https://revistas.javeriana.edu.co/
index.php/MAGIS/article/view/3387
2.601
Reflexión
Guzmán-Valenzuela, C. (2014). Polos epistemológicos: uso
y construcción de teoría en investigación cualitativa en
educación. Magis, Revista Internacional de Investigación en
Educación, 7 (14), 15-28. https://doi.org/10.11144/Javeriana.
M7-14.PEUC
2.539
Investigación
Ruiz, M. & Mora, M. (2009). Una aproximación a las
prácticas de enseñanza de la lectura y la escritura en una
escuela colombiana en 1940. Magis, Revista Internacional
de Investigación en Educación, 2 (3), 15-28. Recuperado a
partir de https://revistas.javeriana.edu.co/index.php/MAGIS/
article/view/3400
2.431
Fuente: elaboración propia
Vale la pena observar dos puntos de la tabla 6. Primero, en casi todas las ocasiones, los más descargados corresponden a artículos publicados en los primeros cinco años de magis. Esto es esperable pues esos
artículos han estado disponibles más tiempo en la plataforma. Solo seis
artículos relativamente recientes figuran en esa lista: los de Duque-Gómez
(2017), Montanares-Vargas y Llancavil-Llancavil (2016), Estévez-Nenninger,
Valdés-Cuervo, Arreola-Olivarría y Zavala-Escalante (2016).
Segundo, es importante notar una clara presencia de las revisiones
en esta lista. Cinco revisiones aparecen en los 20 artículos más descargados, aun cuando esta no es una tipología particularmente frecuente en
magis. Tal como lo presentamos en la tabla 1, las revisiones son apenas el
7,9% del total de artículos publicados en estos 10 años.
Mientras que el número de descargas en PDF puede ser un buen indicador de lectura de un artículo por parte de estudiantes, los registros de
citaciones son un mejor indicador de su uso por parte de investigadores.
En lo que sigue, examinaremos las citaciones a los artículos publicados en
magis en Google Académico y Scopus.
En promedio, cada artículo de magis ha sido citado 11,38 veces en
Google Académico, con grandes diferencias entre los artículos (D. E. =
21,8). En Scopus, el promedio de citaciones es bastante menor (M = 1,0,
D. E. = 1,7).
La tabla 7 muestra los 20 artículos con mayor cantidad de citaciones
en Google Académico. Los resultados revelan que el artículo más citado
en esta plataforma alcanza las 231 citaciones y corresponde, de nuevo, al
artículo de revisión ¿Cómo un liderazgo pedagógico y distribuido mejora
los logros académicos? Revisión de la investigación y propuesta, de Antonio
Bolívar-Botía (2010), que ya había aparecido en el puesto 16 de los artículos más descargados. A buena distancia del anterior, el segundo artículo
más citado es El acoso escolar. De las causas, origen y manifestaciones a la
pregunta por el sentido que le otorgan los actores, de Luis Evelio CastilloPulido (2011), otra revisión, que alcanza 121 citas. En la tabla 6, este artículo aparece como el más descargado.
Al igual que en las descargas, las revisiones tienden también a ser los
artículos más citados. En la lista de los 20 más citados, cinco son revisiones
y cuatro de ellas ocupan los primeros cuatro puestos.
Descargas
magis
1
Número
PÁGINA
Análisis de publicaciones de los primeros 20 números de magis
Volumen
229
Por otro lado, como ya lo habíamos notado en el caso de las descargas, los artículos más citados han sido publicados en los primeros números
de magis, si bien en este caso no hay excepciones. Entre los 20 artículos
más citados en Google Académico no aparece ninguno de los últimos cinco volúmenes. Ver tabla 7.
magis
Tabla 7
Lista de los 20 artículos más citados en Google Académico
VOLUMEN 11 / NÚMERO 23 / ENERO-JUNIO DE 2019 / ISSN 2027-1182 / BOGOTÁ-COLOMBIA / Páginas 221-242
PÁGINA
Volumen
Número
Año
Tipo
Referencia
Revisión
Bolívar-Botía, A. (2010). ¿Cómo un liderazgo pedagógico
y distribuido mejora los logros académicos? Revisión de
la investigación y propuesta. Magis, Revista Internacional
de Investigación en Educación, 3 (5), 79-106. Recuperado
a partir de https://revistas.javeriana.edu.co/index.php/
MAGIS/article/view/3528
231
Revisión
Castillo-Pulido, L. (2011). El acoso escolar. De las causas,
origen y manifestaciones a la pregunta por el sentido que
le otorgan los actores. Magis, Revista Internacional de
Investigación en Educación, 4 (8), 415-428. Recuperado
a partir de https://revistas.javeriana.edu.co/index.php/
MAGIS/article/view/3572
121
Reflexión
Ceballos-Herrera, F. (2009). El informe de investigación
con estudio de casos. Magis, Revista Internacional de
Investigación en Educación, 1 (2), 413-423. Recuperado
a partir de https://revistas.javeriana.edu.co/index.php/
MAGIS/article/view/3394
87
Revisión
Uribe-Álvarez, G. & Camargo-Martínez, Z. (2011).
Prácticas de lectura y escritura académicas en la
universidad colombiana. Magis, Revista Internacional de
Investigación en Educación, 3 (6), 317-341. Recuperado
a partir de https://revistas.javeriana.edu.co/index.php/
MAGIS/article/view/3543
61
Reflexión
Murillo-Torrecilla, F. J. (2008). Los modelos multinivel
como herramienta para la investigación educativa. Magis,
Revista Internacional de Investigación en Educación, 1 (1),
45-62. Recuperado a partir de https://revistas.javeriana.
edu.co/index.php/MAGIS/article/view/3355
57
Investigación
González-Moreno, C.; Solovieva, Y. & Quintanar-Rojas,
L. (2009). La actividad de juego temático de roles en la
formación del pensamiento reflexivo en preescolares.
Magis, Revista Internacional de Investigación en
Educación, 2 (3). 173-190. Recuperado a partir de https://
revistas.javeriana.edu.co/index.php/MAGIS/article/
view/3407
53
Ensayo
Hoyos-Vásquez, G. (2009). Educación para un nuevo
humanismo. Magis, Revista Internacional de Investigación
en Educación, 1 (2), 425-433. Recuperado a partir de
https://revistas.javeriana.edu.co/index.php/MAGIS/article/
view/3395
48
Investigación
Padilla, C. (2012). Escritura y argumentación
académica: trayectorias estudiantiles, factores docentes
y contextuales. Magis, Revista Internacional de
Investigación en Educación, 5 (10), 31-57. Recuperado
a partir de https://revistas.javeriana.edu.co/index.php/
MAGIS/article/view/4164
41
230
3
4
1
3
1
2
1
5
5
8
2
6
1
3
2
10
2010
2011
2009
2011
2008
2009
2009
2012
Citas Google
Continúa
5
1
3
1
4
2
3
1
5
10
2
6
1
7
4
6
2
Año
2010
2012
2009
2011
2008
2011
2010
2011
2009
Tipo
Referencia
Citas Google
Reflexión
Herrera-González, J. D. (2010). La formación de docentes
investigadores: el estatuto científico de la investigación
pedagógica. Magis, Revista Internacional de Investigación
en Educación, 3 (5), 53-62. Recuperado a partir de
https://revistas.javeriana.edu.co/index.php/MAGIS/article/
view/3526
40
Ensayo
Soares-Leite, W. & Nascimento-Ribeiro, C. A. do. (2012).
A inclusão das TICs na educação brasileira: problemas e
desafios. Magis, Revista Internacional de Investigación
en Educación, 5 (10), 173-187. Recuperado a partir de
https://revistas.javeriana.edu.co/index.php/MAGIS/article/
view/4172
40
Investigación
Santos-Rego, M.; Lorenzo-Moledo, M. & PriegueCaamaño, D. (2009). Aprendizaje cooperativo: práctica
pedagógica para el desarrollo escolar y cultural. Magis,
Revista Internacional de Investigación en Educación, 1
(2), 289-303. Recuperado a partir de https://revistas.
javeriana.edu.co/index.php/MAGIS/article/view/3385
39
Investigación
Valdés-Cuervo, A.; Arreola-Olivarría, C.; AnguloArmenta, J.; Carlos-Martínez, E. & García-López, R.
(2011). Actitudes de docentes de educación básica hacia
las TIC. Magis, Revista Internacional de Investigación
en Educación, 3 (6), 379-392. Recuperado a partir de
https://revistas.javeriana.edu.co/index.php/MAGIS/article/
view/3546
37
Revisión
Solaz-Portolés, J. & Sanjosé-López, V. (2008).
Conocimientos y procesos cognitivos en la resolución
de problemas de ciencias: consecuencias para la
enseñanza. Magis, Revista Internacional de Investigación
en Educación, 1 (1), 147-162. Recuperado a partir de
https://revistas.javeriana.edu.co/index.php/MAGIS/article/
view/3361
37
Investigación
Cartolari, M. & Carlino, P. (2011). Leer y tomar
apuntes para aprender en la formación docente: un
estudio exploratorio. Magis, Revista Internacional de
Investigación en Educación, 4 (7), 67-86. Recuperado
a partir de https://revistas.javeriana.edu.co/index.php/
MAGIS/article/view/3557
36
Investigación
Flórez-Romero, R. & Arias-Velandia, N. (2010). Evaluación
de conocimientos previos del aprendizaje inicial de
lectura. Magis, Revista Internacional de Investigación
en Educación, 2 (4), 329-344. Recuperado a partir de
https://revistas.javeriana.edu.co/index.php/MAGIS/article/
view/3516
33
Investigación
Muñoz-González, J.; Ontoria-Peña, A. & MolinaRubio, A. (2011). El mapa mental, un organizador
gráfico como estrategia didáctica para la construcción
del conocimiento. Magis, Revista Internacional de
Investigación en Educación, 3 (6), 343-361. Recuperado
a partir de https://revistas.javeriana.edu.co/index.php/
MAGIS/article/view/3544
32
Investigación
Jarauta-Borrasca, B. & Medina-Moya, J. (2009).
La formación pedagógica inicial del profesorado
universitario: repercusión en las concepciones y prácticas
docentes. Magis, Revista Internacional de Investigación
en Educación, 1 (2), 357-370. Recuperado a partir de
https://revistas.javeriana.edu.co/index.php/MAGIS/article/
view/3390
32
magis
3
Número
PÁGINA
Continúa
Análisis de publicaciones de los primeros 20 números de magis
Volumen
231
Volumen
1
1
10
Año
2008
2012
Tipo
Referencia
Investigación
Soriano de Alencar, E. & de Souza-Fleith, D. (2008).
Criatividade Pessoal: Fatores Facilitadores e Inibidores
Segundo Estudantes de Engenharia. Magis, Revista
Internacional de Investigación en Educación, 1 (1),
113-126. Recuperado a partir de https://revistas.
javeriana.edu.co/index.php/MAGIS/article/view/3359
32
Reflexión
Molina-Natera, V. (2012). Escritura a través del
currículo en Colombia: situación actual y desafíos.
Magis, Revista Internacional de Investigación en
Educación, 5 (10), 93-108. Recuperado a partir de
https://revistas.javeriana.edu.co/index.php/MAGIS/
article/view/4167
30
Investigación
Rodríguez-Jiménez, O. & Murillo-Torrecilla, F. (2011).
Estimación del efecto escuela para Colombia. Magis,
Revista Internacional de Investigación en Educación, 3
(6), 299-316. Recuperado a partir de https://revistas.
javeriana.edu.co/index.php/MAGIS/article/view/3959
30
magis
5
Número
VOLUMEN 11 / NÚMERO 23 / ENERO-JUNIO DE 2019 / ISSN 2027-1182 / BOGOTÁ-COLOMBIA / Páginas 221-242
PÁGINA
Citas Google
232
3
6
2011
Fuente: elaboración propia
Hay algunas coincidencias entre los artículos de las dos listas que
hemos expuesto. Primero, ambas listas comparten todas las revisiones. Segundo, Educación para un nuevo humanismo, el texto de Guillermo HoyosVásquez (2009), aparece entre los 5 más descargados en la plataforma y
es el séptimo más citado en Google Académico, siendo el único ensayo
que se incluye en estas tablas. Igual ocurre con los artículos de MurilloTorrecilla (2008), Los modelos multinivel como herramienta para la investigación educativa, y González-Moreno, Solovieva y Quintanar-Rojas (2012),
La actividad de juego temático de roles en la formación del pensamiento
reflexivo en preescolares, que aparecen entre los 10 más descargados y los
10 más citados en Google Académico.
Otras coincidencias en las tablas son la presencia repetida de algunos autores con más de un artículo reflejado en las tablas. Francisco Javier
Murillo, por ejemplo, aparece con dos artículos entre los 20 más citados en
Google Académico, al igual que José Darío Herrera quien, en la lista de los
20 más descargados, aparece destacado con dos artículos diferentes. Los
dos autores aparecen tanto en la lista de los más descargados como en la
de los más citados. En este sentido, son autores que han aportado a magis
más de un artículo en los 20 números y sus publicaciones han generado un
número destacado de descargas y citaciones.
Por último, vale la pena examinar las citas en la base de Scopus. En
este caso, el promedio de citas es bastante menor que el mostrado en la
base de Google (media = 1,03; D. E. = 1,75). De los artículos publicados
por magis en estos 10 años, el artículo más citado en esta base alcanza apenas 13 citas y coincide con el más citado en Google Académico: ¿Cómo un
liderazgo pedagógico y distribuido mejora los logros académicos? Revisión
de la investigación y propuesta, de Bolívar-Botía (2012). A este le siguen dos
artículos que alcanzan 8 citas. Solo dos artículos, que alcanzan 4 citas, han
sido publicados en los últimos cinco años. En la tabla 8 se presentan los
artículos que han alcanzado, al menos, cuatro citas en la base de Scopus.
Tabla 8
Lista de artículos con cuatro o más citas en la base de Scopus
1
2
2
1
5
4
1
5
3
5
1
4
3
2
10
9
2
10
6
Año
2010
2008
2010
2009
2009
2012
2012
2009
2012
2011
Tipo
Referencia
Citas Scopus
Revisión
Bolívar-Botía, A. (2010). ¿Cómo un liderazgo pedagógico
y distribuido mejora los logros académicos? Revisión de
la investigación y propuesta. Magis, Revista Internacional
de Investigación en Educación, 3 (5), 79-106. Recuperado
a partir de https://revistas.javeriana.edu.co/index.php/
MAGIS/article/view/3528
13
Reflexión
Murillo-Torrecilla, F. J. (2008). Los modelos multinivel
como herramienta para la investigación educativa. Magis,
Revista Internacional de Investigación en Educación, 1 (1),
45-62. Recuperado a partir de https://revistas.javeriana.
edu.co/index.php/MAGIS/article/view/3355
8
Investigación
Gurr-Mark, D.; Drysdale-George, L. & Mulford, B. (2010).
Australian Principal Instructional Leadership: Direct
and Indirect Influence. Magis, Revista Internacional de
Investigación en Educación, 2 (4), 299-314. Recuperado
a partir de https://revistas.javeriana.edu.co/index.php/
MAGIS/article/view/3514
8
Investigación
González-Moreno, C.; Solovieva, Y. & Quintanar-Rojas,
L. (2009). La actividad de juego temático de roles en la
formación del pensamiento reflexivo en preescolares.
Magis, Revista Internacional de Investigación en
Educación, 2 (3), 173-190. Recuperado a partir de https://
revistas.javeriana.edu.co/index.php/MAGIS/article/
view/3407
7
Investigación
Santos-Rego, M.; Lorenzo-Moledo, M. & PriegueCaamaño, D. (2009). Aprendizaje cooperativo: práctica
pedagógica para el desarrollo escolar y cultural. Magis,
Revista Internacional de Investigación en Educación, 1
(2), 289-303. Recuperado a partir de https://revistas.
javeriana.edu.co/index.php/MAGIS/article/view/3385
6
Reflexión
Molina-Natera, V. (2012). Escritura a través del currículo
en Colombia: situación actual y desafíos. Magis, Revista
Internacional de Investigación en Educación, 5 (10), 93108. Recuperado a partir de https://revistas.javeriana.
edu.co/index.php/MAGIS/article/view/4167
5
Investigación
González-Moreno, C. (2012). Formación del pensamiento
reflexivo en estudiantes universitarios. Magis, Revista
Internacional de Investigación en Educación, 4 (9), 595617. Recuperado a partir de https://revistas.javeriana.
edu.co/index.php/MAGIS/article/view/3578
5
Reflexión
Ceballos-Herrera, F. (2009). El informe de investigación
con estudio de casos. Magis, Revista Internacional de
Investigación en Educación, 1 (2), 413-423. Recuperado
a partir de https://revistas.javeriana.edu.co/index.php/
MAGIS/article/view/3394
4
Investigación
Padilla, C. (2012). Escritura y argumentación
académica: trayectorias estudiantiles, factores docentes
y contextuales. Magis, Revista Internacional de
Investigación en Educación, 5 (10), 31-57. Recuperado
a partir de https://revistas.javeriana.edu.co/index.php/
MAGIS/article/view/4164
4
Investigación
Valdés-Cuervo, A.; Arreola-Olivarría, C.; AnguloArmenta, J.; Carlos-Martínez, E. & García-López, R.
(2011). Actitudes de docentes de educación básica hacia
las TIC. Magis, Revista Internacional de Investigación
en Educación, 3 (6), 379-392. Recuperado a partir de
https://revistas.javeriana.edu.co/index.php/MAGIS/
article/view/3546
4
magis
3
Número
PÁGINA
Continúa
Análisis de publicaciones de los primeros 20 números de magis
Volumen
233
2
1
2010
2008
Investigación
4
Investigación
Soriano de Alencar, E. & de Souza-Fleith, D. (2008).
Criatividade Pessoal: Fatores Facilitadores e Inibidores
Segundo Estudantes de Engenharia. Magis, Revista
Internacional de Investigación en Educación, 1 (1), 113126. Recuperado a partir de https://revistas.javeriana.
edu.co/index.php/MAGIS/article/view/3359
4
Investigación
Rodríguez-Jiménez, O. & Murillo-Torrecilla, F. (2011).
Estimación del efecto escuela para Colombia. Magis,
Revista Internacional de Investigación en Educación, 3
(6). Recuperado a partir de https://revistas.javeriana.edu.
co/index.php/MAGIS/article/view/3959
4
Investigación
Conejeros-Solar, M.; Gómez-Arizaga, M. & DonosoOsorio, E. (2013). Perfil docente para alumnos/as con
altas capacidades. Magis, Revista Internacional de
Investigación en Educación, 5(11), 393-411. Recuperado
a partir de https://revistas.javeriana.edu.co/index.php/
MAGIS/article/view/5709
4
Reflexión
Touriñan-López, J. (2008). Teoría de la educación:
investigación disciplinar y retos epistemológicos. Magis,
Revista Internacional de Investigación en Educación, 1
(1), 175-194. Recuperado a partir de https://revistas.
javeriana.edu.co/index.php/MAGIS/article/view/3363
4
Investigación
Salvà-Mut, F.; Oliver-Trobat, M. & Comas-Forgas, R.
(2014). Abandono escolar y desvinculación de la escuela:
perspectiva del alumnado. Magis, Revista Internacional
de Investigación en Educación, 6 (13), 129-142. https://
doi.org/10.11144/Javeriana.M6-13.AEDE
4
Reflexión
Tobin, K. (2013). A Sociocultural Approach to Science
Education. Magis, Revista Internacional de Investigación
en Educación, 6 (12), 19-35. Recuperado a partir de
https://revistas.javeriana.edu.co/index.php/MAGIS/
article/view/7199
4
magis
1
4
Flórez-Romero, R. & Arias-Velandia, N. (2010). Evaluación
de conocimientos previos del aprendizaje inicial de
lectura. Magis, Revista Internacional de Investigación
en Educación, 2 (4), 329-344. Recuperado a partir de
https://revistas.javeriana.edu.co/index.php/MAGIS/
article/view/3516
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PÁGINA
234
3
5
1
6
6
6
11
1
13
12
2011
2013
2008
2014
2013
Fuente: elaboración propia
Varios puntos deben destacarse en esta lista, especialmente en contraste con las dos anteriores. Primero, aunque el artículo más citado es
una revisión, es el único texto de esta tipología que aparece en la lista.
Desaparece acá la preeminencia de las revisiones ya antes observada, para
dar paso a las investigaciones: de los 17 artículos más citados en Scopus 11
son reportes de investigación.
Segundo, sigue apareciendo una clara tendencia a la citación de
algunos autores y algunos temas. Los trabajos de Bolívar-Botía (2010),
González-Moreno, Solovieva y Quintanar-Rojas (2012), Murillo-Torrecilla
(2008) y Ceballos-Herrera (2009), que ya habían aparecido en las listas
anteriores, vuelven a aparecer en esta lista entre los más citados.
En relación con el idioma, es importante anotar que entre las dos
listas previas, los artículos más descargados y los más citados en Google
Académico, no había presencia de artículos en idiomas diferentes al español. Ahora, en la lista de los más citados en Scopus hay un artículo en
Gráfica 2
Promedio de descargas PDF por número (excluido caso atípico)
2000
1800
1600
1400
1200
1000
800
600
400
200
0
Vol. 1 Vol. 1 Vol. 2 Vol. 2 Vol. 3 Vol. 3 Vol. 4 Vol. 4 Vol. 4 Vol. 5 Vol. 5 Vol. 6 Vol. 6 Vol. 7 Vol. 7 Vol. 8 Vol. 8 Vol. 9 Vol. 9 Vol. 10
Núm. 1 Núm. 2 Núm. 3 Núm. 4 Núm. 5 Núm. 6 Núm. 7 Núm. 8 Núm. 9 Núm. 10 Núm. 11 Núm. 12 Núm. 13 Núm. 14 Núm. 15 Núm. 16 Núm. 17 Núm. 18 Núm. 19 Núm. 20
(2008) (2009) (2009) (2010) (2010) (2011) (2011) (2011) (2012) (2012) (2013) (2013) (2014) (2014) (2015) (2015) (2016) (2016) (2017) (2017)
Fuente: elaboración propia
magis
PÁGINA
Análisis de publicaciones de los primeros 20 números de magis
portugués: el de Soriano de Alencar & Souza-Fleith (2008), titulado Criatividade Pessoal: Fatores Facilitadores e Inibidores Segundo Estudantes de
Engenharia y dos artículos en inglés, el de Tobin (2013), A Sociocultural
Approach to Science Education y el de Gurr-Mark, Drysdale-George &
Mulford (2010), titulado Australian Principal Instructional Leadership:
Direct and Indirect Influence. Esto parece señalar que la publicación en
lengua extranjera, si bien no muestra una incidencia en los artículos más
descargados y citados en Google Académico, sí marca una diferencia en la
citación en Scopus.
A fin de contrastar la hipótesis de que los artículos con mayor tiempo
en la plataforma muestran más descargas y más citaciones, se examinan
las medias de descargas y citaciones por número. Los resultados se presentan en las siguientes tres gráficas.
En términos generales, el mayor volumen de descargas se presenta
en el número 8 que casi alcanza 7.000 descargas. Esto se explica, sin embargo, por un solo artículo, al que ya nos habíamos referido. Excluyendo
el artículo El acoso escolar. De las causas, origen y manifestaciones a la
pregunta por el sentido que le otorgan los actores, de Luis Evelio CastilloPulido (2011), una mirada general sobre el promedio de descargas de los
diferentes números muestra variaciones interesantes.
En promedio, hay 1.313 descargas por número; se destacan los números 1, 2, 3, 16 y 17, que superan la media de 1.400 descargas, en especial los números 1, 2 y 17 que superan las 1.600 descargas. El número
de descargas de los tres primeros números es esperable por el tiempo que
han estado disponibles en línea. Sin embargo, resaltan los números 16 y 17
que, a pesar de haber sido publicados recientemente (menos de 4 años),
tienen una media destacada de descargas en sus artículos. Igual tendencia
muestra el número 19, de muy reciente aparición. Tal vez la única característica distintiva de estos números es que incluyen, en su contenido, artículos en idiomas diferentes del español.
235
Gráfica 3
Promedio de citas en Google Académico por número
35
30
25
20
magis
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PÁGINA
236
10
5
0
Vol. 1 Vol. 1 Vol. 2 Vol. 2 Vol. 3 Vol. 3 Vol. 4 Vol. 4 Vol. 4 Vol. 5 Vol. 5 Vol. 6 Vol. 6 Vol. 7 Vol. 7 Vol. 8 Vol. 8 Vol. 9 Vol. 9 Vol. 10
Núm. 1 Núm. 2 Núm. 3 Núm. 4 Núm. 5 Núm. 6 Núm. 7 Núm. 8 Núm. 9 Núm. 10 Núm. 11 Núm. 12 Núm. 13 Núm. 14 Núm. 15 Núm. 16 Núm. 17 Núm. 18 Núm. 19 Núm. 20
(2008) (2009) (2009) (2010) (2010) (2011) (2011) (2011) (2012) (2012) (2013) (2013) (2014) (2014) (2015) (2015) (2016) (2016) (2017) (2017)
Fuente: elaboración propia
En relación con el promedio de citas de artículos de cada número en
Google Académico, se destacan algunos con más de 20 citaciones (números 5, 8 y 6) que, si bien no son los publicados recientemente, tampoco
corresponden a los más antiguos, de manera que presentan una tendencia
destacada en citación en esta plataforma.
Gráfica 4
Promedio de citas en Scopus por número
2.5
2,0
1.5
1,0
0.5
0,0
Vol. 1 Vol. 1 Vol. 2 Vol. 2 Vol. 3 Vol. 3 Vol. 4 Vol. 4 Vol. 4 Vol. 5 Vol. 5 Vol. 6 Vol. 6 Vol. 7 Vol. 7 Vol. 8 Vol. 8 Vol. 9 Vol. 9 Vol. 10
Núm. 1 Núm. 2 Núm. 3 Núm. 4 Núm. 5 Núm. 6 Núm. 7 Núm. 8 Núm. 9 Núm. 10 Núm. 11 Núm. 12 Núm. 13 Núm. 14 Núm. 15 Núm. 16 Núm. 17 Núm. 18 Núm. 19 Núm. 20
(2008) (2009) (2009) (2010) (2010) (2011) (2011) (2011) (2012) (2012) (2013) (2013) (2014) (2014) (2015) (2015) (2016) (2016) (2017) (2017)
Fuente: elaboración propia
Por su parte, la citación en Scopus muestra valores más bajos y tendencias diferentes a los más citados en Google académico. En esta base
de datos, la tendencia es claramente decreciente, sin mayores excepciones. Los números más citados son, en su orden, los números 4 y 1, con
una media de más de 2 citas por artículo, y el número 11, con poco más
de 1,6 citas por artículo. El resto de los números publicados no supera una
media de 1,3 citas por artículo. A partir del volumen 7, la media de citas
Aspectos temáticos
Nivel educativo
En la medida en que magis es una publicación científica dedicada
al ámbito de la educación, la gran mayoría de los artículos que publica se
dirige a uno o varios niveles educativos específicos. Al respecto, la tabla
9 muestra la distribución de los niveles educativos. Tal como se observa,
hay una buena cantidad de artículos, 55, que no se refieren a un nivel
educativo específico (27,1%). El nivel educativo objeto más frecuente en los
artículos examinados es el universitario, que es mencionado en 70 casos
(34,4%). En 54 artículos se menciona el nivel de secundaria (26,6%) y, con
proporciones similares, en 41 se menciona la primaria (20,2%). El preescolar es el nivel más desatendido en las publicaciones de magis, pues solo es
mencionado en 25 casos (12,3%). La educación no formal, por otro lado,
solo es objeto de trabajo en 6 textos (3,0%).
Tabla 9
Distribución de artículos por el nivel educativo al que están dirigidos
Nivel educativo
No.
(%)
No refiere
55
27,1
Preescolar
25
12,3
Primaria
41
20,2
Secundaria
54
26,6
Universitario
70
34,4
Educación No formal
6
3,0
Fuente: elaboración propia
Análisis de coocurrencia de palabras claves
A fin de categorizar las temáticas preferentemente tratadas en los
artículos, se corrió un análisis de conglomerados (cluster analysis) que identifica grupos de palabras claves, en lengua inglesa, que tienden a presentarse de forma simultánea en los artículos publicados. El análisis se hizo
a través del software VOSviewer. Se consideraron las palabras clave que
habían sido mencionadas en, al menos, tres ocasiones.
magis
PÁGINA
Análisis de publicaciones de los primeros 20 números de magis
cae de forma muy acentuada. Solo el volumen 8, número 16, muestra un
mayor número de citas.
En general, no se identifica un impacto destacable de los números
monográficos (8, 12 y 14) en el panorama de las descargas por número.
Sin embargo, en las citaciones en Google Académico y Scopus algunos de
estos sobresalen. En Google Académico, el número 8, monográfico dedicado a la violencia en las escuelas, supera significativamente el promedio
de citaciones de los números anterior y siguiente en este espacio; es el
segundo número con promedio más alto de citaciones en Google académico. Por su parte, el número 14, dedicado al tema de dilemas y desafíos
en investigación cualitativa en educación, muestra el promedio de citación
más alto en Scopus entre los números publicados de 2013 a 2017. El número 12, sin embargo, dedicado a la educación en ciencias, presenta niveles
relativamente bajos en descargas y citaciones, lo cual seguramente estará
relacionado con una comunidad más pequeña y restringida de maestros,
investigadores e interesados en el tema. Estos resultados nos pueden dar
información importante para la elaboración de monográficos en el futuro.
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238
El procedimiento define siete conglomerados que agrupan 53 palabras clave. Cada palabra está representada por un nodo en la gráfica. Los
tamaños de los nodos representan su peso relativo y el color de los nodos
representa el conglomerado al que pertenece.
El primer conglomerado, rojo en la gráfica 5, agrupa 11 palabras, a
saber: bullying, violence, school violence, social representations, teacher
development, teaching practice, school, primary education, youth, environmental education y knowledge. Por sus características, este conglomerado
contiene artículos sobre agresión y violencia en la escuela primaria, en relación cercana con el rol del maestro frente a este fenómeno, en cuanto
recoge elementos relacionados con su desempeño y sus prácticas. Vincula,
además, las representaciones sociales y las condiciones del entorno que
podrían influir en la presencia de fenómenos de violencia en la escuela.
El segundo conglomerado de palabras clave, de color verde en la
gráfica 5, agrupa 10 ítems. Por sus características, se incluyen los artículos
relacionados con aspectos culturales: educación intercultural e indígena en la educación básica, en los que tiende a aparecer la metodología de enseñanza conocida como “aprendizaje cooperativo”, junto con
el aprendizaje activo. Podría pensarse que refieren a investigaciones que
se relacionan con procesos de intervención en comunidades específicas.
Aparecen en este conglomerado referencias geográficas puntuales (México, Latinoamérica).
El tercer conglomerado, azul oscuro en la gráfica 5, agrupa nueve
ítems que se articulan alrededor de la enseñanza y el aprendizaje en la
educación superior, en especial con objetos relacionados con la lectura, la
escritura y el entrenamiento de habilidades en una disciplina concreta. La
práctica reflexiva parece ser una metodología de aparición frecuente en
este conglomerado. Dado que se trata de un tema de alta relevancia en la
literatura en los últimos años, es posible pensar que se agrupan artículos
que recogen experiencias sobre el tema del lenguaje en las universidades,
desde la idea de repensarlas, tensionarlas y reconstruirlas, desde la práctica reflexiva.
El cuarto conglomerado, amarillo en la gráfica 5, agrupa 8 ítems.
Este parece ser de carácter bastante general, pues agrupa conceptos como
educación, ciencia, comunicación y menciones a la metodología de la investigación en la universidad. Hay también referencia a la memoria y a las
emociones, lo que hace pensar en reflexiones generales sobre el conocimiento pedagógico.
El quinto conglomerado, de color violeta en la gráfica 5, contiene 7
ítems y parece girar en torno a temas de política e investigación educativa,
mencionando las palabras epistemología, alfabetización, educación a través
de los medios. Aparecen en este conglomerado referencias a la metodología de investigación y, específicamente, a metodologías de tipo cualitativo.
El sexto conglomerado, azul claro en la gráfica 5, contiene cinco
ítems y se refiere específicamente a temas de educación en ciencias y cultura científica en el nivel secundario. Los conceptos de argumentación y
cultura escolar tienen una fuerte presencia en este grupo de artículos.
Dado que en este conglomerado se ubica el nivel secundario, puede tener
relación con los procesos escolares que buscan dar a los estudiantes las
herramientas necesarias para afrontar la vida universitaria, pues es posible
relacionar la cultura científica con procesos de argumentación académica.
Finalmente, el séptimo conglomerado, de color naranja en el gráfico 5,
contiene solo tres ítems y agrupa los trabajos sobre docentes y prácticas de
enseñanza, en especial dirigidos a preescolar, que como vimos previamente es un nivel poco atendido en los artículos de magis.
Gráfica 5
Red de coocurrencias de palabras clave
magis
En general, vale la pena anotar que ciertos objetos de investigación
aparecen ligados a niveles educativos específicos. Los temas de maestros
y prácticas de enseñanza están ligados a la educación preescolar. La educación básica primaria, por su parte, aparece en relación con temas de
educación intercultural y acoso escolar. En la educación secundaria se han
publicado temas de alfabetización y cultura científica. A su vez, la educación universitaria parece enfatizar en temas de la enseñanza y el aprendizaje de la lectura y la escritura. Lo anterior muestra una cercanía entre lo
abordado en educación secundaria y universitaria.
Análisis de publicaciones de los primeros 20 números de magis
PÁGINA
Fuente: elaboración propia
Sobre la base de la red de palabras que se acaba de presentar, es
posible superponer la información de años de publicación. El resultado se
muestra en la gráfica 6.
Como se observa, la tendencia de los artículos más recientemente publicados se relaciona con las temáticas de la educación intercultural (segundo conglomerado), los temas de juventud, educación primaria y secundaria,
y los de alfabetización y desarrollo profesoral. Las temáticas relacionadas
con educación en ciencias y las reflexiones sobre la naturaleza del saber
educativo parecen concentrarse en los primeros números de magis.
En relación con los cambios de esos asuntos que han sido objeto
constante de los artículos publicados en magis en estos 10 años, los conglomerados muestran algunos temas que, más que cambios, evidencian
transformaciones. Por ejemplo, las preocupaciones en torno al maestro
han tomado formas diversas. Inicialmente, el foco estaba puesto en los
239
magis
procesos de enseñanza que agenciaban, su formación y la práctica reflexiva como espacio de transformación. Estos temas se transforman en unos
más específicos que, hacia los últimos números, abordan directamente el
desarrollo de los profesores. Otro tópico constante que se va acotando
es el relacionado con temas culturales. En los primeros números aparece
como tema amplio “lo cultural”, mientras que, en los últimos números,
aunque el interés permanece, se focaliza en asuntos concretos y poblaciones: aparecen con fuerza la educación intercultural y la educación con
población indígena.
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PÁGINA
Gráfica 6
Fecha de publicación en la red de coocurrencias de palabras clave
Fuente: elaboración propia
Discusión y conclusiones
Los resultados indican una revista que se ha mantenido con buenos
niveles de calidad y con una periodicidad regular. En su mayoría, lo publicado está conformado por reportes o reflexiones de investigación, mientras
que los artículos de revisión bibliográfica y/o bibliométrica resultan ser minoritarios. Probablemente, este sea el momento para hacer un llamado a
la comunidad científica —en general— a considerar los estudios históricobibliográficos como un área que se debe explorar, desarrollar y posicionar
en la investigación en educación.
magis
PÁGINA
Análisis de publicaciones de los primeros 20 números de magis
En términos generales, magis ha publicado, principalmente, reportes
de investigación, pues estos manuscritos representan más del 50% de los
artículos de estos 20 números. En los artículos de investigación se identificó un predominio del enfoque cualitativo del 53,3%, frente a porcentajes
más bajos del enfoque cuantitativo y mixto. Esto es consistente con la tendencia predominante en la investigación educativa, en especial la latinoamericana, que muestra cierto privilegio por este enfoque.
Los indicadores de colaboración no son muy altos. El 43% de los artículos son escritos por un solo autor y el 72% de los artículos proviene de
una institución. Solo en el 22% de los artículos aparecen dos instituciones
y únicamente en el 7% de los casos, estas instituciones están localizadas
en países diferentes. Esto puede estar en relación con una tendencia manifiesta a tratar problemas más bien locales que globales, lo cual es visible
tanto en la dimensión de lo institucional como en la de lo nacional. Aun
así, puede destacarse que en casi el 63% de los artículos aparecen autores
afiliados a instituciones localizadas en países diferentes al de la revista, en
especial España, Chile y México. En este sentido, en línea con su proyecto
inicial, magis ha logrado llegar e impactar a lectores de la región.
A partir de este panorama de los primeros 20 números de magis, se
identifican oportunidades sobre asuntos y tipologías que podrían tener
más fuerza en la revista. El análisis permite reconocer la potencialidad de
los artículos de revisión. Este tipo de artículos se destaca en descargas y
citaciones y, en este sentido, se reconoce como tipología apetecida por
estudiantes e investigadores. Los idiomas diferentes al castellano (inglés
y portugués, según las citaciones) son leídos y citados por quienes tienen
acceso a la base Scopus. En este sentido, podría pensarse en aumentar la
presentación de estos idiomas en los números de magis. En relación con
los niveles educativos, se reconoció el preescolar como el más desatendido
en los artículos de magis, de manera que podría potenciarse la publicación
de artículos que aborden este nivel para presentar resultados más diversos
y abarcadores.
Al adentrarnos en aspectos temáticos, se destacan ciertos grupos
en los artículos publicados en magis que aparecen, curiosamente, ligados
a niveles educativos específicos. Las problemáticas ligadas a los docentes
y las prácticas educativas han aparecido especialmente en relación con la
educación preescolar. Los temas de acoso escolar y violencia en la escuela son de reciente aparición y se examinan en relación con la educación
primaria. En la educación básica, primaria y secundaria aparecen también
temas relacionados con interculturalidad en la escuela y, de igual forma,
figuran entre los artículos de más reciente aparición. Se alcanzan a vislumbrar acá algunos temas que fueron apuestas iniciales de magis.
La educación secundaria, por su parte, está ligada en especial a artículos sobre las temáticas de la educación en ciencias, que han sido publicados, principalmente, en los primeros volúmenes. La educación universitaria
aparece, por su parte, asociada preponderantemente con investigaciones
sobre lectura y escritura en los estudiantes. En este sentido, es posible evidenciar una transición tanto temática como en los niveles educativos, pues
en los primeros números se focaliza sobre la enseñanza de las disciplinas y
su abordaje desde la educación secundaria, mientras que, con el pasar de
los años, la preocupación se concentra específicamente en la educación
universitaria y el foco está puesto en las especificidades de la lectura y la
escritura en los aprendizajes de cada disciplina.
Un aspecto destacable es que, pese a que las revisiones, como tipo
de artículo, representan menos del 10% de los artículos publicados en estos 10 años, tienen un alto impacto en las descargas y en las citaciones de
241
magis
VOLUMEN 11 / NÚMERO 23 / ENERO-JUNIO DE 2019 / ISSN 2027-1182 / BOGOTÁ-COLOMBIA / Páginas 221-242
PÁGINA
242
Google Académico. De los 20 textos más citados, cinco
son revisiones y cuatro están en los primeros puestos.
En los más citados de Scopus, aunque solo aparece una
revisión, es el primer artículo de la lista.
Por otro lado, lo presentado da elementos para
considerar la importancia y utilidad de los números
monográficos. Al respecto, aunque positivos, los resultados pueden tener un dejo agridulce. Algunos de
los monográficos muestran altos índices de citación y
de descargas, y otros, no tanto. Esto nos lleva a considerar que las ventajas en la realización de este tipo
de números estarán en relación directa con el interés
que la temática despierte en una comunidad amplia de
maestros y especialistas. Al plantear un monográfico,
automáticamente se restringe de forma importante la
cantidad de lectores del número a los directamente
relacionados con —e interesados en— el tema específico. Esto, por otro lado, es también positivo pues
este número puede convertirse en referencia obligada
sobre el tema.
Lo anterior permite sugerir la búsqueda de una
solución intermedia que retome las ventajas de la restricción temática, sin que por ello enfrentemos una
pérdida de lectores en el número. La solución, en ese
sentido, puede estar dada por la publicación de dossiers
especializados que no alcancen a cubrir la totalidad del
número. Otras revistas, como la Revista Colombiana
de Educación, han seguido antes, con bastante éxito,
ese camino.
En general, el balance muestra distancias significativas entre las descargas hechas sobre los artículos
y la citación que se hace de ellos. Puede pensarse que
quienes descargan son, en su mayoría, estudiantes
universitarios de los diferentes niveles, que consultan
los artículos como marcos para sus propios trabajos e
investigaciones, o bien maestros en ejercicio, más que
investigadores y autores que escriben y divulgan sus
artículos en bases de alto reconocimiento como Scopus. Podría decirse que los artículos son mayoritariamente descargados por un público no especializado
en publicaciones científicas, que no necesariamente
investiga, pero que está altamente interesado y vinculado con la educación. Es decir, no solo estudiantes
universitarios, sino también profesores de los diferentes niveles que, en el marco de estudios continuos o
por interés propio, consultan determinados artículos
de la revista que llaman su atención, de modo que la
utilidad tiene qué ver más con la reflexión y la práctica propia que con la generación de conocimiento
en una investigación formal. Ello respondería a una
de las apuestas de magis desde sus orígenes: aportar
a la consolidación de una comunidad académica que
piensa la educación, no solo desde las investigaciones
de alto nivel, sino en las reflexiones continuas de los
maestros que ejercen cada día.
Para magis, llegar a los 10 años ha implicado,
además del presente análisis, una revisión interna de
cara a las necesidades de cambio que se han evidenciado a lo largo de los años, así como externa en función de los nuevos requerimientos que ahora se hacen
a las publicaciones seriadas. Después de un proceso
de reflexión, desde el equipo editorial y de la mano
con el comité editorial, hemos reformulado varios
elementos para agilizar los tiempos de respuesta a
los autores, ahorrar y focalizar esfuerzos en la evaluación, y ganar terreno en la visibilidad y la lectura
de los artículos publicados. Esto nos llevó, entre otras
decisiones, a reconsiderar la pertinencia del ejemplar
impreso, que funcionaba más como tradición y colección que como lugar real de lectura. Hacia afuera,
surgen cambios que representan aún retos por abordar, como el ajuste en la concepción del número publicado como unidad que nos ha organizado en este
tiempo, para pensar el proceso de publicación del artículo a medida que este culmina su proceso editorial.
En fin, seguimos aprendiendo y cambiando, para dar
lo mejor a nuestros lectores, autores y revisores, en
estos tiempos desafiantes.
Sobre los autores
Christian Hederich-Martínez es matemático. Magíster
en Educación, Universidad Pedagógica Nacional, UPN, Colombia y Doctor en Psicología, Universidad Autónoma de
Barcelona, UAB, España. Profesor, Universidad Pedagógica
Nacional, UPN, Colombia. Editor de la Revista Colombiana de
Educación. Miembro del comité editorial de magis.
Catalina Roa-Casas es licenciada en Pedagogía Infantil,
Pontificia Universidad Javeriana, Colombia. Magíster en Educación, Pontificia Universidad Javeriana, Colombia. Profesora, Pontificia Universidad Javeriana, Facultad de Educación y
Departamento de Formación, Colombia. Editora de magis.
Referencias
García-Cepero, C. (2008). Documento maestro. Magis,
Revista Internacional de Investigación en Educación. Inédito.
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https://openalex.org/W2037443393
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https://docs.lib.purdue.edu/cgi/viewcontent.cgi?article=1011&context=bmepubs
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English
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Subtle Paranodal Injury Slows Impulse Conduction in a Mathematical Model of Myelinated Axons
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PloS one
| 2,013
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cc-by
| 10,752
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Follow this and additional works at: http://docs.lib.purdue.edu/bmepub Recommended Citation
Babbs, Charles F. and Shi, Riyi, "Subtle Paranodal Injury Slows Impulse Conduction in a Mathematical Model of Myelinated Axons."
(2013). Weldon School of Biomedical Engineering Faculty Publications. Paper 14. http://dx.doi.org/10.1371/journal.pone.0067767 7-3-2013
Subtle Paranodal Injury Slows Impulse
Conduction in a Mathematical Model of
Myelinated Axons. Charles F. Babbs
Purdue University, babbs@purdue.edu
Riyi Shi Charles F. Babbs
Purdue University, babbs@purdue.edu Follow this and additional works at: http://docs.lib.purdue.edu/bmepubs Purdue University
Purdue e-Pubs Purdue University
Purdue e-Pubs Weldon School of Biomedical Engineering Abstract This study explores in detail the functional consequences of subtle retraction and detachment of myelin around the nodes
of Ranvier following mild-to-moderate crush or stretch mediated injury. An equivalent electrical circuit model for a series of
equally spaced nodes of Ranvier was created incorporating extracellular and axonal resistances, paranodal resistances, nodal
capacitances, time varying sodium and potassium currents, and realistic resting and threshold membrane potentials in a
myelinated axon segment of 21 successive nodes. Differential equations describing membrane potentials at each nodal
region were solved numerically. Subtle injury was simulated by increasing the width of exposed nodal membrane in nodes
8 through 20 of the model. Such injury diminishes action potential amplitude and slows conduction velocity from 19.1 m/
sec in the normal region to 7.8 m/sec in the crushed region. Detachment of paranodal myelin, exposing juxtaparanodal
potassium channels, decreases conduction velocity further to 6.6 m/sec, an effect that is partially reversible with potassium
ion channel blockade. Conduction velocity decreases as node width increases or as paranodal resistance falls. The calculated
changes in conduction velocity with subtle paranodal injury agree with experimental observations. Nodes of Ranvier are
highly effective but somewhat fragile devices for increasing nerve conduction velocity and decreasing reaction time in
vertebrate animals. Their fundamental design limitation is that even small mechanical retractions of myelin from very
narrow nodes or slight loosening of paranodal myelin, which are difficult to notice at the light microscopic level of
observation, can cause large changes in myelinated nerve conduction velocity. R (2013) Subtle Paranodal Injury Slows Impulse Conduction in a Mathematical Model of Myelinated Axons. PLoS ONE 8(7): e67767
0067767 tation: Babbs CF, Shi R (2013) Subtle Paranodal Injury Slows Impulse Conduction in a Mathematical Model of Myelinated Axons. P
i:10.1371/journal.pone.0067767 Received January 29, 2013; Accepted May 21, 2013; Published July 3, 2013 Copyright: 2013 Babbs, Shi. This is an open-access article distributed under the terms of the Creative Commons Attributi
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: babbs@purdue.edu * E-mail: babbs@purdue.edu membrane. Charles F. Babbs*, Riyi Shi ences, Center for Paralysis Research, and Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana, Unite Department of Basic Medical Sciences, Center for Paralysis Research, and Weldon School of Biomedical Engineering, Purdue Unive
States of America Department of Basic Medical Sciences, Center for Paralysis Research, and Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana, United
States of America Recommended Citation This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact epubs@purdue.edu for
additional information. Abstract Beyond the paranodal regions are the functionally
important juxtaparanodal zones, where the density of transmem-
brane channels carrying outward potassium current is high. Ring-
like bands of paranodal cell-cell attachments separate and insulate
the bare nodes from the juxtaparanodal regions. Since myelin
inhibits the conduction of ionic current, the action potential tends
to jump from one node to the next along the longitudinal axis of
an axon. This process of jumping, or ‘‘saltatory conduction’’,
boosts the speed of propagation of action potentials along
myelinated axons to tens of meters per second, rather than tens
of centimeters per second typical of unmyelinated axons [5]. Introduction Arrows indicate flow of positive ionic current during depolarization of Node 1 as conduction of the action
potential moves toward Node 2. This sketch is foreshortened in the axial dimension. Anatomically L/s , 1000. (b) Schematic cross section of a
myelinated nerve or fiber tract. Each axon (solid black) is surrounded by a sheath of myelin (white) and in turn surrounded by a sheath of non-
myelinated tissue (shaded) known as endoneurium or neuropil. The mean radius of the sheath of endoneurium is denoted re and the thickness of the
sheath is denoted h. The cross section of endoneurium, 2preh, is much greater than that of the axon. doi:10.1371/journal.pone.0067767.g001
Conduction in Injured Myelinated Axons Conduction in Injured Myelinated Axons Figure 1. Relevant anatomy. (a) Schematic longitudinal section of a myelinated axon. The width of each node of Ranvier is denoted s. The
distance between nodes is denoted L. Arrows indicate flow of positive ionic current during depolarization of Node 1 as conduction of the action
potential moves toward Node 2. This sketch is foreshortened in the axial dimension. Anatomically L/s , 1000. (b) Schematic cross section of a
myelinated nerve or fiber tract. Each axon (solid black) is surrounded by a sheath of myelin (white) and in turn surrounded by a sheath of non-
myelinated tissue (shaded) known as endoneurium or neuropil. The mean radius of the sheath of endoneurium is denoted re and the thickness of the
sheath is denoted h. The cross section of endoneurium, 2preh, is much greater than that of the axon. doi:10.1371/journal.pone.0067767.g001 Figure 1. Relevant anatomy. (a) Schematic longitudinal section of a myelinated axon. The width of each node of Ranvier is denoted s. The
distance between nodes is denoted L. Arrows indicate flow of positive ionic current during depolarization of Node 1 as conduction of the action
potential moves toward Node 2. This sketch is foreshortened in the axial dimension. Anatomically L/s , 1000. (b) Schematic cross section of a
myelinated nerve or fiber tract. Each axon (solid black) is surrounded by a sheath of myelin (white) and in turn surrounded by a sheath of non-
myelinated tissue (shaded) known as endoneurium or neuropil. The mean radius of the sheath of endoneurium is denoted re and the thickness of the
sheath is denoted h. The cross section of endoneurium, 2preh, is much greater than that of the axon. Introduction The majority of the axons in vertebrate nervous systems are
wrapped with insulating layers of back-to-back cell membranes
called myelin. The mechanisms by which myelin speeds axonal
conduction in health and by which damage to myelin leads to loss
of axonal conduction in disease have attracted much attention [1]. Still, the causes of myelin related functional deficits remain
incompletely
understood
[2–4]. Continuing
multidisciplinary
investigation
combining
morphology,
electrophysiology,
and
mathematical modeling may elucidate critical mechanisms and
perhaps guide the development of effective treatments for medical
conditions involving myelin damage, including traumatic injury to
the brain or spinal cord and demyelinating diseases such as
multiple sclerosis. Here we explore in detail the functional consequences of subtle
injury to the well-known anatomic arrangement of the nodes of
Ranvier, coupled with the more recently discovered segregation of
nodal sodium channels from juxtaparanodal potassium channels
[6–8]. Compared to the internodal regions, a node of Ranvier has
a relatively high density of sodium channels, which enable the
generation of action potential at the node. The virtual absence of
sodium channels in the paranodal and juxtaparanodal regions,
together with high electrical resistance of the paranodal region,
blocks axial ionic current beneath the myelin sheath and favors
saltatory conduction. Potassium channels, on the other hand, are
segregated from sodium channels in nodes of Ranvier, residing
predominantly beneath the myelin at the juxtaparanodal area, and
separated from the node by the paranodal region [9]. There is now Myelin forming Schwann cells in the peripheral nervous system
or oligodendrocytes in the central nervous system wrap around the
axon multiple times to create laminated layers of insulating cell
membrane, as shown schematically in Figure 1. Periodic short
gaps in the myelin sheath along the axons having width, s,
approximately 0.3 to 1 micrometer, are the Nodes of Ranvier,
where the density of transmembrane channels carrying inward
sodium
current
is
high
and
where
transmembrane
action
potentials are initiated. Immediately adjacent to the nodes
themselves on either side in the axial dimension are the paranodal
regions, where myelin is tightly attached to the underlying axonal July 2013 | Volume 8 | Issue 7 | e67767 1 PLOS ONE | www.plosone.org Figure 1. Relevant anatomy. (a) Schematic longitudinal section of a myelinated axon. The width of each node of Ranvier is denoted s. The
distance between nodes is denoted L. Introduction doi:10.1371/journal.pone.0067767.g001 second order partial differential equation that describes the
voltages at each node as functions of time and space. Such
equations are known as cable equations, because of their similarity
to cable or transmission line equations [25], and the corresponding
models of
nerve conduction
are
known as
cable models. Subsequent, more detailed mathematical treatment of composite,
myelinated axon models [23,24] leads to expressions essentially
similar to the original solutions of FitzHugh. (Compare, for
example, Basser Eq. (35) and Nygren and Halter Eq. (18) with
FitzHugh Eq. (1)). general agreement that mechanical crush or stretch injury results
in myelin damage, especially in the paranodal region [10,11]. This
idea is consistent with biomechanical modeling showing significant
stress in this area under mechanical insults [12], causing retraction
of myelin away from the node [13,14]. With more severe injury
there is also de-coupling and detachment of paranodal myelin,
opening a low resistance gap or sleeve-like channel between the
node and the juxtaparanodal region, through which increased
potassium ion current may flow [10]. Subtle retraction and detachment of paranodal myelin can have
two electrical effects. The first is increasing bare nodal area, which
increases the electrical capacitance of the node. The second is
loosening of paranodal cell-cell junctions, which reduces the
normally high electrical resistance between the juxtaparanodal
potassium channels and the nodal capacitance. In the present
paper we consider the biophysics of nodal function before and
after traumatic myelin retraction and partial detachment to
explain and predict the effects of such subtle mechanical injury,
which is sufficiently mild that it is clearly visible only at the
electron microscopic level of observation [10]. Our approach is to
employ a mathematical model of ionic current flow that reflects
changes caused by such subtle injury to the nodal and paranodal
regions. The goal of the present paper is to determine quantitatively how
myelinated nerve conduction velocity depends upon the local
integrity of the nodes and paranodal regions at the electron
microscopic level of observation and how nerve conduction might
be slowed or blocked by the known pathological changes to these
structures [2]. We also explore how drug treatments that block
potassium conductance in the juxtaparanodal region may act to
restore conduction in subtly damaged regions. Toward this end it
is insightful to derive from first principles a FitzHugh style cable
model of myelinated axon conduction for the specific purpose of
characterizing the parameters related to subtle injury. Methods The use of mathematical models to study mechanisms of
saltatory conduction is well precedented [15–24]. Classical and
more modern approaches almost universally involve a form of
cable equation or cable model, as originally described by FitzHugh
[15]. This approach regards successive nodes as a chain of leaky
capacitors having voltage sensitive ion channels, and connected in
parallel by extracellular and intracellular resistances between the
nodes. Currents and voltages at each node are computed from a Model of a Myelinated Axon PLOS ONE | www.plosone.org for constants, a and b. Such functions nicely describe the classical waveforms of time-
varying conductance determined experimentally in units of
Seimens per square centimeter. It is easy to show using calculus
that the values of a and b required to produce a peak conductance
G* at time t* after onset of activation are given by the expressions
a~G (e=t )2 and b~2=t, were e is the base of the natural
logarithms <2.781. In turn, values of a and b describing textbook
normal activation functions for sodium and potassium conduc-
tance [5] can be computed as shown in Table 2. Specifically, we
assume for the purpose of this study that the densities of sodium
and potassium of ion channels per square centimeter of membrane
in the regions of the nodes of Ranvier where such channels are
concentrated is similar to that classically described for non-
myelinated axons [6]. To specify extracellular and axonal resistances, Re and Ra, we
note that the resistance of a volume conductor in terms of its
length, cross-section, and resistivity is R~r L
A ,
ð1aÞ ð1aÞ where r is resistivity, L is length, and A is cross sectional area [26]. Resistivity is the intrinsic property of a material that opposes the
flow of steady electric current and is expressed in units of ohm-cm. From the geometry of Figure 1 we can write algebraic expressions
for the internal resistance of an axon, Ra, between nodes as a
function of the axon radius, ra, and also for the external resistance,
Re, of the sleeve of tissue surrounding the myelin, through which
current must flow between two adjacent nodes to complete the
electrical circuit. For Ra and Re we have where r is resistivity, L is length, and A is cross sectional area [26]. Resistivity is the intrinsic property of a material that opposes the
flow of steady electric current and is expressed in units of ohm-cm. From the geometry of Figure 1 we can write algebraic expressions
for the internal resistance of an axon, Ra, between nodes as a
function of the axon radius, ra, and also for the external resistance,
Re, of the sleeve of tissue surrounding the myelin, through which
current must flow between two adjacent nodes to complete the
electrical circuit. Model of a Myelinated Axon A simplified equivalent electrical circuit for a series of equally
spaced nodes of Ranvier, together with associated paranodal and
juxtaparanodal regions, is shown in Figure 2. The intracellular
resistance between nodes along the axons is denoted Ra and is
much larger than electrical resistance of the extracellular current
path between nodes, Re, (not show in Figure 2). The nodal July 2013 | Volume 8 | Issue 7 | e67767 PLOS ONE | www.plosone.org 2 Conduction in Injured Myelinated Axons Time-varying Sodium and Potassium Currents capacitances of the exposed, non-myelinated axon segments at
each node are represented as C~2prasCm, where Cm is the axon
membrane capacitance per unit area, s is the span of bare,
unmyelinated axon in a node of Ranvier, and ra is the radius of the
axon. Definitions of symbols and variables are provided for
reference in Table 1. Local inward sodium currents, iNa0, iNa1, iNa2, … and outward
potassium currents iK0, iK1, iK2, … for nodes 0, 1, 2, … and
nearby juxtaparanodal regions are governed by time varying
transmembrane conductances, GNa0, GNa1, GNa2 … for sodium
and GK0, GK1, GK2 …. for potassium and the respective sodium
and potassium equilibrium potentials [5]. Convenient descriptive
formulas for ion specific conductances as functions of time, t’j,
after onset of activation of node, j, (including both the bare nodal
membrane and functional juxtaparanodal regions) have the form: Because
of
the
relatively
large
distance
between
nodes
compared to the node width, the segregation of ion channels for
inward sodium current and outward potassium current in the
nodal and juxtaparanodal regions does not change or invalidate
the simplified equivalent circuit shown in Figure 2 as a realistic,
model of a myelinated axon segment. However, the potassium
current
sources
in
the
juxtaparanodal
regions
are
a
few
micrometers distant from the sodium current sources in the bare
nodal regions. Potassium current must flow between the axolem-
ma and tightly adherent myelin in the paranodal regions in order
to repolarize the nodal capacitance. This ultrastructural detail may
add a substantial series resistance to the potassium ion ‘‘battery’’,
which is omitted for simplicity in Figure 2 but described in detail in
Appendix S1. G(t’j)~at’2
j e{bt’j
ð2Þ ð2Þ for constants, a and b. for constants, a and b. For Ra and Re we have Using these descriptive functions for time-dependent sodium
and potassium conductance per square centimeter of membrane
area, the corresponding local nodal currents at node, j, and
adjacent paranodal and juxtaparanodal membranes of the model
become iNaj~2prasGNaj(ENa{Vj)
ð3aÞ ð3aÞ and Ra~ raL
pr2
a
and Re~ reL
2preh ,
ð1bÞ ð1bÞ iKj~
Vj{EK
1
2pral GKj zRp
ð3bÞ ð3bÞ where a sleeve of aqueous extracellular fluid of thickness h and
mean radius re surrounds the myelin sheath over distance L and
has extracellular fluid resistivity, re. Noting that extracellular
resistance is about two orders of magnitude less than axonal
resistance leads to a compact model shown in Figure 2. in terms of the axon radius, ra, unstretched node width, s, length of
juxtaparanodal region on both sides of the node, l, local prevailing
transmembrane potential, Vj, and the sodium and potassium Figure 2. Electrical model for multiple nodes of Ranvier. Arrows indicate directions of positive ionic current. Shading indicates foreshortened
myelinated regions. In life the actual distance between nodes (,1000 mm) is much greater than the width of a single node (,1 mm). Current is
denoted by i, capacitance by C, resistance by R, and voltage by V. Lumped ionic currents from sodium and potassium channels in and around each
node are shown as a single current source. doi:10.1371/journal.pone.0067767.g002 Figure 2. Electrical model for multiple nodes of Ranvier. Arrows indicate directions of positive ionic current. Shading indicates foreshortened
myelinated regions. In life the actual distance between nodes (,1000 mm) is much greater than the width of a single node (,1 mm). Current is
denoted by i, capacitance by C, resistance by R, and voltage by V. Lumped ionic currents from sodium and potassium channels in and around each
node are shown as a single current source. doi:10.1371/journal.pone.0067767.g002 July 2013 | Volume 8 | Issue 7 | e67767 3 PLOS ONE | www.plosone.org 3 Conduction in Injured Myelinated Axons Table 1. Nomenclature. for constants, a and b. Symbol
Definition
Units
A
Area
cm2
C
Capacitance of bare membrane in a node of Ranvier
farads
Cm
Specific membrane capacitance per unit area
farads/cm2
d
Axon diameter
cm
e
Base of natural logarithms (2.718…)
G
Membrane conductance per unit area
ohm21 cm22
h
Thickness of a conducting sheath surrounding a myelinated axon
cm
i, i0, i1
Positive ionic current during an action potential
amperes
l
Length of juxtaparanodal region
cm
L
Distance between nodes of Ranvier
cm
n
Number of nodes in a discrete model
p
Circle ratio (3.1415 …)
Ra
Axonal electrical resistance between nodes of Ranvier
ohms
Re
Extracellular electrical resistance between nodes of Ranvier
ohms
Rp
Paranodal electrical resistance between a node and both juxtaparanodal regions of a myelinated axon
ohms
ra
Radius of an axon
cm
re
Radius of a conducting sheath surrounding a myelinated axon
cm
r, re, ra
Resistivity of extracellular or axonal fluid
ohm-cm
s
Span of bare axon in a node of Ranvier in the axial dimension
cm
t
Clock time
sec
t
Clock time of activation of a node
sec
t9
Time after node activation (t 2 t)
sec
V
Transmembrane potential difference
volts
Vth
Threshold potential for initiation of an action potential
volts
v
Nerve conduction velocity
m/sec
doi:10.1371/journal.pone.0067767.t001 Table 1. Nomenclature. doi:10.1371/journal.pone.0067767.t001 Table 2. Constants for descriptive functions for membrane
conductance per unit area, G(t’j)~at’2
j e{bt’j. Coordinates of peak
conductance [5]
Model function constants
t* (sec)
G* (ohm–1cm22)
a (ohm–1cm–2sec–2)
b (sec–1)
Na+
0.0001
0.028
2.16107
26104
K+
0.0005
0.013
3.86105
46103
doi:10.1371/journal.pone.0067767.t002 Table 2. Constants for descriptive functions for membrane
conductance per unit area, G(t’j)~at’2
j e{bt’j. Table 2. Constants for descriptive functions for membrane
conductance per unit area, G(t’j)~at’2
j e{bt’j. equilibrium potentials ENa and EK, as described in [5]. In
calculating potassium current, the juxtaparanodal length, l,
including both sides of the node, is taken as approximately 10
times the normal nodal length based on cytochemical studies of
potassium ion distribution [4,14]. Expression (3b) represents the
current passing through the juxtaparanodal membrane conduc-
tance in series with paranodal resistance, Rp. Rp is the resistance
of the thin sleeve of extracellular matrix material through which
potassium current must flow from the juxtaparanodal regions to
the bare axonal membrane of the local node of Ranvier. Note for
the case Rp = 0 the expression for potassium current is analogous
to that for sodium current. for constants, a and b. If Rp were exceedingly large, then
potassium current would be zero. Details of the treatment of
potassium current in the presence of paranodal resistance and the
estimation of a numerical value for paranodal resistance are
provided in Appendix S1. equilibrium potentials ENa and EK, as described in [5]. In
calculating potassium current, the juxtaparanodal length, l,
including both sides of the node, is taken as approximately 10
times the normal nodal length based on cytochemical studies of
potassium ion distribution [4,14]. Expression (3b) represents the
current passing through the juxtaparanodal membrane conduc-
tance in series with paranodal resistance, Rp. Rp is the resistance
of the thin sleeve of extracellular matrix material through which
potassium current must flow from the juxtaparanodal regions to
the bare axonal membrane of the local node of Ranvier. Note for
the case Rp = 0 the expression for potassium current is analogous
to that for sodium current. If Rp were exceedingly large, then
potassium current would be zero. Details of the treatment of
potassium current in the presence of paranodal resistance and the
estimation of a numerical value for paranodal resistance are
provided in Appendix S1. doi:10.1371/journal.pone.0067767.t002 Conduction in Injured Myelinated Axons Conduction in Injured Myelinated Axons Estimation of other Parameters We consider a textbook normal case as a primary model. Membrane ionic conductances per square centimeter are as listed
in Table 2. From anatomic sections such as those in [28] the radius
of the sleeve of fibrous tissue is about 6 micrometers and the
thickness of the sleeve is about 2 micrometers. Axon diameter
averages 1.0 micrometer [29–31]. The width, s, of a normal node
of Ranvier ranges from about 0.3 micrometer to about 1.0
micrometer, or 0.3 to 1.0 times the diameter of an axon, in various
anatomic specimens [28]. A middle value for the width, s, of a
node is about 0.65 micrometer [10,32]. The span of the
juxtaparanodal region, l, on both sides of a normal node is taken
as 10 times the axon radius [33]. The resistivity of intracellular
fluid is about 200 ohm-cm [31]. The paranodal resistance, Rp, is
estimated as 3.261010 ohms, as explained in Appendix S1. The
specific membrane capacitance of nerve cell membranes is about 1
mircofarad per square centimeter [5]. Using these values to
evaluate parameters, together with normal membrane potentials
[5] (resting membrane potential –85 mV, threshold potential –
50 mV, sodium equilibrium potential +67 mV, potassium equi-
librium potential –95 mV), one can specify the values of all
parameters for a standard normal model. Numerical Integration Increasing or decreasing Dt
without effect on the results confirmed that a sufficiently small
value was chosen for Dt. Wave propagation typically stabilized
over three successive nodes. For active nodes the net current flowing onto a nodal
capacitance is the difference between inflow to and outflow from
the node. Thus, the general structure of equations (5) and (6) below
is: rate of voltage change = transmembrane sodium current inflow,
minus transmembrane potassium current outflow, plus axon
current inflow, minus axon current outflow, all divided by local
capacitance. Ohm’s Law allows calculation of axon current inflow
and outflow from voltages at adjacent nodes. For interior nodes
j = 1, 2, … n –1 dVj
dt ~
ij
Cj
~ iNaj{iKjz Vj{1{Vj
Ra
{ Vj{Vjz1
Ra
1
Cj
:
ð5Þ
For left hand node 0
dV0
dt ~
i0
C0
~ iNa0{iK0{ V1{V2
Ra
1
C0
:
ð6Þ dVj
dt ~
ij
Cj
~ iNaj{iKjz Vj{1{Vj
Ra
{ Vj{Vjz1
Ra
1
Cj
:
ð5Þ ð5Þ Simulation of Injury and Drug Treatment In models of localized crush injury of the spinal cord an impulse
was initiated at node 0. Nodes 1 through 7 had normal
parameters, representing undamaged tissue. A mild form of
stretch or crush injury causing retraction of myelin around
stretched nodes was simulated by increasing the width of exposed
nodal membrane, s, in nodes 8 through 20. Normal nerve
conduction velocity was taken as the inter-nodal distance, divided
by the steady-state wave propagation time between nodes 4 and 5,
namely (t5–t4)/L. Injured nerve conduction velocity was taken as
the inter-nodal distance, divided
by the steady-state
wave
propagation time between nodes 15 and 16, namely (t16–t15)/L. In some simulations a more severe form of stretch or crush injury
was simulated that included both retraction and detachment of
paranodal myelin. Paranodal myelin detachment, that is, radial
separation of paranodal myelin from the underlying axon
membrane, was simulated by decreasing paranodal resistance to
one tenth or one hundredth of its normal value. In other
simulations the effect of the potassium channel blocking drug, 4-
aminopyridine, was simulated by decreasing peak potassium
conductance to 20 percent of its normal value, representing
mild-to-moderate inhibition that would be reasonable to achieve
experimentally. For left hand node 0 dV0
dt ~
i0
C0
~ iNa0{iK0{ V1{V2
Ra
1
C0
:
ð6Þ ð6Þ Numerical Integration us [27], based upon the definition of capacitance and Ohm’s Law. The definition of capacitance is C~dQ=dV, where C is nodal
capacitance and dV is the incremental change in electrical
potential or voltage across the capacitance as charge dQ is
introduced. For current i = dQ/dt flowing during time increment,
dt, we must have dV/dt = i/C. Ohm’s Law, which relates current
to voltage and resistance, is i~(DV)=R, where DV is the
instantaneous difference in voltage across resistance R as current
i occurs. To describe waveforms of membrane potential vs. time at each
node j = 0, 1, 2, … n –1 equations (5) and (6) for dV/dt were
integrated numerically using the simple Euler method, imple-
mented in Visual Basic code within an Excel spreadsheet on an
ordinary personal computer. For each node the membrane
potential Vj at successive time steps Dt was calculated as Vj(tzDt)~Vj(t)z dVj
dt Dt,
ð7Þ ð7Þ Applying these basic concepts with reference to Figure 2
provides a set of governing differential equations that can be used
to describe transmembrane potentials in the chain of nodes. Beginning with the most distant node, n, we set as a boundary
condition using expressions (4), (5), and (6) for dVj/dt. Initial conditions at
t = 0 describe the state of the axon segment at rest, with Vj set to
the resting transmembrane potential for j = 1, 2, … n. Only node 0
is active at time zero (t0 = 0). Nodes j = 1, 2, … n –1 become active
when t.tj. As a boundary condition, the rightmost node, n, in the
chain is clamped at the resting potential and is never activated. dVn
dt
~ 0:
ð4Þ ð4Þ p
g p
A model of 21 nodes (including bare nodal axon and functional
juxtaparanodal membrane) separated by a nominal average
internodal distance, L, of 1 mm represented a hypothetical axon
segment 2 cm in length. The combined bare nodal, paranodal,
and juxtaparanodal regions of each node had a total axial length
on the order of 0.01 mm or about 1 percent of the axial distance
between nodes. Given initial conditions at t = 0, the evolution of
potentials Vj(t) was computed as a ‘‘marching solution’’, for which
stability and accuracy are ensured by using a sufficiently small
value of Dt, such as 0.1 microsecond. Solving for Membrane Potentials at Successive Nodes Solving for Membrane Potentials at Successive Nodes The model of the myelinated axon includes an arbitrary
number ,20 nodal capacitances, connected by equal axonal
resistances, Ra~(raL=(pr2
a)), through which ionic current may
flow, as shown in Figure 2. The capacitances, C~2prasCm,
correspond to the bare membrane areas of successive nodes of
Ranvier. Transmembrane potential is specified as the intracellular
minus extracellular electrical potential at each node. To simulate
myelinated nerve conduction node 0 at the left boundary is
actively depolarized by a suprathreshold stimulus. The successive
nodes 1, 2, … n become active if their membrane potential
exceeds a threshold value, Vth, such as –50 mV. The same conductance functions GNa(t9j) and GK(t9j) are used
for all nodes. However, the time variable, t9j, for each node differs
among nodes, depending on the time of activation. Here we let tj
represent the clock time of activation of nodes j = 0, 1, 2, … n in
the model. In particular, time tj is defined as the clock time, t,
when the transmembrane potential Vj(t) equals or exceeds the
threshold potential Vth for each node j = 0, 1, 2, …. In turn,
t9j = max (0, t2tj). Then the node-specific ionic conductance is
computed for times t.tj as Gj(t’j)~a(t’j)2e{bt’j. For Node 0, the
first node in the chain, t0 = 0, so that activation begins at node 0,
proceeding from left to right in Figure 2. The computational approach used here is an adaptation of that
previously published for modeling the circulatory system by one of July 2013 | Volume 8 | Issue 7 | e67767 PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e67767 4 Normal Nerve Conduction Figure 3 shows successive action potentials in a normal
myelinated axon model. Transmembrane potentials for nodes
numbered 0 through 16 in the series are shown in successive
curves from left to right. A propagated wave of excitation travels
down the chain of nodes from left to right. Nerve conduction
velocity is 19.1 m/sec. July 2013 | Volume 8 | Issue 7 | e67767 PLOS ONE | www.plosone.org 5 Conduction in Injured Myelinated Axons Figure 3. Modeled action potentials at successive nodes of Ranvier in a normal myelinated axon. The dotted horizontal line represents
threshold potential. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; node width 0.65 micrometer; width of juxtaparanodal
region on both sides of a node 5.0 micrometers. The resistivity of intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010 ohms; specific
membrane capacitance of axonal membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential –
50 mV; sodium equilibrium potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g003 Figure 3. Modeled action potentials at successive nodes of Ranvier in a normal myelinated axon. The dotted horizontal line represents
threshold potential. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; node width 0.65 micrometer; width of juxtaparanodal
region on both sides of a node 5.0 micrometers. The resistivity of intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010 ohms; specific
membrane capacitance of axonal membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential –
50 mV; sodium equilibrium potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g003 Figure 4 shows transmembrane sodium and potassium currents
in this normal model as a function of time for node number 6 in
the chain of normal nodes. The peak of potassium conductance is
blunted somewhat, compared to that in an unmyelinated axon,
because of the effect of paranodal resistance, Rp, the anatomic
substrate for which is the thin sleeve of extracellular matrix
material and tight junctions in the paranodal region between the
axolemma and overlying myelin. Recharge current for the node
must pass through this normally thin space to reach the nodal
capacitance. Figure 5. Normal myelinated nerve conduction velocity as a
function of the node width. Normal Nerve Conduction doi:10.1371/journal.pone.0067767.g005 Conduction velocity in otherwise normal myelinated axons
depends on node width, s, measured along the axial dimension, as
shown in Figure 5. Discussion Mathematical modeling can help to organize and distill
knowledge about complex systems, highlight the most important
variables that govern system performance, and suggest testable
hypotheses for future research. The simple computational model
of myelinated nerve conduction presented here provides several
insights into the normal functioning and the failure of myelinated
axons, which remain points of discussion in the 21st century
[35,36]. One critical and underappreciated variable is the width of
the nodes themselves, compared to the diameter of the axon. Another is the paranodal electrical resistance that separates the
high density of sodium channels in the node itself from the
juxtaparanodal potassium channels. The predicted results are similar to the diminished amplitude
and increased latency of compound action potentials observed
experimentally by Shi and Blighty [34]. Because node width, s, is
on the order of only one micrometer normally, subtle damage,
separation, or retraction of the myelin sheath at the nodes of
Ranvier might go unnoticed at the light microscopic level of
observation. Such damage, however, can lead to substantial
degradation in myelinated nerve conduction. When decreased paranodal resistance is included in the model,
such as would occur with decompaction or detachment of
paranodal myelin [10], conduction is further degraded (Figure 7). When node width is stretched three-fold (similar to that observed
in [10]), and paranodal resistance is reduced to one-tenth normal,
the nerve conduction velocity is decreased from 17 m/sec in the
uninjured region on the left to 6.6 m/sec in the injured region on
the right. The amplitude of the action potential is also further
reduced. Evidently, decreased paranodal resistance allows more
outward potassium current to reach the node, diminishing the net
depolarizing current, the rate of rise of the action potential, and
the peak amplitude of the action potential. Note that the 90
percent reduction in Rp represents very subtle injury in this model
since a retraction of the paranodal junction leaving a gap of only
0.1 micron around the circumference of the axon would reduce Rp
by .99.9 percent. Narrower nodes increase conduction velocity (Figure 5) as
previously pointed out in a semi-quantitative way by Giuliodori
and DiCarlo [36]. Simulated Crush Injury Figure 6 illustrates axonal conduction in a model of simulated
crush injury. The widths, s, of nodes 8 through 20 on the right
have been stretched from 0.65 mm to 1.95 mm, simulating the
degree of nodal stretching observed in experimental crush injury
[10]. However, the length of axon over which sodium channels are Figure 5. Normal myelinated nerve conduction velocity as a
function of the node width. doi:10.1371/journal.pone.0067767.g005 Figure 4. Normal ionic currents for node 6 in the myelinated
axon model of Figure 3. doi:10.1371/journal.pone.0067767.g004 concentrated, s*, is not changed in keeping with the pre-injury
segregation of sodium channels in the nodal region. Here s* is
substituted for s in Equation (3a). In the crushed segment (right)
the time between action potentials at successive nodes increases,
indicating slowed impulse conduction. In this example crush injury diminishes action potential
amplitude by about one third and also diminishes the slope of
the rising phase of the action potential, slowing conduction
velocity from 18.8 m/sec in the normal region on the left to
7.8 m/sec in the crushed region on the right. The longer discharge
time required for stretched nodes allows for greater outward
potassium current to develop (Figure 4), and in turn less net
inward positive current inflow during the rising phase of the action
potential. Figure 4. Normal ionic currents for node 6 in the myelinated
axon model of Figure 3. doi:10.1371/journal.pone.0067767.g004 The reduced action potential amplitude after stretch injury
appears to happen for the following reason. The currently PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e67767 6 Conduction in Injured Myelinated Axons Figure 6. Modeled action potentials at successive nodes of Ranvier in a model myelinated axon. Simulated crush injury to nodes on the
right. The dotted horizontal line represents threshold potential. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; normal node
width 0.65 micrometer; injured node width 1.95 micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. The resistivity
of intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010 ohms throughout the model; specific membrane capacitance of axonal
membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential –50 mV; sodium equilibrium
potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g006
Conduction in Injured Myelinated Axons Figure 6. Simulated Crush Injury Modeled action potentials at successive nodes of Ranvier in a model myelinated axon. Simulated crush injury to nodes on the
right. The dotted horizontal line represents threshold potential. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; normal node
width 0.65 micrometer; injured node width 1.95 micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. The resistivity
of intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010 ohms throughout the model; specific membrane capacitance of axonal
membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential –50 mV; sodium equilibrium
potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g006 width, there is complete conduction block in the region of
simulated injury (Figure 8). depolarizing node of Ranvier acts as a constant current source in
the presence of stretch injury. When mild stretch injury increases
node width, the length of axon over which sodium channels are
concentrated is not changed, in keeping with pre-injury segrega-
tion of sodium channels in the nodal region. However, simple
stretch injury increases the nodal surface area, and in turn
increases the nodal capacitance. When the increased nodal
capacitance of the next downstream node is discharged by a
constant current source the rate of change in voltage is reduced
(dV/dt = i/C, where current, i, is constant and capacitance, C, is
increased). The reduced slope between the threshold potential and
the peak of the action potential reflects the increased capacitance. The peak does not reach normal height, because a reduced slope
over a constant duration results in a reduced peak amplitude. Blocked conduction in the simulation of Figure 8 can be
restored by inhibiting peak potassium conductance in all nodes of
the model by 80%, that is, by replacing GKmax = 0.013 S/cm2 with
introducing GKmax = 0.2*0.013 S/cm2. The effect of simulated
drug treatment with a potassium channel blocker is shown in
Figure 9. Conduction is restored. However, conduction velocity
remains low at 7.1 m/sec. Discussion The resistivity of
intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010; injured paranodal resistance 3.26109 ohms; specific membrane capacitance of
axonal membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential –50 mV; sodium equilibrium
potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g007 Figure 7. Modeled action potentials at successive nodes of Ranvier in a model myelinated axon. Simulated crush injury to nodes on the
right. The dotted horizontal line represents threshold potential. In this simulation node width was increased three-fold and paranodal resistance was
decreased to one tenth the normal value. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; normal node width 0.65
micrometer; injured node width 1.95 micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. The resistivity of
intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010; injured paranodal resistance 3.26109 ohms; specific membrane capacitance of
axonal membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential –50 mV; sodium equilibrium
potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g007 myelinated nerve conduction velocity as the distance, L, between
nodes, divided by the time, Dt, it takes for the next downstream
node (e.g. Node 2 in Figure 1) to reach threshold after the
transmembrane potential at a given node (Node 1 in Figure 1)
reaches threshold and nodal sodium channels are fully open. (The
value of Dt can be appreciated graphically with reference to
Figure 3.) As soon as Node 1 becomes a low resistance pathway for
ionic current, downstream Node 2 will depolarize to a value of
about 1/e (37%) of its resting negative membrane potential in one
time constant RaC. The time required to reach threshold is
therefore Dt~aRaC, where a is a constant near 0.5. In turn,
myelinated nerve conduction velocity v~ L
Dt &
L
aRaC ~
1
4raCm
d
s
:
ð9Þ ð9Þ As node width, s, decreases, the myelinated nerve conduction
velocity increases hyperbolically. Further, the node width and
axon diameter are the major anatomic determinants of myelinated
nerve conduction velocity. The direct dependence of conduction
velocity on axon diameter is well established [16,18]. However, the
importance of the node width or the nodal ratio is much less well
appreciated. Discussion The discharge time constant, RaC, which
governs the time required for downstream nodes to be depolarized
from their resting membrane potential to the threshold potential, is
simply RaC~ raL
pr2
a
:2pras Cm~2 raCmL s
ra
~4 raCmL s
d ,
ð8Þ ð8Þ where s/d is the nodal ratio or node width divided by axon
diameter. This expression includes relatively few variables and
holds true as long as paranodal myelin remains tightly coupled to
the underlying axonal membrane, so that paranodal resistance
remains normal. When paranodal resistance if further reduced to 1 percent of the
normal value, together with a three-fold increase in bare nodal The relationship of the discharge time constant (8) to nerve
conduction velocity can be appreciated as follows. Consider PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e67767 July 2013 | Volume 8 | Issue 7 | e67767 7 Conduction in Injured Myelinated Axons Figure 7. Modeled action potentials at successive nodes of Ranvier in a model myelinated axon. Simulated crush injury to nodes on the
right. The dotted horizontal line represents threshold potential. In this simulation node width was increased three-fold and paranodal resistance was
decreased to one tenth the normal value. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; normal node width 0.65
micrometer; injured node width 1.95 micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. The resistivity of
intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010; injured paranodal resistance 3.26109 ohms; specific membrane capacitance of
axonal membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential –50 mV; sodium equilibrium
potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g007 Figure 7. Modeled action potentials at successive nodes of Ranvier in a model myelinated axon. Simulated crush injury to nodes on the
right. The dotted horizontal line represents threshold potential. In this simulation node width was increased three-fold and paranodal resistance was
decreased to one tenth the normal value. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; normal node width 0.65
micrometer; injured node width 1.95 micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. Discussion Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; normal node width 0.65
micrometer; injured node width 1.95 micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. The resistivity of
intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010; injured paranodal resistance 3.26108 ohms; specific membrane capacitance of
axonal membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential –50 mV; sodium equilibrium
potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g009
Conduction in Injured Myelinated Axons Figure 9. Treatment of simulated crush injury to nodes on the right with a potassium channel blocker that reduces peak potassium
conductance in all nodes by 80%. Parameters of injury: 300 percent increase in bare nodal width and a 99% decrease in paranodal resistance. The
dotted horizontal line represents threshold potential. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; normal node width 0.65
micrometer; injured node width 1.95 micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. The resistivity of
intracellular fluid 200 ohm-cm; normal paranodal resistance 3.261010; injured paranodal resistance 3.26108 ohms; specific membrane capacitance of
axonal membrane 1 mircofarad per square centimeter; resting axonal membrane potential –85 mV; threshold potential –50 mV; sodium equilibrium
potential +67 mV; potassium equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g009 and Pryor [37] revealed that the peak latency of action potentials
increased by 35 percent after stretching guinea pig spinal cord
segments. The stretched region in these preparations was about
one third of the total distance over which latency was computed. The modified cable model derived here predicts that myelinated
nerve conduction latency at node 10 in the model of Figure 7
would increase from a normal value of 0.57 sec (Figure 3) to
0.80 sec (Figure 7) a 40 percent increase in latency when the path
from node 0 to node 10 in the model includes three injured nodes
(only one third of the total). Shi and Prior also found that
superfusion with 100 micromolar 4-aminopyridine (a potassium
channel blocker) partially restored the loss in amplitude of the
compound action potential 30 min after stretch injury. However,
there was no significant change in conduction velocity following 4-
aminopyridine treatment. Discussion Since the compound action potential
represents a sum of signals from many axons, these experimental
results are consistent with the model results in Figure 9, showing
that
an
80%
blockade
of
potassium
conductance
restores
conduction with slow velocity following severe stretch injury. The modeling results are also consistent with experimental studies
in other animal models and with preliminary clinical trials of 4-
aminopyridine as a therapeutic agent in spinal cord injury [4]. velocity is located near the elbow of the curve in Figure 5. This
subtle feature hints that mammals are a highly evolved order of
vertebrates. Natural selection would likely favor animals with
higher nerve conduction velocity, quicker reaction times, and
quicker thinking. However, as node spacing gets closer and closer,
problems could arise with quality control in forming nodes to such
close tolerances. For very small values of node span, s, small
absolute change in the separation of myelinated segments on
either side would cause a relatively large change in s and in the
nodal ratio. (Indeed, the magnitude of the derivative dv/ds is
proportional to 1/s2.) Simple movement of peripheral nerves or
very mild concussions in the brain could cause relatively large
changes in nerve conduction velocity, v, and hence in the stability
and predictability of the nervous system. Coordinated complex
movements would become more difficult if conduction time from
brain to muscle, or within the central nervous system, varied
unpredictably. Very narrow nodes would be fast, but they would
also be delicate and perhaps too noisy. In this way the net survival
advantages of quickness might plateau or even degrade as a
function of decreasing node width. We may have reached a near
optimal compromise through natural selection. The model also provides insights into the pathophysiology of
crush injury. The subtle injury involving retraction and fraying of
paranodal myelin increases the effective capacitance of the nodal
membrane by perhaps an order of magnitude or more and also
exposes paranodal and juxtaparanodal regions. Detachment of
paranodal myelin, perhaps only visible at the electron microscopic
level of observation [10], reduces by perhaps an order of
magnitude or more the paranodal resistance through which
repolarizing potassium current must flow to restore the resting
membrane potential after an action potential. These small
anatomical injuries increase early outward potassium current,
opposing inward sodium current at the node. Discussion The sensitive dependence of myelinated nerve conduction
velocity on the nodal width, s, of exposed axon membrane (or the
ratio of nodal width to axon diameter, s/d) has important
biological implications. Interestingly, normal nerve conduction Figure 8. Modeled action potentials at successive nodes of Ranvier in a model myelinated axon. Simulated crush injury to nodes on the
right with 300 percent increase in bare nodal width and a 99% decrease in paranodal resistance. The dotted horizontal line represents threshold
potential. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; normal node width 0.65 micrometer; injured node width 1.95
micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. The resistivity of intracellular fluid 200 ohm-cm; normal
paranodal resistance 3.261010; injured paranodal resistance 3.26108 ohms; specific membrane capacitance of axonal membrane 1 mircofarad per
square centimeter; resting axonal membrane potential –85 mV; threshold potential –50 mV; sodium equilibrium potential +67 mV; potassium
equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g008 Figure 8. Modeled action potentials at successive nodes of Ranvier in a model myelinated axon. Simulated crush injury to nodes on the
right with 300 percent increase in bare nodal width and a 99% decrease in paranodal resistance. The dotted horizontal line represents threshold
potential. Average axon diameter 1.0 micrometer; number of nodes of Ranvier 21; normal node width 0.65 micrometer; injured node width 1.95
micrometer; width of juxtaparanodal region on both sides of a node 5.0 micrometers. The resistivity of intracellular fluid 200 ohm-cm; normal
paranodal resistance 3.261010; injured paranodal resistance 3.26108 ohms; specific membrane capacitance of axonal membrane 1 mircofarad per
square centimeter; resting axonal membrane potential –85 mV; threshold potential –50 mV; sodium equilibrium potential +67 mV; potassium
equilibrium potential –95 mV. The time step for numerical integration was 0.1 microsecond. doi:10.1371/journal.pone.0067767.g008 July 2013 | Volume 8 | Issue 7 | e67767 PLOS ONE | www.plosone.org 8 Conduction in Injured Myelinated Axons Figure 9. Treatment of simulated crush injury to nodes on the right with a potassium channel blocker that reduces peak potassium
conductance in all nodes by 80%. Parameters of injury: 300 percent increase in bare nodal width and a 99% decrease in paranodal resistance. The
dotted horizontal line represents threshold potential. Conclusions The straightforward cable model presented here recapitulates
how the arrangement of myelin covered segments of axons,
punctuated by narrow bare nodes of Ranvier, can produce
saltatory conduction. The function of nodes of Ranvier in speeding
impulse conduction is related to the ultrastructure of the nodes
themselves. Normal myelinated nerve conduction velocity is
inversely related to the node width, s, and to the nodal ratio, s/
d, in the presence of intact paranodal resistance, Rp. The narrow
axial width of the nodes of Ranvier is the key to fast conduction. The biological tradeoff is that even a small mechanical retraction
of myelin from very narrow nodes can cause large changes in
conduction velocity. Very narrow nodes would be very fast, but
exceedingly delicate. Hence evolution may have already found a
near optimal tradeoff between quickness and toughness. Cable models are especially suited to predict outcomes of
biological experiments that measure axonal conduction velocity
because they can be used to simulate wave propagation through
successive nodes. Goldman and Albus [16] described a cable
model and focused on characterizing the relationship between
conduction velocity and fiber diameter in myelinated axons, which
was found to be nearly linear for realistic axon models. Over the
next decade this model was further studied by several groups using
alternative methods for numerical integration and slightly differing
parameter values and to obtain myelinated conductions velocities
between 12 and 22 m/sec and realistic dependence of conduction
velocity upon fiber diameter [17,18]. Modeling also suggests how nodes can malfunction in disease. Subtle mechanical, chemical, or immunological injury to the
nodes of Ranvier, causing small increases in effective node width,
s, and nodal capacitance, C, which could be easily overlooked
using ordinary light microscopes, can contribute to pathology of
conditions such as spinal cord injury and multiple sclerosis that
cause retraction of myelin. The effects of node stretching are
magnified by detachment of paranodal myelin, a process that
decreases paranodal resistance and increases repolarizing potas-
sium current above the normal level. Thus the nodes of Ranvier
are highly effective but somewhat fragile devices for increasing
nerve conduction velocity and decreasing reaction time in
vertebrate animals. Increased nodal capacitance or decreased
paranodal resistance caused by subtle myelin retraction and
detachment cause slowed saltatory conduction and ultimately
conduction block. Conduction in Injured Myelinated Axons Conduction in Injured Myelinated Axons FitzHugh [15]. This approach modeled nodes as strings of leaky
capacitors
having
Hodgkin-Huxley
type
ion
channels,
and
connected in parallel by extracellular and intracellular internodal
resistances. Currents and voltages at each node are computed. FitzHugh derived a second order partial differential equation for
this system to describe voltages at each node as functions of time
and space, and solved the equation numerically. (Much later
Basser [23] and Nygren and Halter [24] used analytical methods
to derive equivalent equations for a composite, myelinated axon,
obtaining expressions essentially isomorphic with the original
solutions of FitzHugh (compare Basser Eq. 35 and Nygren and
Halter Eq. 18 with FitzHugh Eq. 1). Such equations are known as
cable equations, because of their similarity to cable or transmission
line equations [25], and the corresponding models of nerve
conduction are known as cable models. idealized or typical myelinated axon. Noteworthy is the assump-
tion that internodal myelin is an ideal insulator relative to
uninsulated nodes. We ignore internodal leakage currents, which
would likely change nodal currents and voltages by only a few
percent. Supporting Information Figure S1
Electrical model for one side of one node of
Ranvier with potassium conducting regions separated
from the node by high resistance paranodal regions. (TIFF) Appendix S1
Electrical effects of segregated ion chan-
nels. Detailed treatment of the additional complexity of ion
channel segregation in the nodal, paranodal, and juxtaparanodal
regions, leading to quantitative estimation of normal paranodal
resistance Rp&3:2 : 1010 ohms. (DOC) Our model is obviously limited in being a theoretical rather
than an experimental study in which only selected features of the
complete complex biological system are included. Ours is a
deterministic rather than a stochastic model, hence average or
typical values of parameters are used and statistical variation in
axon diameter, axon spacing or internodal width, density and
distribution of ion channels, thickness of the myelin sheath, and so
on are ignored for the sake of answering larger questions about an Conclusions Better understanding of the pathophysiology of
nodal injury may lead to new treatments as well as heightened
awareness of the dangers of subtle neurotrauma in closed head
injury. Blight [19] studied a similar resistance-capacitance network
representing a chain of 20 internodes. In the equivalent electrical
circuit, internodes l-9 and 1 l-20 were each represented by a single
segment, containing the resistance and capacitances of the
internodal axolemma and myelin sheath, separated from the
nodal circuits on either side by the resistance of the axon core. Internode number 10 was broken into 10 shorter sub-segments
such that the importance of paranodal parameters could then be
explored. The model quantitatively reproduced the voltage
response of the axon to injected current pulses and to propagated
action potentials, using Frankenhaeuser-Huxley kinetics. Blight
highlighted the importance of the input resistance of the internode
and the storage of charge at the axolemma. Later Stephanova and
Bostock [20,21] created a distributed-parameter model of the
myelinated human motor nerve fiber, and also highlighted the role
of paranodal resistance. Similarly, McIntyre and coworkers [22]
specifically included paranodal resistance in their model and
studied
the
biophysical
mechanisms
underlying
changes
in
excitability following an action potential. The re-derived cable
equations of Nygren and Halter [24] have sodium channels that
are localized at the node, whereas potassium channels and the
transmembrane sodium/potassium pump are located predomi-
nantly away from the node. In the present study we have retained
the concept of separation of sodium and potassium channels, now
well established as a constant feature of myelinated axons, and we
have added the capability to systematically adjust parameters
related to subtle injury during neurotrauma. Discussion As a result, the time
required to discharge the next downstream node of Ranvier to the
threshold level is increased. In turn, nerve conduction velocity
falls, since it is related to the inter-nodal distance, divided by the
discharge time. py
p
g
p
j
y [ ]
Retraction of myelin around nodes of Ranvier can be produced
by chemical injury as well as by stretch injury. Fu and coworkers
[14], using CARS imaging, reported paranodal myelin splitting
and retraction in response to glutamate excitotoxicity in isolated
rat spinal cord. The nodal ratio in these animals increased from a
normal value near 1.0 to a pathological value near 3.0 after
glutamate. Similarly, Shi and coworkers [32] found that nodal
ratios increased from a control mean of 0.6 to 2.9 twelve hours
after application of 500 micromolar acrolein to isolated guinea pig
spinal
cord. Subtle
nodal
injury,
accompanied
by
slowed
conduction velocity, is also recognized as an important mechanism
in the pathology of multiple sclerosis [3,38]. Hence the mecha-
nisms described in this paper may have relevance beyond the field
of neurotrauma. The modeling approach used in the present study to explore the
particular effects of node width and paranodal resistance builds on
a rich intellectual history. The original model of saltatory
conduction in myelinated nerve fibers was done by Richard The calculated changes in conduction velocity with subtle
paranodal injury in the present paper agree with experimental
observations. Experiments in isolated guinea pig spinal cord by Shi July 2013 | Volume 8 | Issue 7 | e67767 PLOS ONE | www.plosone.org July 2013 | Volume 8 | Issue 7 | e67767 9 References Roper J, Schwarz JR (1989) Heterogeneous distribution of fast and slow
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materials/analysis tools: CFB. Wrote the paper: CFB RS. Suggested
originality of the results: RS. Provided data and measurements for
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Simulations of conduction in uniform myelinated fibers. Relative sensitivity to
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36. Giuliodori M, DiCarlo S (2004) Myelinated vs. unmyelinated 18. McNeal DR (1976) Analysis of a model of excitation of myelinated nerve. IEEE
Trans Biomed Eng 23: 329–337. 37. Shi R, Pryor JD (2002) Pathological changes of isolated sp
response to mechanical stretch. Neuroscience 110: 765–777. 19. Blight A (1985) Computer simulation of action potentials and afterpotentials in
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English
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3D subsurface characterisation of the Belgian Continental Shelf: a new voxel modelling approach
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Geologie & mijnbouw/Geologie en mijnbouw
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cc-by
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Netherlands Journal of
Geosciences
www.cambridge.org/njg Netherlands Journal of
Geosciences
www.cambridge.org/njg Netherlands Journal of
Geosciences 1. Introduction This is an Open Access article,
distributed under the terms of the Creative
Commons Attribution licence (http://
creativecommons.org/licenses/by/4.0/), which
permits unrestricted re-use, distribution, and
reproduction in any medium, provided the
original work is properly cited. To model aggregate resources in 3D, it is important to define deposits with uniform
lithological properties. In a voxel model, 3D interpolation techniques can be implemented to
estimate a representative lithological class (or another property) for each voxel based on the Keywords: 3D stochastic modelling; aggregate resource
estimation; information entropy; North Sea;
Quaternary Author for correspondence:
Vasileios Hademenos,
Email: Vasileios.Chademenos@UGent.be 3D subsurface characterisation of the Belgian
Continental Shelf: a new voxel modelling
approach Vasileios Hademenos1, Jan Stafleu2, Tine Missiaen1,3, Lars Kint4 and
Vera R.M. Van Lancker1,4 Review 1Renard Centre of Marine Geology, Department of Geology, Ghent University, Krijgslaan 281 s8, 9000 Gent, Belgium;
2TNO – Geological Survey of the Netherlands, Princetonlaan 6, 3584 CB Utrecht, the Netherlands; 3Flanders Marine
Institute, Wandelaarkaai 7, 8400 Ostend, Belgium and 4Operational Directorate Natural Environment (RBINS OD
Nature), Royal Belgian Institute of Natural Sciences, Gulledelle 100, 1200 Brussels, Belgium Cite this article: Hademenos V, Stafleu J,
Missiaen T, Kint L, and Van Lancker VRM. 3D
subsurface characterisation of the Belgian
Continental Shelf: a new voxel modelling
approach. Netherlands Journal of Geosciences,
Volume 98, e1. https://doi.org/10.1017/
njg.2018.18 Abstract Modelling of surface and shallow subsurface data is getting more and more advanced and is
demonstrated mostly for onshore (hydro)geological applications. Three-dimensional (3D)
modelling techniques are used increasingly, and now include voxel modelling that often
employs stochastic or probabilistic methods to assess model uncertainty. This paper presents
an adapted methodological workflow for the 3D modelling of offshore sand deposits and aims at
demonstrating the improvement of the estimations of lithological properties after incorpora-
tion of more geological layers in the modelling process. Importantly, this process is driven by
new geological insight from the combined interpretation of seismic and borehole data. Applying 3D modelling techniques is challenging given that offshore environments may be
heavily reworked through time, often leading to thin and discontinuous deposits. Since voxel
and stochastic modelling allow in-depth analyses of a multitude of properties (and their
associated uncertainties) that define a lithological layer, they are ideal for use in an aggregate
resource exploitation context. The voxel model is now the backbone of a decision support
system for long-term sand extraction on the Belgian Continental Shelf. Received: 13 September 2018
Accepted: 22 November 2018 Received: 13 September 2018
Accepted: 22 November 2018 1. Introduction The Belgian Continental Shelf (BCS) is bounded in the north and south by the Dutch and French
parts of the North Sea, on the west by the British part and on the east by the Belgian coast (Fig. 1). The BCS is a sediment-depleted shallow shelf environment comprising a series of sandbanks. There
is no distinct shelf break (De Batist,1989), so there isvery littleaccommodationspaceto accumulate
younger and preserve older sediments. In turn this caused important recycling and redistribution of
the sediment, creating a complex thin and discontinuous Quaternary sediment cover. There is a high demand to exploit the resources within this sedimentary cover (Van Lancker
et al., 2010) and the demand is only increasing due to coastal nourishment projects and new visions
for the development of the marine and coastal zone of Belgium. Availability of sand is critical to
support these initiatives, and therefore the research project TILES was initiated to develop
Transnational and Integrated Long-term Marine Exploitation Strategies (Van Lancker et al.,
2017). Hitherto, no quantitative resource data were available, and also internationally such data
remain scarce, apart from site-related datasets. In a marine aggregate context, it is also important to
have information on admixtures that may adversely affect the quality of the resource (e.g. shells,
mud and gravel content) and/or the environment (e.g. mud; Newell et al., 1998). Therefore, we
opted to develop a three-dimensional (3D) voxel model allowing us to obtain a holistic view
on resource quality and quantity of the Quaternary over wide areas and enabling the addition
of any desired information relevant from a resource or environmental impact perspective. For this application, voxels are a regular grid of rectangular blocks with defined dimensions
(x, y, z) in a Cartesian coordinate system. Each voxel in the model can contain multiple attributes
describing, for example, the stratigraphy, or the spatial variation of lithology in geological units
and other parameters such as uncertainty. Because of their structure, voxels can better define
complex geology and heterogeneities within geological layers (Stafleu et al., 2011). In addition,
voxel models can be created using stochastic techniques that allow the construction of multiple,
equally probable 3D realisations. Furthermore, they facilitate easy querying and analysis: for
example, volume calculations can be performed by selecting and counting the voxels that meet
certain criteria. © Netherlands Journal of Geosciences
Foundation 2019. © Netherlands Journal of Geosciences
Foundation 2019. This is an Open Access article,
distributed under the terms of the Creative
Commons Attribution licence (http://
creativecommons.org/licenses/by/4.0/), which
permits unrestricted re-use, distribution, and
reproduction in any medium, provided the
original work is properly cited. https://doi.org/10.1017/njg.2018.18
Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms rg/10.1017/njg.2018.18
from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available a siteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. 2 2 Vasileios Hademenos et al. Fig. 1. (A) Map showing the depth of the Top-Palaeogene unconformity and the main geomorphological features: the Middle and Offshore Platform, separated by the Middle and
Offshore scarp (De Clercq et al., 2016). These scarps were used to split up the model into regions with similar lithological characteristics. (B) Cross-section showing the extent and
geometry of each stratigraphical unit subdividing the Cainozoic sediments of the BCS. Most of the sandbanks (e.g. Middelkerke, Hinder) have a characteristic internal architecture. Fig. 1. (A) Map showing the depth of the Top-Palaeogene unconformity and the main geomorphological features: the Middle and Offshore Platform, separated by the Middle and
Offshore scarp (De Clercq et al., 2016). These scarps were used to split up the model into regions with similar lithological characteristics. (B) Cross-section showing the extent and
geometry of each stratigraphical unit subdividing the Cainozoic sediments of the BCS. Most of the sandbanks (e.g. Middelkerke, Hinder) have a characteristic internal architecture. quality and reliability of the models becomes crucial. Using
stochastic models, the probability of occurrences of each lithologi-
cal class can be calculated as a first estimate of model uncertainty. To summarise the probabilities per voxel, Wellmann & Regenauer-
Lieb (2012) suggested calculating information entropy as a mea-
sure of model uncertainty. The quality measure thus obtained
can be used to compare different versions of a model. lithological description of boreholes available in the model area
(Van Haren et al., 2016). In many cases, modelling results can
be greatly improved by subdividing the 3D volume into lithostrati-
graphical units that have uniform sediment characteristics. For
example, in the GeoTOP voxel model of the onshore part of the
Netherlands (Stafleu et al., 2011, 2012) the borehole descriptions
were first used to construct 2D bounding surfaces. These surfaces
represented the top and base of each of the lithostratigraphical
units and were used to place each voxel in the model within the
correct unit. Next, the 3D interpolation of lithological class was
performed for each lithostratigraphical unit separately. In this paper, a step-by-step modelling approach is described to
depict the quality and quantity of the available geological resources
in the BCS. For the first time, 3D stochastic modelling is applied to
quantify sand resources in a marine setting making use of both
borehole and seismic data. For this application, the approach is also
new in the sense that it incorporates various levels of geological
knowledge in the modelling process and this is shown to improve
the characterisation of the subsurface. A more detailed case study is
presented to demonstrate how the resolution of a model affects the
depiction of the lithostratigraphy and the volume of the resource. Creating bounding surfaces from borehole data works well if the
spatial data density of the boreholes is relatively high, as is the case in
the GeoTOP model (~10 boreholes per km2 on average). In the BCS
however, borehole density is only about 0.3 per km2. Such low bore-
hole density necessitates the incorporation of other data sources. Most evident are geophysical line data such as shallow 2D/3D seismic
profiles that allow the interpretation of ‘horizons’ to subsequently
allow the generation of bounding surfaces to define the different lith-
ostratigraphical units in 3D modelling (e.g. Bartakovics et al., 2013;
Van Heteren et al., 2014; Jørgensen et al., 2015). https://doi.org/10.1017/njg.2018.18
Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. niversiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. 2.2. Quaternary Our core dataset contained a total of 1770 cores on the BCS extend-
ing to 1 km beyond the border (Fig. 3A) provided by the Royal
Belgian Institute of Natural Sciences (Kint & Van Lancker,
2016). Data originated from the public and private sector and span
several decades (1900–2016). The majority of the cores were rela-
tively shallow, with depths ranging from 0 to 5 m (Fig. 3A). The
spatial distribution of the cores is denser close to the shore, and
sparser further offshore and near the borders (especially towards
France) (Fig. 3A). Metadata were all revised according to
SeaDataNet standards (Schaap, 2017). Thin, discontinuous/heterogeneous Pleistocene and Holocene sedi-
ments overlay the unconformity (Mathys, 2009). The variability of
the geological formations of the Quaternary poses a major challenge
when modelling; the lithostratigraphy content of each sandbank is
unique and interpolation will cause generalisations that may lead
to faulty assumptions about their geological content. To avoid this,
the stratigraphical layers that will be used must be carefully defined. The Pleistocene sediments occur in two main regions of the BCS. On the Offshore Platform, they form the core of the sandbanks
(e.g. Hinder Banks), and closer to the coast they are preserved in
palaeovalleys such as the Ostend Valley (Fig. 1) (De Clercq et al.,
2016, 2018). In the Middle Platform region they were mostly eroded
down to the Palaeogene clays by both the Eemian and the Holocene
transgressions (Mathys, 2009). The Pleistocene sediments originate
mainly from the Eemian interglacial period and comprise mixed
sediments spanning from gravel to clay. There is a lateral
difference in lithology between the nearshore area, where clay to fine
sands predominate (in the palaeovalleys), and the offshore area
where coarser-grained sands with abundant shells are found. p
Due to the diversity of core descriptions and different param-
eterisation schemes dependent on the various project objectives,
all of the descriptions were checked and encoded following
European guidelines on geological data formats (Geo-Seas:
Van Heteren, 2010). For lithology terms, the Wentworth (1922)
classification was used to define sediment classes (e.g. clay, silt,
fine sand, medium sand, coarse sand, gravel). In some cases,
harmonisation of data across the original data sources was
needed to resolve differences in assigning Wentworth classes
to a given grain-size range. 2.1. Palaeogene The BCS is marked by two major geological units greatly varying in
lithology and stratigraphy. These units, respectively the Palaeogene With the advent of 3D models, their increasing complexity and
the variety of methods employed, finding better ways to assess the Netherlands Journal of Geosciences 3 et al. (2011) and expanded with seismic data. It comprises the fol-
lowing steps (Fig. 2): and Quaternary, are bounded by the Top-Palaeogene unconform-
ity (De Clercq et al., 2016). The Palaeogene is a polygenetic layer
composed of compacted clays, sands and sandy clays that were
deposited in a shallow-marine to outer-shelf environment (Le
Bot et al., 2003). The geological units within the Palaeogene range
in age from the upper Palaeogene to the upper Eocene. The layers
dip towards the NE by approximately 1°. Their lithology varies
from west to east, from consolidated clays (Ypresian) to alternating
sequences of silt and clay, but also silty sand, muddy sand and even
calcareous sandstone beds (Le Bot et al., 2005). (1a)
Standardisation and lithological classification of borehole
descriptions
(1b, 3) Delineation of seismic acoustic facies and their seismostra-
tigraphical interpretation
(2)
Stratigraphic interpretation of the boreholes
(4)
Construction of the 2D stratigraphical layer model
(5)
Assignment of lithostratigraphical units to the 3D voxel
model
(6)
3D interpolation of lithological class within each lithostra-
tigraphical unit
(7)
Assessment of the information entropy of the model. The top of the Palaeogene is an angular unconformity
(Fig. 1) representing a hiatus in time between the Lower and
Middle Eocene formations (De Batist, 1989) and the overlying
Quaternary deposits. The depth of the surface varies between 8
and 70 m below lowest astronomical tide (LAT), and its geomor-
phology is characterised by a series of features ranging from
planation surfaces, bounded by scarps and slope breaks, to palae-
ovalleys and elongated depressions (Liu, 1990; Liu et al., 1992;
Mathys, 2009; De Clercq et al., 2016). A detailed description of steps 1 to 7, following an iterative process
as is indicated in Fig. 2, is given below. 2.2. Quaternary For interpolation purposes in the
lithological description, six numerical classes were used, follow-
ing the lithological classification of Vernes & Van Doorn (2005)
ranging from gravel to clay (Table 1). Descriptions on sandy
sediment layers without any further information on lithological
content were characterised as sand and categorised in a separate
numerical class. The Holocene sediments are diverse in origin and composition. They form the major part of the tidal sandbanks of the BCS
(e.g. Trentesaux et al., 1999; Mathys 2009; Van Lancker et al., 2010). Two layers are distinguished, the Lower and Upper Holocene
respectively. In the nearshore area, the Lower Holocene layer
(LHL) is representative of a tidal flat environment (Mathys, 2009)
which was formed around 10,950 cal BP (before present) until it
was submerged around 7500 cal BP. This Lower Holocene layer
was first defined in the Middelkerke Bank (Fig. 1); its sediments
varied from coarse-grained to very-fine sand. The Upper
Holocene layer (UHL) covers the total BCS and forms the most
important sand resource. In the nearshore area, south of the middle
scarp (Fig. 1), it predominantly consists of fine sands related to an
estuarine–marine depositional environment. In the offshore area,
north of the middle scarp (Fig. 1), the layer comprises coarser
material, with medium to coarse sand being typical for the offshore
marine depositional environment. 2.5. Step 1b and 3: Delineation of seismic acoustic facies and
their seismostratigraphical interpretation The available seismic database (Renard Centre of Marine Geology,
Ghent University) comprises over 12,000 km (Fig. 3B) of 2D seis-
mic lines collected during a large number of scientific cruises
from the late 1970s until today. The seismic sources used for the
measurements mainly involved various types of sparkers and
boomers, in combination with a single-channel streamer. The
seismic dataset comprised both digitally recorded data (SEG-Y
format) and older analogue data converted to SEG-Y from scanned
paper rolls. The latter make up one-third of the total database to
roughly 4000 km of seismic profiles (Mathys, 2009). https://doi.org/10.1017/njg.2018.18
Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. siteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. 2.3. Methodology To model offshore aggregate resources, a methodological workflow
was developed based on the voxel modelling approach of Stafleu The converted analogue data often caused serious problems
related to the high uncertainty in geographical location (many lines Fig. 2. Modelling procedure flow chart. Fig. 3. (A). Map showing the seismic network on the BCS against a background of the bathymetry (Flanders Hydrography). (B) Map showing the depth (m) distribution of the co
d t
t
th BCS Th
t
l i
th
iddl
d fi
th
t
t
f th Hi d
B
k
t d
4
Vasileios Hademenos et a Vasileios Hademenos et al. Fig. 2. Modelling procedure flow chart. Fig. 2. Modelling procedure flow chart. Fig. 3. (A). Map showing the seismic network on the BCS against a background of the bathymetry (Flanders Hydrography). (B) Map showing the depth (m) distribution of the core
dataset on the BCS. The grey rectangle in the middle defines the extent of the Hinder Banks case study area. Fig. 3. (A). Map showing the seismic network on the BCS against a background of the bathymetry (Flanders Hydrography). (B) Map showing the depth (m) distribution of the core
dataset on the BCS. The grey rectangle in the middle defines the extent of the Hinder Banks case study area. https://doi.org/10.1017/njg.2018.18
Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/term 5 Netherlands Journal of Geosciences Table 1. Wentworth (1922) and the classification used in the voxel modelling
Wentworth (1922)
μm 2000
Gravel
Gravel
1000
Very coarse sand
Coarse sand
500
Coarse sand
250
Medium sand
Medium sand
125
Fine sand
Fine sand
62.5
Very fine sand
4
Silt
Silt
Clay
Clay Table 1. Wentworth (1922) and the classification used in the voxel modelling depth of the stratigraphic layers. In order to perform this conver-
sion, the sound velocity within each layer was calculated. To
achieve this and to validate the seismic interpretations, borehole
and bathymetric data were incorporated and each seismic reflector
was compared to the lithostratigraphical information from the
cores. This allowed the creation of a velocity model (Fig. 4B)
assigning a laterally varying internal velocity to each layer, rather
than simply providing a constant value for the velocity of
each layer. 2.6. Step 2: Stratigraphic interpretation of the boreholes Only a limited number of boreholes were already assigned a
stratigraphical interpretation, and if available, these interpretations
were mostly made in different projects, each having different qual-
ity requirements (Kint & Van Lancker, 2016). Thus the original
borehole data were far from uniform with respect to stratigraphy,
and therefore we decided to systematically reassign stratigraphical
interpretations to all boreholes in the dataset using both sediment
characteristics and seismic data. Based on geological knowledge
and on an iterative process between step 2 and step 3, the acoustic
facies from the interpreted seismic lines were assigned a strati-
graphical unit exported to the borehole dataset for further use
in the modelling process. For the stratigraphical description of the borehole intervals a
lithostratigraphical unit was assigned based on the conceptual
framework in Fig. 2. The labels in the core data were derived from
the borehole stratigraphical descriptions or, when that information
was unavailable, from the seismic interpretations. A constant
cross-validation between two different types of data was needed. It is noteworthy that only a few cores on the BCS were dated, cre-
ating uncertainty when assigning lithostratigraphical information. A final quality check included the identification of duplicate bore-
holes, gaps or overlapping borehole intervals. Errors were sub-
sequently corrected manually. The bounding surfaces were then combined to create the layer-
based model defining the lithostratigraphical units (see step 5);
cross-cutting between these surfaces has been resolved in seismic
interpretation. The bathymetry (Flanders Hydrography) was used
as the top surface. 2.8. Step 5: Assignment of lithostratigraphical units to the 3D
voxel model Thenextstepistodefinethevolumeinwhichtheinterpolationoflitho-
logical properties will take place. The highest point in the seafloor
bathymetry was 4 m MSL (in the port of Zeebrugge), and the lowest
point in the volume was −70 m MSL (corresponding to the bottom
of the deepest borehole in the dataset). The grid resolution for
modelling (i.e. the size of a single voxel) was set to 200 × 200 × 1 m
(x, y, z), a choice based on data density and scale of the geological
features that were described, while assuring a reasonable speed for 2.7.2. Geological layer creation
Th d
h f
h
h The depth of each seismic horizon was exported per line in a point
format. Next, these points were interpolated by co-kriging using
geostatistical software (ISATIS®), resulting in the creation of 2D
bounding surfaces (grid size 200 × 200 m for the BCS and 100
m × 100 m for the Hinder Banks case study). All maps and models
were vertically referenced to mean sea level (MSL). This reference
level was chosen because it is calculated using onshore fixed points,
while it also serves the need for a unified system between the
Netherlands and Belgium in view of a future cross-border model-
ling programme. Since the Belgian seismic dataset and bathymetry
were referenced to the lowest astronomical tide (LAT) they were
converted to MSL using a grid provided by Deltares. Standard
deviation of the surfaces was estimated using the co-kriging
function and was subsequently used as a measure of uncertainty
in the modelled stratigraphy. https://doi.org/10.1017/njg.2018.18
Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. 2.3. Methodology The laterally varying velocity model was used to
calculate more accurately the Top-Palaeogene surface, the
Top-Pleistocene surface and the Upper/Lower Holocene boun-
dary. If a constant velocity model (e.g. 1500 m s−1) had been used,
the thickness of the Quaternary units would have been locally over-
or underestimated. Additionally, errors in depth caused by local-
ised velocity anomalies due to morphological features such as the
pull-up effect of sandbanks (Fagin, 1996) were addressed using this
dynamic velocity model. Locally, the laterally varying velocity
model based on the core dataset introduces artefacts such as
depressions (e.g. in the Zeebrugge valley) that are not visible in
or not covered by the seismic dataset. Other artefacts include
unrealistically low velocity values in the water body related to
the outcrop of the Palaeogene layer in between the sand banks
(Fig. 4). Nonetheless, the core-based approach leads to better
results than the constant velocity model which generally overesti-
mates the depth of the top Palaeogene surface. Furthermore the
velocity model, due to artefacts inherent in the nature of the dataset
(digitised from paper, seismic lines), does not always refer to a
physical velocity but is often a conversion factor. showed a spatial misplacement). A second problem was related to
the presence of shallow gas preventing seismic penetration (e.g. Missiaen et al., 2002). Other factors, such as bad weather condi-
tions, also resulted in lower data quality. These problems were
addressed either by excluding the problematic line from the dataset
or ignoring the problematic segment. Facies with similar acoustic characteristics were delineated (e.g. Fig. 13 below). This was mostly a seismic interpretation revision of
Mathys (2009). Boundaries were identified between the following
stratigraphical units: Palaeogene, Quaternary, further divided into
Pleistocene, Upper and Lower Holocene (Fig. 1). Acoustic facies
were linked to stratigraphic boundaries. In this process, cross-veri-
fication with the borehole data was essential. niversiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. 2.7. Step 4: Construction of 2D stratigraphical layer models 2.7.1. Time-to-depth conversion 2.7.1. Time-to-depth conversion 2.7.1. Time-to-depth conversion
The horizons identified on seismic profiles were picked in time,
because seismic traces are recorded in two-way travel time of
the signal. A time-to-depth conversion is required to define the 6 Vasileios Hademenos et al. 6 Fig. 4. Map showing the laterally varying velocity model in m s−1 used to calculate the depth of (A) the picked seafloor horizon (water column) and (B) the picked
Top-Palaeogene horizon (Quaternary layer). Fig. 4. Map showing the laterally varying velocity model in m s−1 used to calculate the depth of (A) the picked seafloor horizon (water column) and (B) the picked
Top-Palaeogene horizon (Quaternary layer). the interpolation process. A higher-resolution model of 100× 100 ×
0.5 m was tested at the Hinder Banks. taking into account the previous information, resulting in a prob-
ability between 0 and 1 for each lithological class. The values
are plotted in a cumulative distribution function. Then a random
value between 0 and 1 is drawn and compared to the cumulative
distribution function. The simulated lithological class at the target
voxel corresponds to the rank of the interval to which the random
value belongs. The bounding surfaces, as described in step 4, are now added to
the volume of the model, and the space between them is filled with
voxels. The centres of the voxels (whether above or below a surface)
define the lithostratigraphical unit they belong to. The voxels
are assigned a constant integer value that corresponds to the
lithostratigraphy (e.g. 1 for Nearshore Upper Holocene, 2 for
Offshore Upper Holocene, etc.). Especially in the deeper parts of the model, the neighbourhood
search at a target voxel may end up with no data (neither hard data
from boreholes nor already simulated voxels). The result is then
drawn from proportions. These are the global proportions of each
lithological class observed in the boreholes, which are assumed to
be constant throughout the lithostratigraphical unit (Fig. 5). https://doi.org/10.1017/njg.2018.18
Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. niversiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. 2.9. Step 6: 3D interpolation of lithological class The next step in voxel modelling is to estimate a lithological class
for each voxel, for which the Sequential Indicator Simulation (SIS)
(Goovaerts, 1997; Chilès & Delfiner, 2012) technique was used
(ISATIS®). SIS requires modest computation time and has been
applied earlier in the creation of voxel models in similar geological
settings (Stafleu et al., 2011; Maljers et al., 2015). The SIS method can be extremely useful in relatively homo-
geneous geological units or in cases where good data coverage is
available. However, on the BCS, and especially in the Holocene layer
with its diverse sediment types, it may lead to errors in the form of
so-called ‘flying’ voxels. These comprise voxels in regions of low data
density that have been assigned a lithological class according to the
global proportions (percentage of lithological class in each layer). This problem can be resolved by splitting the layer into smaller,
better-defined sublayers. To allow a good comparison of the results,
parameters such as the size of the neighbourhood (10 km) and the
dimensions of the voxels (200 m × 200 m × 1 m) were kept constant
as new lithostratigraphical layers were added. Borehole data were first migrated to the closest voxel and
considered as hard data afterwards. All the remaining voxels were
scanned using a random path. A neighbourhood is established,
centred on the target voxel, and within this neighbourhood the
procedure searches for the hard data from the boreholes and for
voxels that are already simulated. The neighbourhood is examined
using a variogram model which ensures that data most closely
correlated with the target voxels are assigned the greatest weight. The data are then coded into a set of indicators; hence the name
indicator simulation. For each lithological class, the indicator is
set to 1 if the data belong to the lithological class and to 0 if not. The next step in SIS consists of a co-kriging phase (block kriging) The SIS resulted in 100, statistically equally probable, simulations
of lithological class distributions. From these simulations probabil-
ities of occurrence for each lithological class were calculated giving
an indication of model uncertainty. In addition, the probabilities
were used to compute a ‘most likely’ lithological class model using Netherlands Journal of Geosciences 7 Fig. 5. 2.9. Step 6: 3D interpolation of lithological class Chart representing the global proportions of each lithological class in each lithostratigraphical layer in the process of adding more lithostratigraphical divisions in each
model run (NUH: Nearshore Upper Holocene; OUH: Offshore Upper Holocene). Fig. 5. Chart representing the global proportions of each lithological class in each lithostratigraphical layer in the process of adding m
model run (NUH: Nearshore Upper Holocene; OUH: Offshore Upper Holocene). roportions of each lithological class in each lithostratigraphical layer in the process of adding more lithostratigraphical divisions in each
ocene; OUH: Offshore Upper Holocene). Fig. 5. Chart representing the global proportions of each lithological class in each lithostratigraphical layer in the process of adding more lithostratigraphical divisions in each
model run (NUH: Nearshore Upper Holocene; OUH: Offshore Upper Holocene). and the differences amongst the probabilities (the greater the
differences, the lower the entropy). the averaging method for indicator datasets described by Soares
(1992). However, the 100 individual simulation results remain
available for further use. As suggested in Wellmann & Regenauer-Lieb (2012), informa-
tion entropy can be used as a quality measure of 3D models. In our
study, information entropy is used as a comparative measure
between different runs of the model. This comparison between
the distribution of the information entropy helps quantify the
effect of each layer addition on the model. Moreover, it allows
us to visualise the overall quality of the model and make compar-
isons between the different interpolations. 2.10. Step 7: Assessment of the information entropy of the
model The probabilities of occurrence provide a measure of model uncer-
tainty. Probabilities of an individual voxel can be displayed in a
single bar chart, thus showing its probability distribution and
hence model uncertainty. Similar displays are possible in visualisa-
tions of virtual boreholes (i.e. vertical stacks of voxels). However, in
two- and three-dimensional visualisations (e.g. cross-sections or
3D views) it is not possible to show all probabilities for each voxel
in a single view; the user will always be presented with one of the
probabilities at a time. https://doi.org/10.1017/njg.2018.18
Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. niversiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/njg.2018.18
Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core ter 4. Results Five different units were distinguished defining the lithostrati-
graphical succession in the BCS: Palaeogene, Pleistocene, Lower
and Upper Holocene, the latter with a subdivision into Nearshore
and Offshore. Results are presented on the five model runs starting
from the use of a uniform stratigraphy in the modelling process up
to using all five units. This was done to compare the effects of the
addition of each unit to the model. Additionally, the workflow was
applied in a higher resolution in an area with higher data coverage. Results become progressively more detailed as the introduction of
each new lithostratigraphical layer divides the model into smaller
segments. 4.2.2. Two–layer model (Palaeogene–Quaternary)
The first surface added to the model was the Top-Palaeogene
unconformity (De Clercq et al., 2016). It is the bounding surface
between the Palaeogene and the overlying Quaternary deposits
and constrains the lower boundary of the resource units, and as
such it has a significant impact on the resource calculations. The results of the two-layer model are shown in Fig. 8B. The
Top-Palaeogene layer is now defined as consisting of 56% of clay
(see Fig. 5, in green). Moreover, as the Top-Palaeogene surface
comprises complex geomorphological features, abrupt lateral
changes in the sediment composition are now much better con-
strained. A good example is the Ostend Valley (Fig. 1) cutting into
the underlying clay sediments (Fig. 15). 3.2. Hinder Banks case study: higher-resolution voxel
modelling geological features are clearly visible in each unit, such as the sand-
banks and the platform in the Holocene and the Ostend Valley in
the Pleistocene. Following the steps described above, a case study has been con-
ducted on a smaller area with better data coverage. The case study
area (see Fig. 3A) is located in the Hinder Banks area and com-
prises three major sandbanks (Noordhinder, Westhinder and
Oosthinder). The dense bathymetric, seismic and borehole data
coverage allowed the size of the voxels to be reduced to 100 ×
100 × 0.5 m (x, y, z). The main reasons for choosing a higher voxel
resolution were: (1) to test to what extent voxel size affects resource
volume calculations; (2) to evaluate whether a higher resolution
allows a better depiction of the different layers within the sand-
banks; and (3) to compare the effects of different voxel sizes on
the assigned lithological classes and probabilities of occurrence. The layers that are used for this test include the bathymetry,
Top-Palaeogene and Top-Pleistocene; all three layers were interpo-
lated at a resolution of 100 × 100 m, similar to the voxel size. The
latter was achieved by re-interpolating the points from the seismic
interpretation (cf. step 4). The Hinder Banks area is the main target
for sand dredging in the years to come (Mathys et al., 2011); as
such, an accurate estimate of the resource volume and lithology
is crucial. 4.2. Stepwise incorporation of geological knowledge g
y
(
g
p y)
In the single-layer model, the volume in which the 3D interpola-
tion of lithological class takes place is bounded only by the bathym-
etry (in MSL) at the top and a horizontal boundary at −70 m MSL. The lithological classes of the borehole dataset were used without
stratigraphical interpretation. The results for the single-layer model are shown in Fig. 8A. The
model seems to work well in the area around Zeebrugge due to the
good data coverage and where the model clearly shows the transition
from Palaeogene clays to Quaternary sands. Although no strati-
graphic information was added to the model, the model still correctly
predicts clay in the depth intervals that contain Palaeogene layers,
and sand in the depth intervals that contain Quaternary layers. In other parts of the BCS, with much lower data coverage, the litho-
logical class ‘sand’ is wrongly propagated into the Palaeogene layer
due to the global proportion of sands (see Fig. 5, in blue) in the bore-
hole dataset. The model uncertainty map on the right shows high
uncertainties (red voxels) in many areas. 3.1. Stepwise improvement by adding layers One of the aims of this study was to demonstrate how adding
more geological information to the modelling process would
improve the lithological characterisation of the lithostratigraph-
ical units. As such, each borehole interval was attributed different
levels of stratigraphy. For example, in the second run of the
model when only two layers were used, the labels in the borehole
descriptions are ‘Palaeogene’ and ‘Quaternary’, while in the
last interpolation using five layers the core dataset contains the
full set of relevant lithostratigraphical information (Nearshore
Upper Holocene, Offshore Upper Holocene, Lower Holocene,
Pleistocene, Palaeogene). To deal with this problem, Wellmann & Regenauer-Lieb (2012)
proposed the use of information entropy as a measure of uncer-
tainty in 3D models. The information entropy of a voxel is a single
value ranging from 0 to 1 that can be calculated from each of the
probabilities of lithological classes. An entropy value of 0 means
that there is no uncertainty, whereas a value of 1 occurs when
all lithological classes have the same probability. Values in between
0 and 1 account for both the number of lithological classes with a
probability higher than 0 (the more classes, the higher the entropy) Vasileios Hademenos et al. 8 4.1. Lithological and stratigraphical framework The 2D bounding surfaces, that were created in step 4 of the meth-
odology section, are shown in Fig. 6. When overlaid, they form the
top and bottom of each unit that will be used in the different inter-
polations. The space between them is filled with voxels. The vox-
elised lithostratigraphical units and their extent can be seen in
Fig. 7. 4.2.3. Three-layer model (Palaeogene–Pleistocene–Holocene)
The second added surface is the Top-Pleistocene surface, allowing
differentiation of Quaternary deposits in terms of their lithological
composition. The patchy and lithologically mixed Quaternary
layer comprises the infill of the palaeovalleys in the nearshore area
of the BCS, and forms the core of the sandbanks in the offshore area
(see Fig. 1). The results of the three-layer model for the BCS are
shown in Fig. 8C. In the Ostend Valley, for example, fine sands
can now be distinguished from overlying clay sediments. Results show that the Quaternary cover is very thin and its sedi-
ments are accumulated mainly in the sandbanks. Each sandbank is
unique in its stratigraphical and lithological content. This
makes the modelling procedure more challenging because these
geological features and their internal structure must be taken into
consideration when splitting a lithostratigraphical unit. Fig. 7
shows the results of the lithostratigraphical characterisation of
the borehole dataset, as discussed in step 5. https://doi.org/10.1017/njg.2018.18
Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. 4.2.4. Four-layer model (Palaeogene – Pleistocene – Lower
Holocene – Upper Holocene) 4.2.4. Four-layer model (Palaeogene – Pleistocene – Lower
Holocene – Upper Holocene) The level of detail even in the 200 × 200 m resolution of the
surfaces allowed the robust modelling of the different geological
units described previously, taking into account the stratigraphical
boundaries and features (Fig. 6) of the Quaternary cover. For
example the extent of the Ostend valley is 10 × 5 km; its internal
features can be described well by a 200 × 200 m resolution model. Other resolutions have been tested for the BCP model, though
based on the data density and calculations time the 200 × 200
m resolution was decided upon as the most effective. All of these In the nearshore, one extra layer was added to account for the litho-
logical differentiation in the Lower Holocene which is here related
to a fine-grained tidal-flat environment (Mathys, 2009). Further
offshore, the Holocene deposits are coarser. The main purpose
of adding this layer was to demonstrate the sensitivity of the model
in describing a lithologically varying layer with very few cores
crossing it. The results of the four-layer model are shown in
Fig. 8D. 9 Netherlands Journal of Geosciences 9 Fig. 6. Views of the modelled bounding surfaces used in the voxel modelling. (A) Top-Palaeogene, (B) Top-Pleistocene, (C) Top-Lower Holocene and (D) Top-Upper Holocene
(bathymetry), the latter with a subdivision into Nearshore and Offshore defined by the Middle Scarp. Fig. 6. Views of the modelled bounding surfaces used in the voxel modelling. (A) Top-Palaeogene, (B) Top-Pleistocene, (C) Top-Lowe
(bathymetry), the latter with a subdivision into Nearshore and Offshore defined by the Middle Scarp. g surfaces used in the voxel modelling. (A) Top-Palaeogene, (B) Top-Pleistocene, (C) Top-Lower Holocene and (D) Top-Upper Holocene
ion into Nearshore and Offshore defined by the Middle Scarp. Fig. 6. Views of the modelled bounding surfaces used in the voxel modelling. (A) Top-Palaeogene, (B) Top-Pleistocene, (C) Top-Lower Holocene and (D) Top-Upper Holocene
(bathymetry), the latter with a subdivision into Nearshore and Offshore defined by the Middle Scarp. 4.2.5. Five-layer model (Palaeogene – Pleistocene – Lower
Holocene – Nearshore Upper Holocene – Offshore Upper
Holocene) the Holocene layer of the three-layer model (Top-Pleistocene
surface). These ‘flying’ voxels were introduced by the SIS as the
global proportions of the clay–silt percentages were forced through
the entire model (see Fig. 5). https://doi.org/10.1017/njg.2018.18
Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. re. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/te 5. Case study with higher resolution of the voxel model The analysis of the results of the case study gives a detailed over-
view of the lithological properties of the area. The 100 × 100 × 0.5 m
voxel model shows a predominance of medium sand (Fig. 10). A
major difference with the 200 × 200 × 1 m resolution five-layer
model is the detailed lithological variation within the sandbanks. The 100 × 100 × 0.5 m was chosen based on the data density. The sandbanks are characterised by a Pleistocene core of coarse sand
and gravel, mixed with clay and silts. The silt is mainly found in the
upper part of the Pleistocene layer forming a transition boundary
towards the Offshore Upper Holocene layer. Coarse sand and gravel
populate the space between the sandbanks as well. Moreover, the
Palaeogene layer is now composed almost completely of clay since
the limited number of cores that penetrate the Palaeogene layer in
the Hinder Banks have a global proportion of 92% clay. For the BCS, the Middelkerke Bank (localisation, see Fig. 1) was
the first sandbank from which the succession of geological layers
was defined (Trentesaux et al., 1999). Fig. 13 shows the original
seismic line and its interpretation, based on the combination with
the boreholes. The same information was now used in the voxel
modelling. Compared to the previous interpretations, we are
now able to model the lithological distribution, as well as the
related information entropy of the model (Fig. 14). The higher val-
ues of information entropy can be seen in the centre of the sand-
bank associated with the uncertainty caused by the lack of cores. By visualising and querying the different models, the effects of
the changes are discussed and evaluated. As a first example, the
effect of a better parameterisation of the Top-Palaeogene is
shown in Fig. 15. This was most striking for the delineation of
the Ostend palaeovalley in the nearshore zone. In the single-layer
model, fine sand voxels extended horizontally and masked
the boundary of the valley. With the addition of the Top-
Palaeogene surface, a distinct V-shape of the palaeovalley became
apparent and allowed showing an infill of the valley with fine-sand
(yellow) voxels above the Palaeogene clay layers. A second example
illustrates the dramatic change that the addition of layers can make
to the distribution of the lithological classes (Fig. 16). This is most
evident on the level of the sandbanks (e.g. 4.2.4. Four-layer model (Palaeogene – Pleistocene – Lower
Holocene – Upper Holocene) Clay–silt percentages are significantly
higher in the nearshore area, because of the nearby estuary of the
Scheldt river. In order to separate the two regions (with and
without Scheldt influence), the Middle Scarp (see Fig. 1) was used )
In the final model, the Upper Holocene layer was further split
(laterally) into two smaller areas. This was done primarily because
of the increasing presence of medium sand further offshore as well
as to reduce the presence of ‘flying’ clay voxels that were present in )
In the final model, the Upper Holocene layer was further split
(laterally) into two smaller areas. This was done primarily because
of the increasing presence of medium sand further offshore as well
as to reduce the presence of ‘flying’ clay voxels that were present in 10 Vasileios Hademenos et al. Fig. 7. Fence diagram of voxelized lithostratigraphical units in the BCS. The borehole dataset is colour-coded following their lithostratigraphical interpretation. The blue line
represents the extent of the modelled area. ostratigraphical units in the BCS. The borehole dataset is colour-coded following their lithostratigraphical interpretation. The blue line Fig. 7. Fence diagram of voxelized lithostratigraphical units in the BCS. The borehole dataset is colour-coded following their lithostra
represents the extent of the modelled area. of the higher-resolution model is the fact that the lithostratigraph-
ical units are much better defined, as shown by the Pleistocene
inner core of the sandbanks. Additionally, some features were bet-
ter delineated, e.g. the fine sand in the top zone of one of the sand-
banks which is present in both models. as proxy where the depositional environment changes from a near-
shore estuarine–marine to an offshore marine environment. The
results of the five-layer model are shown in Fig. 8E. Another way of querying the model is the creation of cross-
sections (Fig. 9). This type of data visualisation allows inspection
of the in-depth distribution of the different assigned variables. Following the geological cross-section of Fig. 1, we can see all five
lithostratigraphical units with the assigned lithology and informa-
tion entropy. Cross-sections like these give a first glimpse of the
areas where we are confident of the assigned lithology (blue colour
in bottom figure), e.g. the fine sand above the Ostend Valley and
the coarse sand tips off the Hinder Banks. 6.1. Importance of geological knowledge in voxel modelling Modelling the subsurface geology of shallow marine environments
is highly challenging because of the complex depositional environ-
ments that often vary on short spatial scales. Detailed interpolation
of lithological information is hence critical and requires the best
available knowledge on the geological layers constraining different
resource qualities. https://doi.org/10.1017/njg.2018.18
Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. niversiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. 5. Case study with higher resolution of the voxel model Hinder Banks) where
in the second run of the model the base of the sandbank is
better defined because of the addition of the Top-Palaeogene To compare the resource volumes between the different resolu-
tion models of the Hinder Banks, resource quantity was queried in
the first 2 m below seafloor (Fig. 11). The different resolutions do
not affect the total volume of the queried voxels. However, there is
a difference when comparing the lithologies of the two models
(Fig. 12). This difference is due to the fact that in the 100 × 100 ×
0.5 m resolution model the core dataset used is confined to a buffer
zone around the modelled area, while in the 200 × 200 × 1 m
resolution model the core dataset of the entire BCS (Fig. 3) is used. This clearly influences the voxels assigned with gravel, since the
percentage of gravel is much higher in the cores around the
Hinder Banks compared to the BCS dataset. Another advantage Netherlands Journal of Geosciences 11 Fig. 8. Top view of the different runs of the model. Left: lithoclass. Right: distribution of the entropy. (A) Uniform stratigraphy (no bounding surfaces defining the stratigraphy)
B) One bounding surface (Top-Palaeogene). (C) Two bounding surfaces (Top-Palaeogene and Top-Pleistocene), defining three lithostratigraphical layers of which only the
Pleistocene is shown here. (D) Three bounding surfaces (Top-Palaeogene, Top-Pleistocene and Lower Holocene) defining four lithostratigraphical layers of which only the
Lower Holocene is shown here. (E) Four bounding surfaces (Top-Palaeogene, Top-Pleistocene, Lower Holocene, Nearshore Upper Holocene, Offshore Upper Holocene), defining ig. 8. Top view of the different runs of the model. Left: lithoclass. Right: distribution of the entropy. (A) Uniform stratigraphy (no bounding surfaces defining the stratigraphy). B) One bounding surface (Top-Palaeogene). (C) Two bounding surfaces (Top-Palaeogene and Top-Pleistocene), defining three lithostratigraphical layers of which only the
Pleistocene is shown here. (D) Three bounding surfaces (Top-Palaeogene, Top-Pleistocene and Lower Holocene) defining four lithostratigraphical layers of which only the
ower Holocene is shown here. (E) Four bounding surfaces (Top-Palaeogene, Top-Pleistocene, Lower Holocene, Nearshore Upper Holocene, Offshore Upper Holocene), defining
ve lithostratigraphical layers of which the Nearshore (bottom) and Offshore Upper Holocene (top) are shown here. Fig. 8. Top view of the different runs of the model. Left: lithoclass. Right: distribution of the entropy. (A) Uniform stratigraphy (no bounding surfaces defining the stratigraphy). (B) One bounding surface (Top-Palaeogene). 5. Case study with higher resolution of the voxel model (C) Two bounding surfaces (Top-Palaeogene and Top-Pleistocene), defining three lithostratigraphical layers of which only the
Pleistocene is shown here. (D) Three bounding surfaces (Top-Palaeogene, Top-Pleistocene and Lower Holocene) defining four lithostratigraphical layers of which only the
Lower Holocene is shown here. (E) Four bounding surfaces (Top-Palaeogene, Top-Pleistocene, Lower Holocene, Nearshore Upper Holocene, Offshore Upper Holocene), defining
five lithostratigraphical layers of which the Nearshore (bottom) and Offshore Upper Holocene (top) are shown here. Fig. 8. Top view of the different runs of the model. Left: lithoclass. Right: distribution of the entropy. (A) Uniform stratigraphy (no bounding surfaces defining the stratigraphy). (B) One bounding surface (Top-Palaeogene). (C) Two bounding surfaces (Top-Palaeogene and Top-Pleistocene), defining three lithostratigraphical layers of which only the
Pleistocene is shown here. (D) Three bounding surfaces (Top-Palaeogene, Top-Pleistocene and Lower Holocene) defining four lithostratigraphical layers of which only the
Lower Holocene is shown here. (E) Four bounding surfaces (Top-Palaeogene, Top-Pleistocene, Lower Holocene, Nearshore Upper Holocene, Offshore Upper Holocene), defining
five lithostratigraphical layers of which the Nearshore (bottom) and Offshore Upper Holocene (top) are shown here. 12 Vasileios Hademenos et al. Fig. 9. Cross-section of the final model (for location, see Fig. 1). Top: lithostratigraphical units. Middle: lithological class. Bottom: model entropy on the lithological class. 0
indicates low and 1 high uncertainty. Fig. 9. Cross-section of the final model (for location, see Fig. 1). Top: lithostratigraphical units. Middle: lithological class. Bottom: mo
indicates low and 1 high uncertainty. Fig. 9. Cross-section of the final model (for location, see Fig. 1). Top: lithostratigraphical units. Middle: lithological class. Bottom: model entropy on the lithological class. 0
indicates low and 1 high uncertainty. Fig. 10. High-resolution voxel model (100 × 100 × 0.5 m) of the Hinder Banks. Left: lithological class. Right: model entropy for the lithological class, shown only for the Quaternary. Fig. 10. High-resolution voxel model (100 × 100 × 0.5 m) of the Hinder Banks. Left: lithological class. Right: model entropy for the lithological class, shown only for the Quaternary. 00 × 100 × 0.5 m) of the Hinder Banks. Left: lithological class. Right: model entropy for the lithological class, shown only for the Quaternary. Fig. 10. High-resolution voxel model (100 × 100 × 0.5 m) of the Hinder Banks. Left: lithological class. Right: model entropy for the lithologi surface. https://doi.org/10.1017/njg.2018.18
Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. niversiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/njg.2018.18
Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. rg/10.1017/njg.2018.18
from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available a 5. Case study with higher resolution of the voxel model In the third run of the model the introduction of the
Pleistocene unit allowed us to define a core of lithologically mixed
sediments. Finally, the fifth run of the model allowed us to differ-
entiate layers of different lithological classes in the main body of
the sandbanks once the offshore Upper Holocene unit was defined. The final figures illustrate that more data allow the creation of
higher-resolution models that constrain better the stratigraphical
layers and their lithological properties. Offshore Upper Holocene layer, as depicted in Fig. 15B, there is sig-
nificantly less clay–silt in the global proportions (Fig. 5) when split
from the Upper Holocene. As a result of the lower percentages, the
majority of ‘flying voxels’ (Fig. 15A) are no longer present. To quantify the added value of incorporating geological knowl-
edge in the voxel modelling procedure, the model uncertainty cal-
culations, performed on each of the model runs, are now discussed
and compared (Schweizer et al., 2017). Fig. 17 shows distribution
curves of the model uncertainty with each new addition of a geo-
logical layer. The addition of geological layers also allowed the reduction of the
number of ‘flying voxels’, that were introduced as a consequence of
the SIS method. In areas with low data density, SIS draws the litho-
logical class from the global proportions per lithostratigraphical unit. By splitting the model volume into separate lithostratigraphical
units, these percentages change according to the lithological content
of the boreholes belonging to the lithostratigraphical units. In the In the single-layer model (first run – blue line) the mean of the
model uncertainty distribution is close to 0.7 (Fig. 17A). Adding
the Quaternary layer (second run – red line), the mean is close
to 0.5, indicating an improvement of the modelling of the
Palaeogene layer. The different runs of the model did not have 13 Netherlands Journal of Geosciences Fig. 11. Queried volumes of the first 2 m of sediment in the Hinder Banks area. (A) 200 × 200 × 1 m resolution. (B) 100 × 100 × 0.5 m resolution. the same pronounced effect on the model uncertainty distribution
of the Pleistocene layer (Fig. 17B). Also, the addition of the Lower
Holocene layer did not show any improvement in the model
uncertainty distribution of the model (Fig. 17C). 5. Case study with higher resolution of the voxel model This can be
attributed to the fact that this layer is poorly describe
and that there are no distinct differences in th
content that the addition of the lithostratigraphi
describe. Fig. 11. Queried volumes of the first 2 m of sediment in the Hinder Banks area. (A) 200 × 200 × 1 m resolution. (B) 100 × 100 × 0.5 m resolution. Fig. 12. Queried volumes comparison of the first 2 m of sediment in the Hinder Banks area. Fig. 13. Example of a seismic reflection profile and interpreted seismostratigraphical units. From Trentesaux et al. (1999: Fig. 3, p. 256). ps://doi.org/10.1017/njg.2018.18
wnloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cam Fig. 11. Queried volumes of the first 2 m of sediment in the Hinder Banks area. (A) 200 × 200 × 1 m resolution. (B) 100 × 100 × 0.5 m resolution. Fig. 11. Queried volumes of the first 2 m of sediment in the Hinder Banks area. (A) 200 × 200 × 1 m resolution. (B) 100 × 100 × 0.5 m resolution. Fig. 12. Queried volumes comparison of the first 2 m of sediment in the Hinder Banks area. Fig. 13. Example of a seismic reflection profile and interpreted seismostratigraphical units. From Trentesaux et al. (1999: Fig. 3, p. 256). Fig. 12. Queried volumes comparison of the first 2 m of sediment in the Hinder Banks area. Fig. 12. Queried volumes comparison of the first 2 m of sediment in the Hinder Banks area. Fig. 13. Example of a seismic reflection profile and interpreted seismostratigraphical units. From Trentesaux et al. (1999: Fig. 3, p. 256). 13. Example of a seismic reflection profile and interpreted seismostratigraphical units. From Trentesaux et al. (1999: Fig. 3, p. 256). attributed to the fact that this layer is poorly described in the cores
and that there are no distinct differences in the lithological
content that the addition of the lithostratigraphical layer can
describe. the same pronounced effect on the model uncertainty distribution
of the Pleistocene layer (Fig. 17B). Also, the addition of the Lower
Holocene layer did not show any improvement in the model
uncertainty distribution of the model (Fig. 17C). This can be 14 Vasileios Hademenos et al. Fig. 14. Left: lithostratigraphical units. Middle: lithological class. 5. Case study with higher resolution of the voxel model Right: model uncertainty, as queried from the voxel model along the same cross-section of Trentesaux et al. (1999; see Fig. 13). For values see legend of Fig. 9. Fig. 14. Left: lithostratigraphical units. Middle: lithological class. Right: model uncertainty, as queried from the voxel model along the same cross-section of Trentesaux et al. (1999; see Fig. 13). For values see legend of Fig. 9. Fig. 15. The effect of adding the Top-Palaeogene bounding surface in the area of the Ostend Valley, a buried valley in the nearshore area. (A) Uniform stratigraphy model. (B) One-
layer model. Fig. 15. The effect of adding the Top-Palaeogene bounding surface in the area of the Ostend Valley, a buried valley in the nearshore area. layer model. Fig. 15. The effect of adding the Top-Palaeogene bounding surface in the area of the Ostend Valley, a buried valley in the nearshore area. (A) Uniform stratigraphy model. (B) One-
layer model. laeogene bounding surface in the area of the Ostend Valley, a buried valley in the nearshore area. (A) Uniform stratigraphy model. (B) One- Fig. 15. The effect of adding the Top-Palaeogene bounding surface in the area of the Ostend Valley, a buried valley in the nearshore area. (A) Uniform stratigraphy model. (B) One-
layer model Fig. 16. Cross-section in the area of the Hinder Banks showing the distribution of lithoclasses of the different runs of the 200 × 200 × 1 m model. (A) One-layer model (no bound
surfaces). (B) One bounding surface (Top-Palaeogene). (C) Two bounding surfaces (Top-Palaeogene and Top-Pleistocene). (D) Four bounding surfaces (Top-Palaeogene, T
Pleistocene, Lower Holocene, Nearshore Upper Holocene, Offshore Upper Holocene). Followed by the final results from the 100 × 100 × 0.5 m resolution model. (E) litholog
class, and (F) lithostratigraphical units. Fig. 16. Cross-section in the area of the Hinder Banks showing the distribution of lithoclasses of the different runs of the 200 × 200 × 1 m model. (A) One-layer model (no bounding
surfaces). (B) One bounding surface (Top-Palaeogene). (C) Two bounding surfaces (Top-Palaeogene and Top-Pleistocene). (D) Four bounding surfaces (Top-Palaeogene, Top-
Pleistocene, Lower Holocene, Nearshore Upper Holocene, Offshore Upper Holocene). Followed by the final results from the 100 × 100 × 0.5 m resolution model. (E) lithological
class, and (F) lithostratigraphical units. Fig. 16. 5. Case study with higher resolution of the voxel model Cross-section in the area of the Hinder Banks showing the distribution of lithoclasses of the different runs of the 200 × 200 × 1 m model. (A) One-layer model (no bounding
surfaces). (B) One bounding surface (Top-Palaeogene). (C) Two bounding surfaces (Top-Palaeogene and Top-Pleistocene). (D) Four bounding surfaces (Top-Palaeogene, Top-
Pleistocene, Lower Holocene, Nearshore Upper Holocene, Offshore Upper Holocene). Followed by the final results from the 100 × 100 × 0.5 m resolution model. (E) lithological
class, and (F) lithostratigraphical units. 15 Netherlands Journal of Geosciences Fig. 17. Distribution of the model entropy on the lithological class for each layer queried for different runs of the model. 0 indicates low and 1 high uncertainty. Fig. 17. Distribution of the model entropy on the lithological class for each layer queried for different runs of the model. 0 indicates low and 1 high uncertainty. he model entropy on the lithological class for each layer queried for different runs of the model. 0 indicates low and 1 high uncertaint Fig. 17. Distribution of the model entropy on the lithological class for each layer queried for different runs of the model. 0 indic with high uncertainty help us to understand the limitations of the
datasets and will guide the planning of new data acquisition
surveys. Adding the Nearshore Upper Holocene layer resulted in a more
distinctive peak in the model uncertainty spectrum (Fig. 17D –
green line). The high values of model uncertainty (0.6–0.8) are
caused by the complex geology of the Scheldt estuary. The presence
of laminated layers consisting of clay and fine sand makes it diffi-
cult for the method to predict the lithology of the voxels in the
region, at least at the scale of the present model. With increasing use of the BCS, both by the public and private
sector, there are ample opportunities to validate and apply the
model for different purposes, ideally by incorporating new data. Some of these applications may require a higher vertical resolution,
which will require more tests. With the 1 m vertical scale resolu-
tion, certain features such as the clay–silt–fine-sand laminated
layering in the estuarine deposits are easily overlooked, especially
in regions with high lithological heterogeneity. A case study close
to the port of Zeebrugge has been planned where the resolution will
be decreased to 0.7–0.4 m. 5. Case study with higher resolution of the voxel model In this area, it is also planned to incor-
porate additional data, such as cone penetration tests, making the
model more valuable for geotechnical applications. The Offshore Upper Holocene layer shows two distinct peaks
(0.5 and 0.65) in the model uncertainty spectrum in the second
and third runs of the model (Fig. 17E – red, light blue and purple
lines). When the Holocene layer is split in the fifth run of the model
(green line), the model uncertainty improves (peak around 0.4). This is because the offshore sandbanks have a better data coverage
than the areas in the gullies between the sandbanks, where the
information entropy is higher (0.5–0.7). Last but not least, it needs emphasising that the model is the
backbone of a resource decision support system (Van Lancker
et al., 2017). As such, users have direct access through the
TILES website to the available geological information and can
query resource quality and quantity, in combination with environ-
mental and socio-economic datasets, in view of different applica-
tions, now and in the future. 6.2. Future perspectives The model provides the uncertainty (information entropy) of the
lithological class in each voxel based on the calculations of the SIS
method. However, this uncertainty is now solely related to statis-
tical calculations, whilst there is also uncertainty imposed by the
dataset itself. Data-related uncertainties include those originating
from the seismic lines (bad quality, misplacement) and borehole
descriptions (mislabelling, insufficient interpretations, inadequate
metadata) datasets and may have an adverse effect on the confi-
dence we have in the resulting model. These uncertainties have
already been quantified and incorporated in the model as flags,
and procedures are now evaluated on how to propagate those
uncertainties in the voxel models (De Tré et al., 2018). The areas https://doi.org/10.1017/njg.2018.18
Downloaded from https://www.cambridge.org/core. Universiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. niversiteit Gent, on 30 Apr 2019 at 15:54:41, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. 7. Conclusions A 3D voxel model was created depicting in detail stratigraphical
and lithological information of the subsurface of the BCS. The
3D environment allows easy viewing of geological properties
providing spatial context to certain features or heterogeneities in
the subsurface, which is highly valuable in resource management. Vasileios Hademenos et al. 16 Geological knowledge was incorporated in a stepwise approach
and gave a level of detail in the model that would not have been
achieved by only using available coring data. Moreover, the step-
wise approach allowed us to monitor the effect of each layer addi-
tion on the model and its effectiveness in describing the geological
features. In addition, model uncertainty, calculated as information
entropy, was added to the voxels, providing insight into how suc-
cessful the model is in unambiguously estimating lithological
classes. Because of the automated workflow, new data can be incor-
porated easily and results can be compared and evaluated against
previous versions of the model. Goovaerts, P., 1997. Geostatistics for natural resources evaluation. Oxford
University Press (New York): 483 pp. Goovaerts, P., 1997. Geostatistics for natural resources evaluation. Oxford
University Press (New York): 483 pp. Jørgensen, F., Høyer, A., Sandersen, P., He, X. & Foged, N., 2015. Combining
3D geological modelling techniques to address variations in geology, data
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Vera Van Lancker 0000-0002-8088-9713 Author ORCIDs. Vera Van Lancker 0000-0002-8088-9713 Author ORCIDs. Vera Van Lancker 0000-0002-8088-9713 Acknowledgements. This paper is a contribution to the Brain-be project TILES
(Transnational and Integrated Long-term marine Exploitation Strategies), funded
by Belgian Science Policy (Belspo) under contract BR/121/A2/TILES, and the
Flemish research project SeArch (IWT SBO, contract nr. 120003). The research
is fully supported by the ZAGRI project, a federal Belgian programme for con-
tinuous monitoring of sand and gravel extraction, paid from private revenues. Contributing EU projects have been EMODnet Geology (MARE/2008/03;
MARE/2012/10; EASME/EMFF/2016/1.3.1.2 – Lot 1/SI2.750862)), Geo-Seas
(FP7, Grant 238952) and ODIP (FP7, Grant 312492). Underlying data have been
acquired during numerous campaigns on RV Belgica, RV Simon Stevin and many
Dutch research and monitoring vessels. For Belgian waters, ship-time was granted
by Belspo / RBINS ODNature and Flanders Marine Institute. Nikki Trabucho,
TNO, is acknowledged for the graphical design of Figures 2 and 5. The
Federal Public Service Economy, Continental Shelf department is thanked for
continuous support. Results, datasets, findings and the final version of the models
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information entropy as a quality measure for 3-D geological models. Tectonophysics 526–529: 207–216. Wentworth, C.K., 1922. A scale of grade and class terms for clastic sediments. The Journal of Geology 30: 377–392. Van Lancker, V.R.M., Bonne, W., Garel, E., Degrendele, K., Roche, M., Van
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2016) Van Lancker, V., Francken, F., Kint, L., Terseleer, N., Van den Eynde, D., De
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g
isatis-geostatistics-software (accessed on April 2015)
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TILES: http://www.odnature.naturalsciences.be/tiles/ (accessed on July 2018)
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O filme de busca e a construção ensaística de uma memória familiar emoldurada pelas montanhas mineiras
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* Adriano Medeiros da Rocha: Universidade Federal de Ouro Preto, Instituto de Ci-
ências Sociais Aplicadas, curso de Comunicação Social-Jornalismo. 35420000 - Ma-
riana, Minas Gerais, Brasil. E-mail: adrianomedeiros.audiovisual@gmail.com
Eduardo Henrique Moreira: Mestrando. Universidade Federal de Ouro Preto, Instituto
de Ciências Sociais Aplicadas (ICSA), Programa de Pós-Graduaçao em Comunicação.
35420-000, Mariana, Minas Gerais, Brasil. E-mail: eduardomoreira.op@gmail.com O filme de busca e a construção ensaística de uma memória
familiar emoldurada pelas montanhas mineiras Adriano Medeiros da Rocha & Eduardo Henrique Moreira* Seu Carlito, narrativas sobre um comerciante da roça (Brasil, 20
70min30seg)
Direção e Montagem: Eduardo Moreira
Produção: Eduardo Moreira e José Carlos Moreira
Fotografia e Som Direto: Eduardo Moreira e Thiago Novais
Trilha Sonara: Matheus Ferro Seu Carlito, narrativas sobre um comerciante da roça (Brasil, 2016
70min30seg)
Direção e Montagem: Eduardo Moreira
Produção: Eduardo Moreira e José Carlos Moreira
Fotografia e Som Direto: Eduardo Moreira e Thiago Novais
Trilha Sonara: Matheus Ferro O início, o fim, e o meio... Doc On-line, n. 24, setembro de 2018, www.doc.ubi.pt, pp. 294-306. DOI: 10.20287/doc.d24.ac05 DOI: 10.20287/doc.d24.ac05 O início, o fim, e o meio... A produção de um documentário envolve a captação de algum tipo matéria
prima temática. Nesse processo, como também na edição, o cineasta expõe sua
visão do tema abordado em seu filme, ele enquadra sua história. Na concep-
ção de Sheila Bernard (2008), os filmes documentários se baseiam em fatos,
e é disso que vem o seu poder e sua diferenciação do cinema de ficção. Par-
tindo desse princípio, podemos entender os documentários como uma forma
subjetiva de representação do mundo derivada das decisões do cineasta. Esses
filmes são, portanto, carregados de sentidos próprios de quem os faz. De acordo com Bill Nichols (2005: 47), os documentários não são uma re-
produção da realidade, mas são representações do mundo que vivemos. Assim
como fazem os outros meios de comunicação, no processo de produção de um
documentário, os fatos são representados através da abordagem escolhida pelo
diretor e sua equipe. Assim, essa abordagem emerge de uma série de escolhas. Consuelo Lins e Cláudia Mesquita (2008: 58) consideram que a rede de
escolhas que envolvem a produção documental é o “mais puro” artifício que
gera o filme. Para os autores, os documentários “não brotam do coração do
real, espontâneos, naturais, recheados de pessoas e situações autênticas”, eles
dependem da dedicação e envolvimento de seus realizadores Sheila Curran Bernard (2008: 47) afirma que as redes de escolhas definem
como as histórias são contadas nos documentários. Para ela, essa rede envolve O filme de busca e a construção ensaística de uma memória familiar... 295 escolhas criativas acerca da estrutura do filme, de perspectiva, de equilíbrio, de
estilo, de elenco, entre outras. A autora entende que as escolhas caracterizam
a abordagem do filme. Dê a um grupo de cineastas alguma aparelhagem e a mesma história a ser
contada em linhas gerais, e você acabará tendo em mãos filmes bastante di-
ferentes em estilos, ponto de vista, enfoque e, na verdade, diversos em muito
mais do que isso. Essas diferenças caracterizam a abordagem: como apresen-
tar a história na tela. (Bernard, 2008: 47) Cada filme documentário tem sua abordagem, ou seja, a rede de escolhas
criativas feitas para sua realização. Portanto, como afirma Nichols (2005: 48),
são todos diferentes, pois “não adotam um conjunto fixo de técnicas, não tra-
tam apenas de um conjunto de questões, não apresentam apenas um conjunto
de formas ou estilos”. O início, o fim, e o meio... Bernard considera a maior parte dos documentários
como sendo também narrativas e, em sua opinião, isso significa, pura e sim-
plesmente, que eles contam histórias. Para a autora, a forma como estas obras
contam as histórias e quais histórias contam são os elementos que separa esses
filmes em subcategorias de gêneros ou estilo (Bernard, 2008:15). Mesmo com as subcategorias que caracterizam alguma(s) abordagem(s), a
prática está sujeita a constantes mudanças. Neste universo, há uma constante
busca por abordagens alternativas e transformações no uso da linguagem. Re-
fletindo sobre essa questão, Bernard ressalta que: Ao discutir abordagem, tenha consciência de que os cineastas, conforme se
mencionou, não raro combinam diferentes abordagens. Alguns usam narra-
ção (ou texto na tela, que desempenhe o mesmo papel), mas a usam com
parcimônia. Outros combinam voice-over com narração, ou o roteiro com
voice-over em lugar da narração. Alguns filmes fabricam situações que então
se desdobram na tela, tornando-se, portanto, observacionais. (Bernard, 2008:
51) Bill Nichols (2005) argumenta que os documentários representam ques-
tões, aspectos, características e problemas encontrados no mundo histórico por
meio de sons e imagens que compões seus discursos, o que suscita a questão
da voz que cada filme tem. O fato de documentários não serem uma reprodução da realidade da a eles
uma voz própria. Eles são uma reprodução do mundo, e essa representação
significa uma visão singular do mundo. A voz do documentário é, portanto,
o meio pelo qual esse ponto de vista ou essa perspectiva singular se dá a
conhecer. (Nichols, 2005: 73) O fato de documentários não serem uma reprodução da realidade da a eles
uma voz própria. Eles são uma reprodução do mundo, e essa representação
significa uma visão singular do mundo. A voz do documentário é, portanto,
o meio pelo qual esse ponto de vista ou essa perspectiva singular se dá a
conhecer. (Nichols, 2005: 73) Cada documentário tem sua voz fílmica, ou seja, uma natureza própria. Essa voz também pode ser entendida como uma impressão digital que atesta a
individualidade do cineasta, ou até mesmo o poder de decisão de seus financia- 296 Adriano Medeiros da Rocha & Eduardo Henrique Moreira dores. Sendo a maneira como o cineasta traduz seu ponto de vista e estabelece
uma relação com mundo histórico, a voz fílmica remete ao estilo do filme. O início, o fim, e o meio... Quando assistimos a documentários participativos, esperamos testemunhar o
mundo histórico da maneira pela qual ele é representado por alguém que nele
se engaje ativamente, e não por alguém que observa discretamente, recon-
figura poeticamente ou monta argumentativamente esse mundo. O cineasta
despe o manto do comentário com voz-over, afasta-se da meditação poética,
desce do lugar onde pousa a mosquinha na parede e torna-se um ator social
(quase) como qualquer outro. (Nichols, 2005: 154) Quando assistimos a documentários participativos, esperamos testemunhar o
mundo histórico da maneira pela qual ele é representado por alguém que nele
se engaje ativamente, e não por alguém que observa discretamente, recon-
figura poeticamente ou monta argumentativamente esse mundo. O cineasta
despe o manto do comentário com voz-over, afasta-se da meditação poética,
desce do lugar onde pousa a mosquinha na parede e torna-se um ator social
(quase) como qualquer outro. (Nichols, 2005: 154) Documentários participativos simulam o que é, para o cineasta, estar numa
determinada situação e como ela consequentemente se altera. A experiência
extraída do encontro entre o cineasta e o tema será o conteúdo do filme. O
cineasta e tem a possibilidade de se posicionar como mentor, crítico, interro-
gador, colaborador ou provocador, ou mesmo como fio condutor da história. (Nichols, 2005 :155). Silvio Da-Rin (2006) acredita que, nesse modo ensaístico e interativo, “o
cineasta dispõe de novos recursos para recusar o papel de agenciador oculto
de imagens sonoras e visuais, podendo exibir-se como um ser humano impli-
cado” (Da-Rin, 2006: 155 e 156). Além dessas características, Dan-Rin aponta
também para o antiilusionismo que se manifestou de forma colateral no estilo
interativo que permeia o filme ensaio. No modo interativo, o antiilusionismo se manifesta de forma colateral, apesar
da contribuição inaugural de ChroniquedùnÉté. Um dos fatores que marcou
a novidade radical foi a liberdade com que os equipamentos de filmagem e os
membros da equipe eram exibidos. (...) Fundava-se ali a tendência de deslocar
o documentarista dos bastidores para a superfície do filme, substituindo a
voz off incorpórea por um corpo humano visível que interage com os atores
sociais. (Da-Rin, 2006: 183) No modo interativo, o antiilusionismo se manifesta de forma colateral, apesar
da contribuição inaugural de ChroniquedùnÉté. Um dos fatores que marcou
a novidade radical foi a liberdade com que os equipamentos de filmagem e os
membros da equipe eram exibidos. O início, o fim, e o meio... Para Bill Nichols (2005: 135), ao definir traços característicos semelhantes
nas vozes individuais de filmes e cineastas, podemos estabelecer grupos que
compartilham vozes semelhantes. Essas vozes compartilhadas referem-se aos
gêneros de documentário. Nichols divide os gêneros do documentário em seis modos: poético, expo-
sitivo, observativo, participativo, reflexivo e performático. Apesar de determi-
narem uma estrutura de afiliação frouxa, eles “estabelecem as convenções que
um determinado filme pode adotar e propiciam expectativas específicas que os
espectadores esperam ver satisfeitas” (Nichols, 2005: 135). Por meio desta pesquisa, buscar-se-á investigar e compreender o tipo ou
modo de documentário que Consuelo Lins e Cláudia Mesquita (2008: 55) con-
sideram como ensaio fílmico, ou seja, aquele formato que remete a uma forma
híbrida, sem regras nem definição exata, mas que articula modos de abordagem
e composições variados, objetos e discursos heterogêneos. Para Bill Nichols
(2005: 170) essas características mencionadas pelas autoras acima se aproxi-
mam ao modo que ele denomina performático. Este último teórico do cinema
entende que filmes desse estilo dão mais ênfase às características subjetivas da
experiência e da memória, se afastando do relato objetivo. Para ele, o signifi-
cado é claramente um fenômeno subjetivo, carregado de afetos. Um carro, um revólver, um hospital ou uma pessoa terão significados diferen-
tes para pessoas diferentes. Experiência e memória, envolvimento emocional,
questões de valor e crença, compromisso e princípio, tudo isso faz parte de
nossa compreensão dos aspectos do mundo que mais são explorados pelo do-
cumentário (...). O Documentário performático sublinha a complexidade de
nosso conhecimento do mundo ao enfatizar suas dimensões subjetivas e afe-
tivas. (Nichols, 2005: 169) Já Sílvio Da-Rin (2006: 183) aponta como elemento principal desse sub-
gênero a auto-reflexidade, que constitui uma busca de alternativas às insufici-
ências e às limitações identificadas nos diversos modos de representação em
lidar criticamente com o ilusionismo cinematográfico. Os conceitos sobre este tipo de filme são variados e nem sempre conduzem
a um consenso. Contudo, para esta pesquisa, adotaremos a perspectiva de que
o ensaio fílmico possui sua hibridez justificada em suas variações demasiadas -
transições e transformações nas quais gêneros e estilos não se limitam, mas se
complementam. A participação também acontece nesse tipo de filme, e é uti-
lizada de diversas maneiras. Esses traços, segundo Nichols, são características
que passam a aparecer nos documentários que ele denomina participativos. 297 O filme de busca e a construção ensaística de uma memória familiar... (...) é o desempenho de Emiko Omori na reconstituição da história reprimida
da experiência de sua própria família, nos campos de confinamento de nipo-
americanos durante a Segunda guerra Mundial, que dá forma a Coelho na
lua (1999). Em Diário inconcluso (1983), Marilu Mallet propõe uma estru-
tura ainda mais explícita, na forma de diário, ao fazer o retrato de sua vida
de exilada chilena em Montreal, casada com o cineasta canadense Michael
Rubbo. (...) Esses filmes fazem do cineasta uma pernona tão nítida quanto O início, o fim, e o meio... (...) Fundava-se ali a tendência de deslocar
o documentarista dos bastidores para a superfície do filme, substituindo a
voz off incorpórea por um corpo humano visível que interage com os atores
sociais. (Da-Rin, 2006: 183) Conforme Nichols (2005: 158), há casos em que o cineasta se distancia da
postura investigativa para adotar uma relação mais receptiva e reflexiva frente
aos acontecimentos que o filme deseja contar ou que envolvem este profis-
sional e seu tema. Essa escolha leva o filme a se aproximar do diário e do
testemunho pessoal. Nesse tipo de narrativa é comum encontrarmos a voz na
primeira pessoa predominando na estrutura global do filme. Seria o engaja-
mento participativo do cineasta, agora também como sujeito registrado, um
dos principais elos para se conseguir a atenção do público. 298 Adriano Medeiros da Rocha & Eduardo Henrique Moreira qualquer outra de seus filmes. Como testemunho ou confissão, muitas vezes,
estes manifestam um poder revelador. (Nichols, 2005: 158 e 159) O crítico de cinema afirma que a intensidade emocional e a expressividade
subjetiva do modo performático tomaram forma nos anos 80 e 90 e foram
desenvolvidos principalmente por grupos que possuíam forte atenção para a
esfera social e as questões comunitárias. De acordo com Cláudia Mesquita e
Consuelo Lins (2008), essas características ensaísticas no Brasil são notáveis
em filmes dos anos 80, como Mato eles? (1982), de Sergio Bianchi e Ilha das
Flores (1989), de Jorge Furtado. Os filmes desse estilo são carregados de emoção e significados que dão a
eles características expressivas de uma dada perspectiva. Desse modo, os docu-
mentários performáticos dirigem aos espectadores uma representação pessoal
e pontual de sujeitos específicos, incluindo o cineasta. (Nichols, 2005: 43). Dialogando com Nichols, Silvio Da-Rin entende que o cineasta assimila os re-
cursos retóricos desenvolvidos ao longo da história do documentário e produz
uma inflexão deles sobre si mesmos. Não satisfeito em simplesmente expor argumentos sobre seu objeto, o cine-
asta passa a engajar-se em um metacomentário sobre os mecanismos que dão
forma a este argumento. No lugar da ênfase absoluta sobre os personagens e
os fatos do mundo histórico, o próprio filme afirma-se como fato no domínio
da linguagem. O início, o fim, e o meio... (Da-Rin, 2006: 170) Para Nichols, o estilo performático, ou o ensaio-fílmico, mistura livre-
mente as técnicas expressivas que dão textura e densidade àquela ficção que
ainda era presente nos modos poético e expositivo, mas que foi sendo deixada
de lado quando o modo observacional priorizou a filmagem direta do encon-
tro social, ou seja, possibilidades audiovisuais como planos de ponto de vista,
números musicais, representações de estado subjetivo da mente, retrocessos,
fotogramas congelados. Nichols (2005: 171) ainda ressalta que os filmes desse estilo "nos envol-
vem menos com ordens ou imperativos retóricos do que com a sensação re-
lacionada com sua nítida sensibilidade”. Em sua opinião, a sensibilidade do
cineasta busca estimular a do espectador, ou seja, é a carga afetiva aplicada no
filme que indiretamente nos envolve na sua representação do mundo histórico. Um dos métodos utilizados para a realização dos documentários de ensaio
é o que o crítico cineasta Jean-Louis Comolli (2008) chama de dispositivos de
escritura. Consuelo Lins e Cláudia Mesquita entendem que o dispositivo de
escritura está relacionado à criação, pelo realizador, de um artifício ou proto-
colo produtor de situações a serem filmadas ou a um tipo de maquinação de
uma lógica, de um pensamento, que atribui às condições, regras e/ou limites O filme de busca e a construção ensaística de uma memória familiar... 299 para que esses filmes aconteçam. Na opinião das autoras, a adoção de um dis-
positivo nega diretamente a ideia de documentário como obra que aprende a
essência de uma temática ou realidade fixa e preexistente. Não se trata, porém, de um procedimento produtor que gera efeitos seme-
lhantes em todo o filme, mas que é criado a cada obra, imanente, contingente
às circunstâncias de filmagem, e submetido às pressões do real. A simples
adoção de um dispositivo não garante, em suma, o sucesso de um filme; tudo
depende da sua adequação ao assunto eleito, mas sobretudo do trabalho con-
creto de filmagem, que a maquinação anterior não dispensa. (Lins & Mes-
quita, 2008: 57) O documentarista Eduardo Coutinho, ao refletir sobre dispositivo fílmico,
utilizava o termo prisão para caracterizar o conjunto de regras auto impostas
que delimitam o processo de realização de seus documentários. O dispositivo
utilizado pelo cineasta, pelo menos até Edifício Master (2002), era de ordem
espacial: filmar numa única locação (Lins e Mesquita, 2008). O início, o fim, e o meio... Porém, esse tipo
de abordagem só foi evidenciado no decorrer dos anos 2000 pelos documentá-
rios Um passaporte húngaro (2002), de Sandra Kougut, e 33 (2003), de Kiko
Goffman, os quais Consuelo Lins e Cláudia Mesquita denominam filmes de
busca. Neles, o motivo da realização do documentário deixa de ser a alteridades clás-
sica para se relacionar a aspectos da experiência pessoal e da subjetividade dos
próprios realizadores. Nos dois filmes, os diretores interagem com persona-
gens e situações como sujeitos interessados, protagonistas de um processo de
busca pessoal. (Lins & Mesquita, 2008: 51) Lins e Mesquita notam que as histórias registradas não preexistem à fil-
magem, mas são produzidas por um agir do documentarista, portanto, eles são
também o fio condutor de seus filmes. Segundo Lins e Mesquita, o crítico
Bernadet aposta no híbrido pessoa-personagem: Como notou Jean Claude Bernadet: “O documentarista determina um pro-
jeto, sabe de onde parte, sabe o que gostaria de alcançar, mas não pode prever
os resultados a que chegará nem o percurso que terá de cumprir. (...) Essas
pessoas-personagens têm objetivos, enfrentam obstáculos, alcançam seus ob-
jetivos ou não, exatamente como nos filmes de ficção”. (Lins & Mesquita,
2008: 52) A partir do diálogo teórico apresentado anteriormente, pode-se entender
que os filmes de busca são documentários dos modos participativo e perfor-
mático, ou então são filmes de ensaio, que possuem um dispositivo que faz
o cineasta sair em busca de um objetivo. Nesse tipo de documentário, é es-
tabelecida uma conexão entre o domínio privado e o domínio público, como
acontece em Um passaporte húngaro (2002). Dessa forma, o cineasta faz sua
representação do mundo histórico de maneira subjetiva através da exibição de 300 Adriano Medeiros da Rocha & Eduardo Henrique Moreira uma busca íntima, marcando a diferença desse documentário em relação à ex-
posição da vida privada a que se assiste diariamente na televisão. Lins e Mesquita apontam também como traço marcante dos documentários
de busca a utilização da entrevista com uso deslocado, sem reproduzir a tradi-
cional dicotomia sujeito-objeto. Para elas, as entrevistas aparecem nos filmes
de busca como “instrumento para obtenção de informações no processo con-
creto de pesquisa e busca empreendido pelos realizadores.” (Lins & Mesquita,
2008: 52). O início, o fim, e o meio... Inspirados em conceitos encontrados nos filmes de busca e no mecanismo
da pessoa-personagem, os realizadores Eduardo Moreira e Thiago Novais tam-
bém decidiram ir a campo para desenvolver o primeiro média metragem da
dupla, em 2016. O documentário, ou ensaio fílmico, que realizaram também
pode ser identificado como um filme de busca. Um dos realizadores da obra
– Eduardo Moreira – é o personagem condutor da história numa busca pela
memória do seu avô, Carlos Moreira. O dispositivo escolhido para a gravação
é a trajetória da equipe saindo da cidade de Ouro Preto com destino ao sub-
distrito de Piranga onde viveu Carlos, o Manja Léguas. Durante o percurso,
a dupla passou pelos distritos ouropretanos: Santa Rita de Ouro Preto, Ban-
deiras e Santo Antônio. Nas várias paradas a dupla entrevistou filhos e netos
do patriarca da família Moreira. Também nessas localidades foi feito o regis-
tro em vídeos das paisagens, situações cotidianas e objetos que representam
a cultura e o estilo de vida da zona rural mineira. Da mesma maneira, foram
registradas cenas do cotidiano dos entrevistados que remetem, ou representam,
elos de relacionamento com Carlos Moreira. Segundo Bernard (2008: 185) é
preciso haver imagens de cobertura suficientes para dar opções para a edição. Pense em rodar o seu documentário como você faria com um drama: no âm-
bito de determinada cena você desejará tomadas amplas, tomadas médias,
close-ups e cortes, para garantir que as tomadas sejam suficientemente longas
e estáveis para serem usadas. Você quer ser capaz de criar cenas visuais que
proporcionem contexto e alguma outra informação à história. (Bernard, 2008:
185) O percurso regido pelo dispositivo adotado representa um mergulho feito
pelo personagem central na história do avô de Eduardo. Através da vontade
de resgatar parte significativa de uma memória de sua família, o neto de Car-
los, que só o conheceu pelas histórias que ouvia do seu pai, dos tios e da
falecida avó, decidiu embarcar em uma viagem para o Manja Léguas e do-
cumentar essa busca. A cada parada, um ou mais encontros com os tios e
primos, que contaram e demonstraram, através das suas narrativas, quem foi
Carlos dentro daquela memória coletiva. As interações tiveram um caráter
mais íntimo/pessoal por se tratar de um encontro em família para compartilhar O filme de busca e a construção ensaística de uma memória familiar... 301 a memória da mesma. O início, o fim, e o meio... Esse tipo de encontro para conversas sobre temas fa-
miliares, normalmente, é uma situação rotineira no cotidiano dos habitantes da
zona rural. A partir do conteúdo das entrevistas houve a tentativa de apresentar
os aspectos imagéticos e também emocionais desses encontros. Para que os entrevistados não se sentissem muito incomodados com a pre-
sença da(s) câmera(s) durante os diálogos, primeiramente a equipe buscou
constituir um tempo de interação com cada um deles. Logo no momento da
chegada ao ambiente de moradia ou trabalho de cada entrevistado o registro já
começava a ser feito, a câmera era logo apresentada ao entrevistado e o equipa-
mento registrador seguia o caminho. Na sequência, o entrevistado apresentava
a sua casa/trabalho e, aos poucos, era estabelecida uma relação mais amena
com a câmera. Após esse período de aproximação, a entrevista em profundi-
dade era realizada. Ao se aproximar e estabelecer um tempo de convívio com seus parentes
do interior, o ator social central se aprofundou cada vez mais da história de
sua família. A cada encontro era promovida também uma nova descoberta que
remetia ao passado da família e atestava contestações de seu presente. O uni-
verso de expectativas e impressões geradas pelos encontros foi ilustrado pelas
imagens de uma segunda câmera, que colocava o pesquisador, literalmente,
como agente participativo. Esse registro foi tentado no calor do momento em
cada uma das chegadas e partidas. As experiências obtidas durante o trajeto pelas estradas repletas de pai-
sagens diferenciadas e localidades típicas da zona rural mineira e também de
cada um dos encontros com os familiares do personagem que buscava a his-
tória do seu avô foram registrados em duas perspectivas: em primeira pessoa,
pelo próprio personagem e sua câmera, e em terceira pessoa por um com-
panheiro de viagem – Thiago Novais. O objetivo foi ter um ponto de vista
além do que era registrado pelo personagem central que fez a busca. Essas
imagens de cobertura explicitam detalhes dos cenários, dos personagens e da
natureza dos encontros, na tentativa de ilustrar o universo cultural e simbólico
que os envolve. Conforme o entendimento de Bernard (2008: 185), é impor-
tante garantir as informações visuais necessárias para o registro da busca do
personagem central, a personalidade dos entrevistados e os encontros. O início, o fim, e o meio... Note que você está filmando casualmente tudo que for possível: está asse-
gurando que possui informação visual passível de transmitir a informação
narrativa básica: o que, onde, como. Quer estabelecer tempo, lugar e pessoas,
buscando imagens que possam lhe fazer cortar informações verbais. Atente
nos detalhes narrativos que revelam personagens: pode ser um cigarro es-
quecido enquanto queima ou a pilha de garrafas de licor no lixo reciclável. Busque tomadas que mostrem como as pessoas se comportam umas em rela-
ção às outras e com que habilidade manuseiam suas ferramentas de trabalho. 302 Adriano Medeiros da Rocha & Eduardo Henrique Moreira Você pode querer humor. E conforme foi mencionado, precisa estar certo de
ter uma série suficiente de ângulos, tomadas e cortes que possibilitarão ao
editor condensar horas de material em um filme final que conte uma história
coerente e visualmente satisfatória. (Bernard, 2008: 185) A dupla partiu da hipótese de que a identidade de Carlos Moreira, presente
na memória daqueles que o conheceram, é resultado dos seus feitos em vida. Histórias sobre o patriarca da família Moreira são contadas e recontadas há
cerca de meio século. Na família do caixeiro viajante as lembranças da sua
trajetória já são repassadas para a geração dos seus bisnetos. Nas comunidades
e distritos próximos ao Manja Léguas, até mesmo os filhos e netos dos que
conviveram com Carlos em vida reconhecem sua importância na história da
região. Inicialmente, a vontade da dupla foi entender como se dá a construção da
identidade do indivíduo. Em seguida também houve a necessidade de com-
preender o funcionamento da memória coletiva das famílias e da identidade
familiar. Como o filme dialoga sobre memórias de uma família do interior,
questões ligadas à complexidade do ambiente e das relações sociais encontra-
das na zona rural mineira também permeiam a obra. No caminho teórico que embasou a pesquisa, a dupla de realizadores uti-
lizou alguns conceitos chave, tais como identidade e família. Com base em
Anthony Giddens (1938), pode-se afirmar que as ações do indivíduo são obser-
vadas e compreendidas por ele próprio e que são sustentadas por suas convec-
ções e estados emocionais. O meio que cerca o indivíduo, a época e sociedade
em que vive, os atributos culturais que lhe são dados, tudo isso se relaciona
diretamente com as referidas questões que relacionam sua existência. O início, o fim, e o meio... Esses
fatores envolvem também o processo de construção de significados baseados
nos conjuntos de atributos culturais inter-relacionados que levam a definição
da(s) identidade(s) do indivíduo (Castells, 1942: 22). Nesse sentido, Manuel
Castells afirma que para um determinado indivíduo, ou ainda um ator coletivo,
pode haver identidades múltiplas. Porém essa pluralidade é apontada pelo au-
tor como uma “[...] fonte de tensão e contradição tanto na auto-representação
quanto na ação social” devido à confusão existente entre o que é a identidade
“[...] e o que tradicionalmente os sociólogos têm chamado de papéis, e con-
juntos de papéis”. (Castells, 1942: 22). Do ponto de vista sociológico, toda identidade é construída. As experi-
ências adquiridas pelo indivíduo ao longo de sua vida são processadas pelas
instituições sociais nas quais ele está inserido e assim sua identidade é esta-
belecida. Nessa linha de raciocínio, Castells lança a hipótese de que quem
constrói as identidades são os determinantes do conteúdo simbólico das iden- O filme de busca e a construção ensaística de uma memória familiar... 303 tidades coletivas e seu significado para aqueles que com ela se identificam ou
dela se excluem. A construção de identidades vale-se da matéria prima fornecida pela histó-
ria, geografia, biologia, instituições produtivas e reprodutivas, pela memória
coletiva e por fantasias pessoais, pelos aparatos de poder e revelações de cu-
nho religioso. Porém, todos esses materiais são processados pelos indivíduos,
grupos sociais e sociedades, que reorganizam seu significado em função de
tendências sociais e projetos culturais enraizados em sua estrutura social, bem
como em sua visão de tempo/espaço. Havendo aqui a hipótese de que, em li-
nhas gerais, quem constrói a identidade coletiva, e para quê essa identidade
é construída, são em grande medida os determinantes do conteúdo simbólico
dessa identidade, bem como de seu significado para aqueles que com ela se
identificam ou dela se excluem. (Castells, 1942: 23) Castells (1942: 26) considera que a construção da identidade do indivíduo
consiste no seu projeto de vida. Nesse ponto, o indivíduo que assume um pa-
pel dentro da sociedade é apenas um sujeito. O autor entende que sujeitos não
são indivíduos, mesmo considerando que são constituídos a partir de indiví-
duos. Quando o indivíduo tem um projeto de vida diferente “talvez com base
em uma identidade oprimida, porém expandindo-se no sentido da transforma-
ção da sociedade como prolongamento desse projeto” ele atesta sua identidade
(Castells, 1942: 26). O início, o fim, e o meio... Em um recorte espacial mais micro, Ana Rojas Acosta e Maria Amália
Faller Vitale (2008) sugerem que as famílias sejam abordadas como “algo que
se define por uma história que se conta aos indivíduos, ao longo do tempo,
desde que nascem, por palavras, gestos, atitudes ou silêncios, e que será por
eles reproduzida e ressignificada [...]” (Acosta & Vitale, 2008: 26 e 27). Cada
família faz isso de maneira particular, dados os seus distintos lugares e mo-
mentos. Ao construir a sua própria história, ou o seu próprio mito, a família ex-
pressa o significado e a explicação da realidade que viveu. Ela se utiliza dos
elementos objetivos e subjetivos da cultura em que vivem seus indivíduos. A
delimitação simbólica da família acontece por meio dos referenciais sociais e
culturais da determinada época e sociedade. Sendo assim, para Acosta e Vitale,
trabalhar com famílias requer a abertura para uma escuta, a fim de localizar os
pontos de vulnerabilidade, e também os recursos disponíveis. Pensar a família como uma realidade que se constitui pelo discurso sobre
si própria, internalizado pelos sujeitos, é uma forma de buscar uma definição
que não se antecipe à sua própria realidade, mas que nos permita pensar como
ela se constrói, constrói sua noção de si, supondo evidentemente que isto
se faz em cultura, dentro, portanto, dos parâmetros coletivos do tempo e do
espaço em que vivemos, que ordenam as relações de parentesco (entre irmãos,
entre pais e filhos, entre marido e mulher. (Acosta & Vitale, 2008: 26 e 27) 304 Adriano Medeiros da Rocha & Eduardo Henrique Moreira Considerando que o ser humano se constitui simbolicamente, ou seja, se
constitui a partir dos elementos culturais, pode-se entender que não há reali-
dade humana exterior à cultura. Dessa mesma forma, a construção simbólica
da família é feita com a partir da sua memória coletiva. O discurso social que
a define reflete nas diferentes famílias como um espelho. Cada família faz
sua própria tradução desse discurso e o reflete com a sua imagem filtrada pela
singularidade das experiências vividas. Partindo da noção de que a identidade da família é definida pela sua própria
história, é possível considerar que a memória coletiva familiar está muito atre-
lada ao papel dos avós. Esses atores sociais representam um elemento-chave
nos processos de identificação e, portanto, na construção do sentido de perten-
cimento entre os membros de uma família. O início, o fim, e o meio... Acosta e Vitale (2008) afirmam
que, quando se pesquisa ao menos três gerações de uma família, a figura dos
avós revela bem essa dimensão. Através deles é possível recuperar a história
familiar, social, atingindo várias gerações. Tanto aqueles que conheceram os avós pessoalmente, como os que só os
conheceram por fotografias e histórias, tem neles a representação de figuras
presentes no imaginário. As inserções sociais, culturais e as relações de gê-
nero definem a trajetória desses personagens. Recordamos deles através da
posição que ocuparam na família relacionada ao nosso próprio lugar (Acosta
& Vitale, 2008). Porém essas posições podem ir se modificando ao longo da
vida. Acosta e Vitale defendem que a herança simbólica transmitida pelos
avós, entendida também como os seus legados geracionais, compõe nossa me-
mória coletiva familiar. Afinal, eles contribuem ou contribuíram de diversas
formas na vida cotidiana das suas famílias. A importância dos avós para a família é inegável. Com base em Kaës
(2001), as autoras Acosta e Vitale (2008) afirmam que “o pensamento psica-
nalítico tem tradicionalmente apontado o papel simbólico da figura dos avós –
vivos ou mortos – na função de assegurar aos netos uma identidade enraizada
no tempo imemorial”. Segundo elas, os avós proporcionam uma identidade
genealógica para a família. Nesse caso, a criança está no centro das trans-
missões inconscientes dessa “vida psíquica” que transpassam as gerações. De
acordo com Acosta e Vitale, eles são os personagens que se movimentam de
acordo com as gerações, mas que permanecem em nossa memória “como figu-
ras cristalizadas” em determinado momento do percurso. A herança simbólica
que é transmitida por eles se mantém ao longo de nossas vidas pode ser recri-
ada no processo de continuidade e descontinuidade dos bens simbólicos que
recebemos. O filme de busca e a construção ensaística de uma memória familiar... 305 No filme Seu Carlito: narrativas sobre um comerciante da roça a dupla
de realizadores promove a busca pela identidade de Carlos Moreira e acaba
por encontrar elementos significativos para entender o processo de construção
da identidade do indivíduo, mas também da memória coletiva da família e da
própria comunidade, sobretudo ambientada na zona rural de Ouro Preto. Para
explorar a trajetória de um personagem desse meio foi necessário ir atrás da-
queles que ainda reproduzem as narrativas sobre ele. O início, o fim, e o meio... Como a identidade do
indivíduo é moldada por suas ações em vida, é possível acessar as informa-
ções presentes na memória coletiva e resgatar os elementos que estruturam a
identidade do personagem pesquisado. da identidade do indivíduo, mas também da memória coletiva da família e da
própria comunidade, sobretudo ambientada na zona rural de Ouro Preto. Para
explorar a trajetória de um personagem desse meio foi necessário ir atrás da-
queles que ainda reproduzem as narrativas sobre ele. Como a identidade do
indivíduo é moldada por suas ações em vida, é possível acessar as informa-
ções presentes na memória coletiva e resgatar os elementos que estruturam a
identidade do personagem pesquisado. As narrativas da época em que o comerciante viveu no Manja Léguas fo-
ram cuidadosamente mapeadas pelos realizadores. Cada um dos atores sociais
apresentou fragmentos e versões das várias histórias sobre o avô de Eduardo. O realizador, e também personagem do filme, Eduardo Moreira, teve a opor-
tunidade de imergir nos resquícios da realidade rural em que seu avô viveu e,
através dos encontros e conversas com seus tios e primos mais velhos, consegui
acessar e registrar as narrativas sobre seu avô. Em sua viagem para o Manja,
passou pelos cenários onde aconteceu boa parte das histórias que ouviu. A
experiência proporcionou ao realizador conhecer a trajetória do seu avô e as
narrativas inspiradoras daqueles que ainda guardam suas lembranças. A cada
encontro recebeu de seus familiares as lições, lembranças e memórias que o
patriarca deixou como herança, traduzindo-a, cuidadosamente, neste filme do-
cumentário de buscas e virtudes. Giddens, A. (2002). Modernidade e identidade. Rio de Janeiro: Zahar. Acosta, A. R. & Vitale, M. A. F. (2008). Famílias: redes, laços e políticas
públicas. 4 ed. São Paulo: Cortez/Instituto de Estudos Especiais/PUC-SP.
Bernard, S. C. (2008). Documentário: Técnicas para uma produção de alto
impacto. Rio de Janeiro: Elsevier.
Castells, M. (2001). O Poder da Identidade: Paraísos comunais: Identidade e
significado na sociedade em rede, vol. II. São Paulo: Paz e Terra.
Comerford, J. C. (2003). Como uma família: Sociabilidade, territórios de
parentesco e sindicalismo rural. Rio de Janeiro. RelumeDumará.
Comolli, J.-L. (2008). Ver e poder: A inocência perdida – Cinema, Televisão,
Ficção, documentário. Minas Gerais: Editora UFMG.
Da-Rin, S. (2004). Espelho Partido: Tradição e Transformação do Documen-
tário. Rio de Janeiro: Azougue Editorial.
Giddens, A. (2002). Modernidade e identidade. Rio de Janeiro: Zahar. Referências bibliográficas Acosta, A. R. & Vitale, M. A. F. (2008). Famílias: redes, laços e políticas
públicas. 4 ed. São Paulo: Cortez/Instituto de Estudos Especiais/PUC-SP. Bernard, S. C. (2008). Documentário: Técnicas para uma produção de alto
impacto. Rio de Janeiro: Elsevier. Acosta, A. R. & Vitale, M. A. F. (2008). Famílias: redes, laços e políticas
públicas. 4 ed. São Paulo: Cortez/Instituto de Estudos Especiais/PUC-SP. Bernard, S. C. (2008). Documentário: Técnicas para uma produção de alto
impacto. Rio de Janeiro: Elsevier. Castells, M. (2001). O Poder da Identidade: Paraísos comunais: Identidade e
significado na sociedade em rede, vol. II. São Paulo: Paz e Terra. Comerford, J. C. (2003). Como uma família: Sociabilidade, territórios de
parentesco e sindicalismo rural. Rio de Janeiro. RelumeDumará. Comolli, J.-L. (2008). Ver e poder: A inocência perdida – Cinema, Televisão,
Ficção, documentário. Minas Gerais: Editora UFMG. Comolli, J.-L. (2008). Ver e poder: A inocência perdida – Cinema, Televisão,
Ficção, documentário. Minas Gerais: Editora UFMG. Da-Rin, S. (2004). Espelho Partido: Tradição e Transformação do Documen-
tário. Rio de Janeiro: Azougue Editorial. Da-Rin, S. (2004). Espelho Partido: Tradição e Transformação do Documen-
tário. Rio de Janeiro: Azougue Editorial. Giddens, A. (2002). Modernidade e identidade. Rio de Janeiro: Zahar. Lins, C. & Mesquita, C. (2008). Filmar o Real: sobre documentário brasileiro
contemporâneo. Rio de Janeiro: Jorge Zahar. Nichols, B. (2005). Introdução ao documentário. São Paulo: Papirus. Lins, C. & Mesquita, C. (2008). Filmar o Real: sobre documentário brasileiro
contemporâneo. Rio de Janeiro: Jorge Zahar.
Nichols, B. (2005). Introdução ao documentário. São Paulo: Papirus. Lins, C. & Mesquita, C. (2008). Filmar o Real: sobre documentário brasileiro
contemporâneo. Rio de Janeiro: Jorge Zahar.
Nichols, B. (2005). Introdução ao documentário. São Paulo: Papirus. Lins, C. & Mesquita, C. (2008). Filmar o Real: sobre documentário brasileiro
contemporâneo. Rio de Janeiro: Jorge Zahar. Nichols, B. (2005). Introdução ao documentário. São Paulo: Papirus. 306
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Design and Acquisition of EOG Based Interactive Communications for ALS Patients
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International journal of applied mathematics, electronics and computers
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cc-by-sa
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Accepted : 12/05/2017 Published: 21/08/2017 DOI: 10.18100/ijamec.2017Special Issue30460 Abstract: Amyotrophic lateral sclerosis (ALS) is a motor neuron disease caused by loss of function of spinal cord and brain stem
nerve cells. Loss of function in nerve cells leads to weakness and explosion (atrophy) in the muscles. The outcome of the weakness in the
muscles needs the help of someone. Despite the limitations of the movement, studies on methods of increasing the daily quality of life
ALS patients are continuing. Electrooculogram (EOG) signals were taken from an instrumentation amplifier with 48dB gain and 107dB
CMMR ratio. A 16Hz Low Pass Filter and a 50Hz Notch Filter were used to increase the signal to noise ratio. EOG signals are digitized
with 10bit resolution ADC and applied to ATmega328 microcontroller. So, the software of microcontroller determines the horizontal and
vertical movements of the eye. In this way, the interactive PC software was controlled by the EOG signals. EOG-based interactive
software which was developed using the C # programming language has provided to patients' daily requirements, social media accounts
etc. The system has been tested on healthy subjects and it has been seen that people can control the software by eye movements. As a
result, system will be useful not only for ALS patients, but also for permanent or partially bedridden patient (MS, Hemiplegia, etc.)
groups. And also, while the quality of life in the patients' own habitats is being raised, they will also be able to benefit from health
services within the scope of home health services. Keywords: Amyotrophic Lateral Sclerosis (ALS), Atrophy, Electrooculogram (EOG), Home Health Care, Interactive movements are recorded with the EMG signal [3]. Bulling (2009)
and the working group have designed EOG eyewear which
provide daily environmental awareness and sensitivity. Real-time
EOG signals can be received with the specially designed glasses
and transmitted to the computer interface via Bluetooth. 8 games
designed in the computer interface were requested to play with 11
eye movements and at the same time the EOG signals obtained
were analyzed by signal processing techniques [4]. In 2012,
Mingmin Yan and others developed a mouse-controlled system
using EOG signals for ALS patients. The position of the eye was
determined using signal processing techniques on the received
EOG signals [5]. Ayvaz and Gürkan (2014) have designed an
EOG circuit with 3 electrodes in order to control the actuators in
their work [6]. Advanced Technology and Science
Original Research Paper Advanced Technology and Science
Original Research Paper International Journal of
Applied Mathematics,
Electronics and Computers
ISSN:2147-82282147-6799 www.atscience.o International Journal of
Applied Mathematics,
Electronics and Computers
ISSN:2147-82282147-6799 www.atscience.o www.atscience.org/IJAMEC Original Research Paper Accepted : 12/05/2017 Published: 21/08/2017 In the other work they did in 2015, they developed
the EOG based Human Computer Interface application. The
system was tested on 10 people, the results of performing the
desired eye movement commands were recorded and processed in
MATLAB environment. [7-8]. Mondal et al (2015) have
implemented EOG based prototype data system design. The
signals from this low-cost EOG prototype demonstrated the
success of the signal they received compared to the EOG module
in the BIOPAC MP36 system [9]. In 2016, Tamura and others
created EOG and EMG based computer interfaces. By applying
signal processing techniques on the signals they have acquired
with the interface they have created they have distinguished the
right-left commands on the EOG sign [10]. In this study, it is
aimed to realize a system that will enable EOG based interactive
software to control many commands from daily necessities of
ALS patients to social media accounts. In this way
communication between the patient and the companion is ensured 1,2,3 Biomedical Engg., Afyon Kocatepe University, Afyonkarahisar –
03200, TURKEY
* Corresponding Author: Email: nozkan@aku.edu.tr
Note: This paper has been presented at the 5th International Conference
on Advanced Technology & Sciences (ICAT'17) held in İstanbul (Turkey),
May 09-12, 2017. Design and Acquisition of EOG Based Interactive Communications for
ALS Patients Neşe ÖZKAN*1, Ali IŞIK2, Uğur FİDAN3 IJAMEC, 2017, Special Issue , 1–4 | 1 2. Materials and Methods In general, home care services are offered at the professional
level or by family members in the home or living environment of
the individual, in order to maintain, raise and rehabilitate the
health of the individual. 29280 dated February 27, 2015 by the
Ministry of Health of the Republic of Turkey, "Health Services at
Home by the Ministry of Health and its Affiliates" regulates the
scope and content of home health services [11]. These services
aim to protect the quality of life and the social dignity of the
individual in a wide range of needs, including both health and
social services. Home care services are generally expressed as
services that take place in institutional care and reduce or delay
the need to stay in institutions [12]. ALS and paralyzed patients
are often in need of treatment within the scope of this regulation. Fig. 2. EOG measuring circuit block diagram. When the eyes move in different directions, the reciprocating eye
muscles are stimulated. Thus, horizontal and vertical eye
movements can be observed simultaneously. In Figure 3, EOG
signs appear in the muscle groups surrounding the eye during
vertical movements. Fig. 3. Vertical Eye Movements and EOG Sign. 2.2. EOG Based Interactive Communication System Fig. 3. Vertical Eye Movements and EOG Sign. ALS disease is known to Aran Duchenne until 1840 and is known
in many countries by the name Charcot. The first clinical and
pathological description of ALS was made by Jean Martin
Charcot (1874). In a case study of 25 patients, they defined ALS
on the basis of various clinical findings such as weakness -
melting and pathological reflexes in the muscles [13]. ALS
disease is a central nervous system disease caused by loss of
function of motor cells (neurons) in the spinal cord and brain Fig. 3. Vertical Eye Movements and EOG Sign. 2.2. EOG Based Interactive Communication System Figure 4 shows the block diagram of the EOG based interactive
communication system design. The system consists of 3 basic
parts. The first part forms the EOG measuring circuit, which
translates eye movements into electrical action. It constitutes the
second part of the digital process controller (DSP) system which
converts from electrical signals to position information according
to 360° and converts the position information into mouse control
signals and transfers the information to the PC via telemetry
method. 1. Introduction ALS disease is a central nervous system disease caused by loss of
function of motor cells (neurons) in the spinal cord and brain
stem. Loss of function in the nerve cells leads to weakness and
eruption (atrophy) in the muscles. This situation removes the
movement functions of ALS patients and leaves them in need of
someone's help. Although there are limitations of the movement,
studies on the methods that increase the daily life quality of ALS
patients who are not different from healthy individuals as
consciousness continue. In the literature, Barea et al. (2002) developed the human-
machine interface to control the wheelchair with EOG signals [1]. Kherlopian et al. (2006) developed a computer-based system
capable of automatic analysis and processing of EOG signals. Signal processing techniques have been used to reduce noise in
the EOG signal during work and to eliminate unwanted signals
caused by muscle movements around the eye. The right, left, and
center positions of the eye were determined by asking them to
print on the screen by eye movements with 13 words and 5 words
with 6 letters [2]. In another work, they created a computer
keyboard interface and implemented a virtual keyboard that can
be controlled with EOG and EMG marks. When the eye gaze is
obtained with the EOG signal during operation, the user's muscle IJAMEC, 2017, Special Issue , 1–4 | 1 This journal is © Advanced Technology & Science the S / N ratio of the measuring system, the EOG signals are
passed through the Notch and band pass filter. and interactive communication with the patient's social
environment will be strengthened. While the quality of life is
being upgraded within the patients' own living spaces,
technological infrastructure will be provided for health services
within the scope of home health services. Fig. 2. EOG measuring circuit block diagram. Fig. 2. EOG measuring circuit block diagram. 2. Materials and Methods The last part is interactive PC software that will enable
communication between the patient and the environment. Developed using the C # programming language on the Visual
Studio .NET platform, EOG based interactive software enables
patients to control many commands from everyday needs to
social media accounts. stem. Figure 1 shows how the muscular movements due to
deformation in the nerve cells in ALS disease are restricted. Fig. 4. EOG Based Interactive Communication System stem. Figure 1 shows how the muscular movements due to
deformation in the nerve cells in ALS disease are restricted. This journal is © Advanced Technology & Science 3. Results In order to ergonomically place the EOG electrodes on the
measurement surface, the eyewear frame was designed in the
Solid Works program and was manufactured using the
MassPortal Pharaoh ED model 3D printer (Fig. 5). Fig. 5. Produced Eyewear model Fig. 7. Eye Movements Fig. 7. Eye Movements Fig. 5. Produced Eyewear model EOG-based interactive software developed using the C #
programming language The Lenovo Idea center model all in one
is run on the Lenovo PC (Figure 8) with i5 processor with 21.5"
1920 *1080 resolution monitor capability. In order to enable
interactive communication with the external environment of the
patient, the software includes modules for Patient related,
Conversation, Communication, Swift messages, Media and
Social media. The distance between the monitor and the EOG
measurement system was set to 80 cm. It was desirable for the
person to control the modules on the software with eye
movements. Table 1 shows the functions to be controlled and the
control periods. During the test, the person controlled all modules
in the software with eye movements. The average duration of
access to the modules is 3.24 seconds and the module selection
time is 4.46 seconds. The realized system open circuit diagram is shown in Figure 6. Ag / AgCl electrodes were placed around the orbicularis oculi
muscles. Electrooculogram (EOG) signals were taken from an
instrumentation amplifier with 48dB gain and 107dB CMMR
ratio. A 16Hz Low Pass Filter and a 50Hz Notch Filter were used
to increase the signal to noise ratio. EOG signals are digitized
with 10bit resolution ADC and applied to ATmega328
microcontroller. So, the software of microcontroller determines
the transvers and sagittal movements of the eye. The movement
information of the eye was sent to the computer with NRF24L01
transceiver at 2.4GHz operating frequency with 9.6Kb/s data
transmission speed. This way, the interactive PC software was
controlled by the EOG signals. Fig. 6. Circuit diagram of the system Fig. 8. Implemented Interface Program Fig. 6. Circuit diagram of the system Fig. 8. Implemented Interface Program Fig. 8. Implemented Interface Program Fig. 6. Circuit diagram of the system Table 1. Accessing and Controlling Modules with EOG Table 1. Accessing and Controlling Modules with EOG Table 1. 2.1. Measurement of Electrooculogram (EOG) Signals Eye movements are provided by three pairs of muscles, mainly
medial-lateral recti muscle, superior-inferior recti muscle and
superior-inferior muscular muscle. The stimulus from the cerebral
nerves induces potential tension in the ocular muscles with
frequency components between 0.5uV and 0.3Hz to 40Hz. These
potential changes are detected by Ag / AgCl electrodes and
amplified by instrumentation amplifiers (Figure 2). To increase Fig. 4. EOG Based Interactive Communication System IJAMEC, 2017, Special Issue , 1–4 | 2 Fig. 7. Eye Movements This journal is © Advanced Technology & Science IJAMEC, 2017, Special Issue , 1–4 | 3 3. Results Accessing and Controlling Modules with EOG
Modules
Arrival Time (s)
Departure Time (s)
Conversation
6.16
8.18
Communication
0.84
3.03
Swift messages
2.87
3.14
Media
1.16
1.88
Social media
5.18
6.11 The Analog Discovery 2 data logger, manufactured by Digilent,
was connected to both outputs of the EOG circuit to test that the
hardware was tracking the movements of the eyeball. It is
desirable for the person to make eye movements in accordance
with the commands specified at a distance of about 80 cm. Figure
7 shows the response of the eye sphere to the sagittal and
transverse axis commands. During the eye blinking movements,
there are 180° phase different signals between the signals from
both channels. The obtained data was analyzed by signal
processing techniques and converted to a coordinate data to be
detected by the monitor and transferred to the PC. IJAMEC, 2017, Special Issue , 1–4 | 3 This journal is © Advanced Technology & Science 4. Discussion and Conclusions Amyotrophic Lateral Sclerosis (ALS) is a motor neuron disease
which is caused by loss of function of the spinal cord and brain
stem nerve cells. Loss of function in the nerve cells leads to
weakness and eruption (atrophy) in the muscles. This situation
eliminates the movement functions so patient needs to the
assistance of a companion. Although there are limitations of the
movement, studies on the methods that increase the daily life
quality of ALS patients who are not different from healthy
individuals as consciousness continue. In this study, the
interactive PC software was controlled by the EOG signals. EOG-
based interactive software which was developed using the C #
programming
language
has
provided
to
patients'
daily
requirements, social media accounts etc. The system has been
tested on healthy subjects and it has been seen that people can
control the software by eye movements. As a result, system will
be useful not only for ALS patients, but also for permanent or
partially bedridden patient (MS, Hemiplegia, etc.) groups. And
also, while the quality of life in the patients' own habitats is being
raised, they will also be able to benefit from health services
within the scope of home health services. [12] M. Yılmaz, F. Sametoğlu, G. Akmeşe, A. Tak, B. Yağbasan, S.
Gökçay, S. Erdem, “In-Home Health Services as an Alternative Form of
Presentation of Patient Care,” Istanbul Med Journal, 2010.
[13] F. Koç and E. Tan, “Amyotrophic Lateral Sclerosis,” TND
Neuromuscular Diseases Scientific Working Group. Acknowledgements This study is supported by TÜBİTAK BİDEB Authority under
the project numbered 2209/A program 1919B011601485. We
would like to thank TUBITAK Scientific Human Resource
Support Program for their support in conducting this research. IJAMEC, 2017, Special Issue , 1–4 | 4 This journal is © Advanced Technology & Science [12] M. Yılmaz, F. Sametoğlu, G. Akmeşe, A. Tak, B. Yağbasan, S.
Gökçay, S. Erdem, “In-Home Health Services as an Alternative Form of
Presentation of Patient Care,” Istanbul Med Journal, 2010. [13] F. Koç and E. Tan, “Amyotrophic Lateral Sclerosis,” TND
Neuromuscular Diseases Scientific Working Group. References [1] R. Barea, L. Boquete, M. Mazo, E. López, “System for assisted
mobility using eye movements based on electrooculography,” IEEE
Transactions on Neural Systems and Rehabilitation Engineering, 2002,
10(4), 209-218. [2] A. R. Kherlopian, J. P. Gerrein, M. Yue, K. E. Kim, J.W. Kim, M. Sukumaran, P. Sajda, “Electrooculogram based system for computer
control using a multiple feature classification model,” In Engineering in
Medicine and Biology Society, 2006, pp. 1295-1298. gy
y
pp
[3] H. S. Dhillon, R. Singla, N. S. Rekhi, R. Jha, “EOG and EMG based
virtual keyboard: A brain-computer interface,” In Computer Science and
Information Technology, 2009, pp. 259-262. [4] A. Bulling, D. Roggen, G. Tröster, “Wearable EOG goggles:
Seamless sensing and context-awareness in everyday environments,”
Journal of Ambient Intelligence and Smart Environments, 2009, 1(2),
157-171. [5] M. Yan, H. Tamura, K. Tanno, “Development of mouse cursor control
system using electrooculogram signals and its applications in revised
hasegawa dementia scale task,” In World Automation Congress (WAC),
2012, pp. 1-6. [6] A. Ayvaz, G. Ertaş, “Design of an EOG Based Electronic Circuit to
Control Actuators,” National Congress of Medical Technology’14,
Turkey, 2014, 22-25. [7] S. Gürkan, P. A. Öner, A. İstanbullu, S. Doğan, “Designing a Portable
Data
Acquisition
System
for
Human-Computer
Interface
Applications,” National Congress of Medical Technology’15, Turkey,
2015, 165-168. [8] S. Gürkan, G. Gürkan, A. Kaya, A. B. Uşaklı, “A Novel Eye-Blınk
Based Easy Communıcatıon Devıce for Amyotrophıc Lateral Sclerosıs
Patıents: Eyepad®,” 2015. [9] C. Mondal, M. K. Azam, M. Ahmad, S.K. Hasan, M.R. Islam,
“Design and implementation of a prototype electrooculography based data
acquisition
system,”
In Electrical
Engineering
and
Information
Communication Technology (ICEEICT), 2015 International Conference
on, pp. 1-6. , pp
[10] H. Tamura, M. Yan, K. Sakurai, K. Tanno, “EOG-sEMG Human
Interface
for
Communication,” Computational
Intelligence
and
Neuroscience, 2016. [11] Ministry of Health and its Affiliates Implementing Regulation On
The Presentation of Home Health Care Services, 27.02.2015. This journal is © Advanced Technology & Science
|
https://openalex.org/W2606506288
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https://www.nature.com/articles/srep46601.pdf
|
English
| null |
The pretreatment lymphocyte to monocyte ratio predicts clinical outcome for patients with hepatocellular carcinoma: A meta-analysis
|
Scientific reports
| 2,017
|
cc-by
| 6,035
|
The pretreatment lymphocyte
to monocyte ratio predicts
clinical outcome for patients with
hepatocellular carcinoma: A meta-
analysis received: 23 September 2016
accepted: 21 March 2017
Published: 18 April 2017 Wei Song1,*, Chuan Tian2,*, Kai Wang1, Run-jin Zhang1 & Shu-bing Zou1 The lymphocyte-to-monocyte ratio (LMR) has been reported to predict clinical outcomes in multiple
malignancies. The aim of this study was to assess the prognostic role of pretreatment LMR in
hepatocellular carcinoma (HCC). A total of seven studies comprising 2,738 patients were included in the
meta-analysis. Pooled results showed that elevated LMR was significantly associated with increased
overall survival (OS) (HR: 0.31, 95% CI: 0.20–0.47, p < 0.001), disease-free survival (DFS)/recurrence-
free survival (RFS) (HR: 0.57, 95% CI: 0.49–0.67, p < 0.001). The favorable prognostic impact of high
LMR on OS was observed in all subgroup with different sample size, type of publication, NOS score, and
the cut-off value of LMR. In addition, low LMR was significantly correlated with TNM stage and BCLC
stage. We therefore conclude that elevated pretreatment LMR could be a favorable prognostic factor for
clinical outcomes in patients with HCC. According to the American Cancer Society, hepatocellular carcinoma (HCC) is the fifth most common malig-
nancy and the second cause of cancer-related mortality in the United States in 20151,2. Around the world, there
were an estimated 500,000 to 1 million people died of HCC per year3. Liver resection is the most effective therapy
for the majority of patients with resectable HCC, but unfortunately most of patients are not eligible for curative
resection at the time of diagnosis4. In addition, postoperative recurrence rate for patients with early stage HCC
can be high at 50% to 70% after liver resection5. Therefore, the overall prognosis of HCC patients remains poor. It is vital to develop novel potential biomarker to select appropriate treatment strategies and predict prognosis.ll Systemic inflammatory responses can largely influence the formation and recurrence of HCC6–8. Several
inflammatory biomarkers, such as neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR),
and platelet-to-lymphocyte ratio (PLR) are identified as prognostic indicators in a wide variety of solid tumors9–12. These tests are simple and inexpensive to perform, and they are readily available in daily oncologic practice. LMR
is a readily available, routinely measured, and inexpensive inflammatory biomarker, which can be easily applied
into in daily oncologic practice. Accumulating evidence shows that lymphocytes are known to play a crucial role
in suppressing HCC progression through immunoselection in an immunosuppressive network, which dictates
immune responses to tumors13. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Search strategies.
We se Search strategies. We searched MEDLINE, EMBASE, PubMed, and Cochrane databases from inception
up to February 2017. Search terms included “HCC” or “hepatocellular carcinoma” or “liver cancer” or “liver
tumor” or “liver neoplasms” or “liver cell carcinoma”, “LMR” or “lymphocyte monocyte ratio” or “lymphocyte to
monocyte ratio” or “lymphocyte-to-monocyte ratio” or “lymphocyte-monocyte ratio”, “survival” or “prognostic”
or “prognosis” or “recurrence” or “clinical outcome”. The references of eligible studies, relevant systematic reviews
and meta-analyses in this field were manually retrieved. Inclusion criteria. The criteria for inclusion were listed as follows: (1) the diagnosis of HCC were confirmed
by pathology; (2) studies assessing the association of pretreatment LMR with OS, DFS/RFS, or CSS; (3) the cut-off
value of LMR was reported; and (4) studies supplied sufficient information for calculating hazard ratio (HR) and
95% confidence interval (CI). Exclusion criteria. The exclusion criteria were as follows: (1) reviews, letters, case reports, and comments; (2)
reporting insufficient data for calculating an HR and 95% CI; and (3) overlapping or duplicate data. Data extraction and Assessment of risk of bias. The two reviewers independently reviewed all eligible
studies and extracted data. Any disagreement was resolved by a third reviewer. The following information was
captured using data abstraction forms:i Study characteristics included first author’s name, year of publication, study design (prospective or retrospec-
tive), type of publication, country, ethnicity, survival analysis methods (multivariate, univariate), and time of
follow-up. Patient characteristics included patient ages and genders, number of patients, disease stage, treatment,
and cut-off value. Outcome measures included HRs for OS, DFS, RFS, or CSS as well as their 95% CIs. HRs were
extracted from multivariate or univariate analyses or estimated from Kaplan-Meier survival curves20.hh The study quality was evaluated in accordance with the Newcastle-Ottawa Scale (NOS)21. This included
an assessment of subject selection, comparability of groups, and clinical outcome. A total of nine items were
extracted and each item scored 1. The maximum score is 9 and those studies with a NOS score ≥7 were consid-
ered as high-quality studies. Statistical analysis. The meta-analysis was conducted using RevMan 5.3 software (Cochrane Collaboration,
Copenhagen, Denmark). Heterogeneity between studies was estimated using Cochrane’s Q statistic and I2 statistic22. A p-value < 0.1 for the Q-test or I2 > 50% indicated significant heterogeneity. When there was no statistically
significant heterogeneity, we used the fixed-effects model for pooling the results; otherwise, the random-effects
model was applied. The pretreatment lymphocyte
to monocyte ratio predicts
clinical outcome for patients with
hepatocellular carcinoma: A meta-
analysis A low lymphocyte count is associated with the systemic inflammatory responses
and is known to promote cancer progression through effects upon cell-mediated immunity14. Conversely, acti-
vated circulating monocytes can secrete multiple proinflammatory cytokines, which are involved in tumor devel-
opment and progression15,16. As a consequence high monocyte counts are associated with microvascular invasion
and poor prognosis in patients with HCC17. Moreover, a high LMR, which is defined as absolute lymphocyte
counts divided by monocyte counts, is reported to correlate with favorable prognosis in patients with HCC18,19. 1Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Nanchang University, No.1 Minde Road,
Nanchang, China. 2Department of Nuclear Medicine, Guizhou Provincial People’s Hospital, Guiyang, China. *These
authors contributed equally to this work. Correspondence and requests for materials should be addressed to S.-B.Z. (email: zousb999@163.com) 1 Scientific REporTS | 7:46601 | DOI: 10.1038/srep46601 www.nature.com/scientificreports/ Nevertheless, there has been no meta-analysis to assess the correlation between pretreatment LMR and the survival
of HCC patients. Therefore, we conducted a meta-analysis to assess the effects of pretreatment LMR on OS and DFS/
RFS in HCC. In addition, the correlations between LMR and the clinicopathological features was also examined. Search strategies.
We se HRs and their 95% CIs were searched in the original articles or extrapolated using meth-
ods described by Tierney and Parmar20,23. The log HR and standard error (SE) were used for aggregation of the
survival results23. The associations between LMR and clinicopathologic features were expressed as odds ratios
(ORs) and its 95% CIs. P < 0.05 was defined as statistically significant. Subgroup analyses were conducted for: the
sample size, type of publication, NOS score, and the cut-off value of LMR. Sensitivity analyses were carried out to
evaluate result stability excluding each study. Results Search results. Our search strategy yielded 63 potentially relevant records. After duplicates removal (21
records), 42 articles were screened for eligibility. Of these, 32 were excluded through titles and abstracts, leaving
10 articles for further evaluation. Subsequently, 3 articles did not meet the inclusion criteria and were therefore
excluded. Eventually, seven studies, comprising a total of 2,507 patients, were considered eligible for the present
meta-analysis18,24–29. The PRISMA flow diagram of the study selection process was shown in Fig. 1. Characteristics of the included studies. According to the publication type, 6 and 1 studies were pub-
lished in full-text and abstract forms, respectively. All included studies were from China and were published
between 2014 and 2017. The sample sizes ranged from 208 to 1,020. There were six studies for OS, and all for DFS/
RFS. The cut-off values for LMR ranged from 0.83 to 3.77. All included studies consisted of two groups: high and
low LMR. HR and 95% CI was extracted directly from the seven studies. In methodological quality of studies,
the NOS scores of the included studies ranged from 5 to 8. Detailed patient characteristics and methodological
quality are shown in Table 1. Meta-analysis. LMR and OS in HCC. Six of the included studies reported the data of LMR and OS in HCC. Overall, elevated LMR had a significant association with increased OS (HR: 0.31, 95% CI: 0.20–0.47, p < 0.001)
with significant heterogeneity (p = 0.002, I2 = 74%; Fig. 2). Meta-analysis. LMR and OS in HCC. Six of the included studies reported the data of LMR and OS in HCC
Overall, elevated LMR had a significant association with increased OS (HR: 0.31, 95% CI: 0.20–0.47, p < 0.001)
with significant heterogeneity (p = 0.002, I2 = 74%; Fig. 2).i i
To detect the potential heterogeneity, subgroup analyses stratified by sample size, type of publication, NOS
score, and the cut-off value of LMR (Table 2). Subgroup analysis revealed that high LMR predicted increased OS
in patient with HCC, regardless of the sample size (≥300 and <300), type of publication (full-text and abstract),
NOS score (≥7 and <7), and the cut-off value of LMR (≥3 and <3). Scientific REporTS | 7:46601 | DOI: 10.1038/srep46601 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ LMR and DFS/RFS in HCC. All included studies reported the data of LMR and DFS/RFS in HCC. Results A combined
analysis demonstrated that elevated LMR was significantly correlated with increased DFS/RFS (HR: 0.57, 95% CI:
0.49–0.67, p < 0.001), with no significant heterogeneity between studies (p = 0.21, I2 = 28%; Fig. 3). Figure 1. Flow diagram of the study selection process. Author
Year
Country
Type of
publication
Follow-up
(months)
Treatment
No. of
patients
Stage
Cut-off
value
Survival
analysis
Analysis
NOS
score
Li
2017
China
Full-text
33 (6–85)
Surgery
253
Mixed
3.0
OS/RFS
MV/UV
8
Liao
2016
China
Full-text
44 (1.5–84)
Surgery
387
Mixed
3.3
OS/RFS
MV
8
Lin (1)
2015
China
Full-text
34.8
(1.7–106.6)
Surgery
210
Mixed
3.23
OS/RFS
MV/UV
7
Lin (2)
2014
China
Abstract
34 (2–106)
Surgery
210
—
3.2
OS/RFS
MV/UV
—
Tang
2016
China
Full-text
38
(1.5–82.2)
Surgery
208
Mixed
0.83
OS/RFS
MV/UV
5
Wu
2016
China
Full-text
45.5 (2–93)
Surgery
450
Mixed
3.77
OS/RFS
MV/UV
7
Yang
2017
China
Full-text
NA
Surgery
1020
Mixed
3.23
DFS
MV/UV
6
Table 1. Characteristics of the studies included in the meta-analysis. OS: overall survival; DFS: disease-free
survival; RFS: recurrence-free survival; MV: multivariate; UV: univariate NA: not available. Figure 1. Flow diagram of the study selection process. Figure 1. Flow diagram of the study selection process. Author
Year
Country
Type of
publication
Follow-up
(months)
Treatment
No. of
patients
Stage
Cut-off
value
Survival
analysis
Analysis
NOS
score
Li
2017
China
Full-text
33 (6–85)
Surgery
253
Mixed
3.0
OS/RFS
MV/UV
8
Liao
2016
China
Full-text
44 (1.5–84)
Surgery
387
Mixed
3.3
OS/RFS
MV
8
Lin (1)
2015
China
Full-text
34.8
(1.7–106.6)
Surgery
210
Mixed
3.23
OS/RFS
MV/UV
7
Lin (2)
2014
China
Abstract
34 (2–106)
Surgery
210
—
3.2
OS/RFS
MV/UV
—
Tang
2016
China
Full-text
38
(1.5–82.2)
Surgery
208
Mixed
0.83
OS/RFS
MV/UV
5
Wu
2016
China
Full-text
45.5 (2–93)
Surgery
450
Mixed
3.77
OS/RFS
MV/UV
7
Yang
2017
China
Full-text
NA
Surgery
1020
Mixed
3.23
DFS
MV/UV
6
Table 1. Characteristics of the studies included in the meta-analysis. OS: overall survival; DFS: disease-free
survival; RFS: recurrence-free survival; MV: multivariate; UV: univariate NA: not available. Table 1. Characteristics of the studies included in the meta-analysis. OS: overall survival; DFS: disease-free
survival; RFS: recurrence-free survival; MV: multivariate; UV: univariate NA: not available. LMR and DFS/RFS in HCC. All included studies reported the data of LMR and DFS/RFS in HCC. Discussion Hepatocellular carcinoma (HCC) is mainly caused by viral infections such as hepatitis B virals (HBV) and hepati-
tis C virals (HCV)3. Mounting studies indicated that infiltration of inflammatory cells in the tumor microenviron-
ment significantly affected the biological behavior of HCC25. In the tumor microenvironment, the inflammatory
immune cells have been implicated in HCC progression30,31. The pretreatment LMR, as a promising inflammatory
biomarker, is reportedly linked to prognosis in patients with HCC18,19,28. Therefore, LMR may serve as a promis-
ing prognostic factors for HCC patients. To our knowledge, this is the first meta-analysis to investigate the prog-
nostic effect of pretreatment LMR in HCC.i f
p
Our meta-analysis provides strong evidence that the presence of high LMR significantly increases OS and
DFS/RFS in patients with HCC. Stratified analysis demonstrated that high LMR was significantly correlated with
increased OS in patient with HCC, regardless of the sample size, type of publication, NOS score, and the cut-off
value of LMR. In addition, we further assessed the correlation between pretreatment LMR and clinicopathologi-
cal features. The pooled data showed low LMR was linked with TNM stage and BCLC stage. Therefore, pretreat-
ment LMR may serve as a promising prognostic biomarker for estimating HCC prognosis. Furthermore, we also
performed sensitivity analysis and found that no study had a significant effect on the observed effect size (pooled
HR), indicating the robustness of our findings.h gi
g
The exact molecular mechanisms responsible for the prognostic impact of LMR in HCC are unclear. It has
been suggested that cross-talk between the inflammatory response and tumor progression play a critical role in
the initiation and progression of HCC14,32,33. In the tumor microenvironment, inflammatory infiltrates have a
large influence on the biological behavior of HCC34,35. Tumor-infiltrating lymphocytes (TILs), as representative
component of the immune microenvironment, are implicated in several stages of HCC progression, and TIL
phenotypes may be a predictor for favorable prognosis36,37. Conversely, low lymphocyte counts might result in
an insufficient immunological reaction, which lead to inferior survival in multiple cancers38,39. In addition, infil-
trated CD4+ and CD8+ T cells interaction among each other are essential to the antitumor immune response
by inducing tumor cell apoptosis40,41. Monocytes infiltrating tumor tissue are also involved in HCC development
and progression42. Activated monocytes in HCC microenvironments can trigger and polarize T-cell responses
and faciliate inflammation-induced tumor development43. Tumor-associated macrophages (TMAs) are derived
from circulating monocytes. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Fi
2 F
t l t f
th
i ti
b t
LMR
d OS Figure 2. Forest plots for the association between LMR and OS. Figure 2. Forest plots for the association between LMR and OS. Figure 2. Forest plots for the association between LMR and OS. Subgroup
No. of
studies
No. of
patients
Effects
model
HR (95% CI)
P value
Heterogeneity
I2 (%)
Ph
Overall
6
1718
Random
0.31 (0.20–0.47)
<0.001
74
0.002
Sample size
≥300
2
837
Random
0.23 (0.08–0.65)
0.005
84
0.01
<300
4
881
Random
0.34 (0.19–0.61)
<0.001
77
0.005
Cut-off for LMR
≥3
5
1510
Random
0.28 (0.17–0.48)
<0.001
78
0.001
<3
1
208
—
0.43 (0.26–0.71)
0.001
—
—
Type of publication
Full-text
5
1508
Random
0.27 (0.17–0.44)
<0.001
74
0.004
Abstract
1
210
—
0.35 (0.24–0.50)
0.006
—
—
NOS score
≥7
4
1300
Random
0.28 (0.17–0.48)
<0.001
78
0.001
<7
1
208
—
0.43 (0.26–0.71)
0.001
—
—
Table 2. Pooled hazard ratios (HRs) for OS according to subgroup analyses. Table 2. Pooled hazard ratios (HRs) for OS according to subgroup analyses. Results A combined
analysis demonstrated that elevated LMR was significantly correlated with increased DFS/RFS (HR: 0.57, 95% CI:
0.49–0.67, p < 0.001), with no significant heterogeneity between studies (p = 0.21, I2 = 28%; Fig. 3). LMR and DFS/RFS in HCC. All included studies reported the data of LMR and DFS/RFS in HCC. A combined
analysis demonstrated that elevated LMR was significantly correlated with increased DFS/RFS (HR: 0.57, 95% CI:
0.49–0.67, p < 0.001), with no significant heterogeneity between studies (p = 0.21, I2 = 28%; Fig. 3). LMR and clinicopathological features. To further explore the impact of LMR on the clinical features in HCC, we
identified 9 clinical factors in HCC. The pooled analysis demonstrated that low LMR was significantly correlated
with TNM stage (III-IV vs. I-II; HR = 1.78, 95% CI: 1.19–2.68, P = 0.005) and BCLC stage (B/C vs. A; HR = 1.70,
95% CI: 1.21–2.39, P = 0.002). Whereas no significant association was found with gender, AFP, liver cirrhosis,
tumor differentiation, tumor number, tumor size, and vascular invasion. The correlation between LMR expres-
sion and clinicopathological parameters of HCC is shown in Table 3. Sensitivity analysis. In order to assess the influence of single studies on the overall estimate, the sensitivity anal-
ysis was performed. Each single study was removed each time to estimate the influence of individual data sets
on the combined HR for OS. The results showed that no study had a significant effect on the observed effect size
(pooled HR), indicating the robustness of our findings. Scientific REporTS | 7:46601 | DOI: 10.1038/srep46601 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Forest plots for the association between LMR and DFS/RFS. Figure 3. Forest plots for the association between LMR and DFS/RFS. Characteristics
No. of studies
No. of patients
OR (95% CI)
p
Heterogeneity
I2 (%)
Ph
Gender (male vs. female)
3
913
1.16 (0.64–2.09)
0.62
54
0.11
AFP (>400 ng/mL vs. <400 ng/mL)
2
660
1.61 (0.81–3.20)
0.17
73
0.05
Liver cirrhosis (yes vs. no)
3
913
1.66 (0.97–2.83)
0.06
52
0.12
Differentiation (low vs. moderate/high)
3
913
1.04 (0.74–1.46)
0.82
0
0.88
Tumor number (multiple vs. single)
2
660
0.96 (0.62–1.50)
0.87
0
0.61
Tumor size (>5 cm vs. <5 cm)
3
913
1.36 (0.79–2.35)
0.27
69
0.04
TNM stage (III-IV vs. I-II)
2
703
1.78 (1.19–2.68)
0.005
33
0.29
BCLC stage (B/C vs. A)
2
703
1.70 (1.21–2.39)
0.002
0
0.38
Vascular invasion (pos vs. neg)
2
660
1.47 (0.48–4.49)
0.50
84
0.01
Table 3. Meta-analysis of the association between LMR and clinicopathological features of HCC. Figure 3. Forest plots for the association between LMR and DFS/RFS. Characteristics
No. of studies
No. of patients
OR (95% CI)
p
Heterogeneity
I2 (%)
Ph
Gender (male vs. female)
3
913
1.16 (0.64–2.09)
0.62
54
0.11
AFP (>400 ng/mL vs. <400 ng/mL)
2
660
1.61 (0.81–3.20)
0.17
73
0.05
Liver cirrhosis (yes vs. no)
3
913
1.66 (0.97–2.83)
0.06
52
0.12
Differentiation (low vs. moderate/high)
3
913
1.04 (0.74–1.46)
0.82
0
0.88
Tumor number (multiple vs. single)
2
660
0.96 (0.62–1.50)
0.87
0
0.61
Tumor size (>5 cm vs. <5 cm)
3
913
1.36 (0.79–2.35)
0.27
69
0.04
TNM stage (III-IV vs. I-II)
2
703
1.78 (1.19–2.68)
0.005
33
0.29
BCLC stage (B/C vs. A)
2
703
1.70 (1.21–2.39)
0.002
0
0.38
Vascular invasion (pos vs. neg)
2
660
1.47 (0.48–4.49)
0.50
84
0.01
Table 3. Meta-analysis of the association between LMR and clinicopathological features of HCC. Figure 3. Forest plots for the association between LMR and DFS/RFS. Characteristics
No. of studies
No. of patients
OR (95% CI)
p
Heterogeneity
I2 (%)
Ph
Gender (male vs. female)
3
913
1.16 (0.64–2.09)
0.62
54
0.11
AFP (>400 ng/mL vs. <400 ng/mL)
2
660
1.61 (0.81–3.20)
0.17
73
0.05
Liver cirrhosis (yes vs. no)
3
913
1.66 (0.97–2.83)
0.06
52
0.12
Differentiation (low vs. moderate/high)
3
913
1.04 (0.74–1.46)
0.82
0
0.88
Tumor number (multiple vs. single)
2
660
0.96 (0.62–1.50)
0.87
0
0.61
Tumor size (>5 cm vs. www.nature.com/scientificreports/ <5 cm)
3
913
1.36 (0.79–2.35)
0.27
69
0.04
TNM stage (III-IV vs. I-II)
2
703
1.78 (1.19–2.68)
0.005
33
0.29
BCLC stage (B/C vs. A)
2
703
1.70 (1.21–2.39)
0.002
0
0.38
Vascular invasion (pos vs. neg)
2
660
1.47 (0.48–4.49)
0.50
84
0.01
Table 3. Meta-analysis of the association between LMR and clinicopathological features of HCC. Table 3. Meta-analysis of the association between LMR and clinicopathological features of HCC. Table 3. Meta-analysis of the association between LMR and clinicopathological features of HCC. cancers45,46. Given this background, the observed favorable of high pretreatment LMR on OS of cancer patients
may reflect the critical function of LMR in an inflammatory tumor microenvironment that inhibits tumor
progression. cancers45,46. Given this background, the observed favorable of high pretreatment LMR on OS of cancer patients
may reflect the critical function of LMR in an inflammatory tumor microenvironment that inhibits tumor
progression. Nevertheless, there were several limitations to the present study. First, excessive heterogeneity existed among
studies. Subgroup analyses didn’t find the potential sources of heterogeneity. In addition, we performed sensitivity
analysis. The results showed that no study had a significant effect on the observed effect size. Second, the cut-off
value for LMR was not unified in each study. Third, all included studies were retrospective analysis. Fourth, all
included studies were from China, which limited our conclusions for other ethnic populations.i In conclusion, our findings demonstrated that an elevated pretreatment LMR is associated with favorable
outcomes in patients with HCC. Discussion TAMs can accelerate HCC cell proliferation, tumor-associated angiogenesis, and
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carcinoma patients. Int. J. Scientific REporTS | 7:46601 | DOI: 10.1038/srep46601 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Competing Interests: The authors declare no competing financial interests. Competing Interests: The authors declare no competing financial interests. Competing Interests: The authors declare no competing financial interests. Scientific REporTS | 7:46601 | DOI: 10.1038/srep46601 6 www.nature.com/scientificreports/ How to cite this article: Song, W. et al. The pretreatment lymphocyte to monocyte ratio predicts clinical
outcome for patients with hepatocellular carcinoma: A meta-analysis. Sci. Rep. 7, 46601; doi: 10.1038/srep46601
(2017). How to cite this article: Song, W. et al. The pretreatment lymphocyte to monocyte ratio predicts clinical
outcome for patients with hepatocellular carcinoma: A meta-analysis. Sci. Rep. 7, 46601; doi: 10.1038/srep46601
(2017). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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Built structures influence patterns of energy demand and CO2 emissions across countries
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Nature communications
| 2,023
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cc-by
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Built structures influence patterns of energy
demand and CO2 emissions across countries Helmut Haberl
1,5
, Markus Löw1,5, Alejandro Perez-Laborda2,5, Sarah Matej
1,
Barbara Plank
1, Dominik Wiedenhofer
1, Felix Creutzig
3,4,
Karl-Heinz Erb
1 & Juan Antonio Duro2 Helmut Haberl
1,5
, Markus Löw1,5, Alejandro Perez-Laborda2,5, Sarah Matej
1,
Barbara Plank
1, Dominik Wiedenhofer
1, Felix Creutzig
3,4,
Karl-Heinz Erb
1 & Juan Antonio Duro2 Built structures, i.e. the patterns of settlements and transport infrastructures,
are known to influence per-capita energy demand and CO2 emissions at the
urban level. At the national level, the role of built structures is seldom con-
sidered due to poor data availability. Instead, other potential determinants of
energy demand and CO2 emissions, primarily GDP, are more frequently
assessed. We present a set of national-level indicators to characterize patterns
of built structures. We quantify these indicators for 113 countries and statis-
tically analyze the results along with final energy use and territorial CO2
emissions, as well as factors commonly included in national-level analyses of
determinants of energy use and emissions. We find that these indicators are
about equally important for predicting energy demand and CO2 emissions as
GDP and other conventional factors. The area of built-up land per capita is the
most important predictor, second only to the effect of GDP. Increasing global temperature driven by growing greenhouse gas
(GHG) emissions is a major global concern1. GHGs mainly result
from the energy-related combustion of fossil fuels2. The question
emerges, which factors drive energy demand and emissions, and to
what extent they are malleable. This question is relevant at many
levels, from products to individuals, households, and cities up to
continental and even global totals. We here focus on countries,
because many decision-making processes occur at the national level. A widespread approach for cross-country analyses classifies the
factors influencing resource use and emissions into population,
affluence, and technology3–5. These factors are captured in the
STIRPAT (Stochastic Impacts by Regression on Population, Affluence
and Technology) framework3, derived from the classical IPAT
(impact = population × affluence × technology) approach5,6. Increasing global temperature driven by growing greenhouse gas
(GHG) emissions is a major global concern1. GHGs mainly result
from the energy-related combustion of fossil fuels2. The question
emerges, which factors drive energy demand and emissions, and to
what extent they are malleable. This question is relevant at many
levels, from products to individuals, households, and cities up to
continental and even global totals. Article https://doi.org/10.1038/s41467-023-39728-3 1Institute of Social Ecology, University of Natural Resources and Life Sciences, Vienna, Austria. 2Economics Department and Eco-SOS, Universitat Rovira i
Virgili, Tarragona, Spain. 3Mercator Research Institute on Global Commons and Climate Change, EUREF 19, 10829 Berlin, Germany. 4Technical University
Berlin, Straße des 17 Junis 135, 10623 Berlin, Germany. 5These authors contributed equally: Helmut Haberl, Markus Löw, Alejandro Perez-Laborda.
e-mail: helmut.haberl@boku.ac.at Built structures influence patterns of energy
demand and CO2 emissions across countries The distance from the vertical axis either to the right (positive corre-
lation) or to the left (inverse correlation) depicts the additional
explanatory power of the respective indicator over a model con-
sidering only GDP and DENS. Several indicators provide additional
explanations over GDP and DENS alone. The area of built-up land (both
BLcap and BLfract) is positively correlated with TFC and CO2, as are the
density of rail and road infrastructures, especially in rural areas. UPOP
is inversely correlated with TFC and CO2, as is urban road density
(not significant) and BLmono. PGAS, which had not been significant in
the bivariate correlations, emerges as an important factor, which is
also observed for RDurban and other indicators. UPdens lose importance,
most likely due to its high correlation with GDP. Despite these insights from urban studies, material stock pat-
terns are seldom considered in debates on national-level analyses of
factors determining levels of energy demand and emissions, as well
as their possible decoupling from GDP11,15. A systematic review of the
empirical literature on these questions15,37 revealed only one study38
considering material stock patterns in analyzing transport-related
emissions. Hence little is known about the effects of material stock
patterns on energy demand and CO2 emissions beyond the city level. This results from a scale mismatch: maps of material stock patterns
provide fine-grained spatial detail30, often focused on specific
regions, that cannot be included in national-level analyses of factors
driving energy demand and emissions. For cross-country analyses,
consistent indicators need to be developed from spatial data and
then aggregated at the national level in a manner which preserves key
information on patterns and supports comparative analyses across
countries and world regions. In this work, we develop national-level indicators of character-
istics of built structures that, based on urban studies, can be potential
determinants of resource use and emissions. We quantify them for 113
countries comprising 91.2% of the world population and 97.3% of
global GDP. We analyze two independent variables: (1) yearly total per-
capita final energy consumption (abbreviated as TFC) and (2) yearly
per-capita CO2 emissions (abbreviated as CO2). We test material stock
pattern indicators against other variables thathave been widely used in
national-level analyses of determinants of energy use and emissions,
here denoted as conventional factors. Built structures influence patterns of energy
demand and CO2 emissions across countries The price of gasoline (abbreviated as PGAS) is an energy price
indicator that is strongly related to settlement patterns21. Some studies
used urban population as a percent of the total population (abbre-
viated as UPOP) as a development indicator22. p
In terms of their Pearson coefficients in bivariate correlations, several
indicators of material stock patterns are as strongly correlated with
TFC and CO2 as the conventional factors (Fig. 2a). GDP is positively
correlated with both TFC and CO2. HDD and the fraction of the urban
population also show the expected pattern, while PGAS and DENS
are largely uncorrelated. Almost all material stock pattern indicators
are correlated with both TFC and CO2. The extent of built-up land
(BLcap and BLfract) is positively correlated with both TFC and CO2, as are
total and rural road density and the dispersion of built-up land and
most railway-related indicators. The correlation coefficients of BLcap
with TFC and CO2 are both ~0.7; BLcap is the second-best predictor of
both TFC and CO2 after GDP. As expected from the urban literature,
urban population density (UPdens) is inversely correlated with CO2
and energy, whereas the share of urban population (UPOP) is strongly
positively correlated with CO2 and TFC. Inverse relations prevail
for BLmono, BLcomp, and the urban-to-rural relations of infrastructure
density. At the urban scale, the influence of population density and the
spatial layout of urban areas on cities’ resource demand has been
widely studied20,23–28. There are several reasons why the extent and
spatial layout (density and form) of built structures—henceforth
denoted as material stock patterns29, i.e., the spatial patterns of
societies’ material stocks in infrastructures and buildings—could affect
energy demand and CO2 emissions. The accumulation of material
stocks requires massive amounts of resources such as steel-reinforced
concrete, mortar, bricks, timber, plastics, glass, gravel, or sand30–33,
which are associated with high GHG emissions34. Heating, cooling, and
lighting of buildings and production processes in industrial plants
require much energy20,23,26,35, as does the mobility of goods and people
on roads and railways33,36. Semi-partial correlations of the material stock pattern indicators
controlled for GDP and population density (DENS) are shown in Fig. 2b. The part of each material stock pattern indicator correlated with GDP
and DENS is removed, revealing the strength of the linear correlation
between TFC or CO2 and the remaining part of the respective variable. Built structures influence patterns of energy
demand and CO2 emissions across countries We here focus on countries,
because many decision-making processes occur at the national level. A widespread approach for cross-country analyses classifies the
factors influencing resource use and emissions into population,
affluence, and technology3–5. These factors are captured in the
STIRPAT (Stochastic Impacts by Regression on Population, Affluence
and Technology) framework3, derived from the classical IPAT
(impact = population × affluence × technology) approach5,6. be reduced while GDP is growing. This may be possible e.g., through
more efficient technologies, but the debate is so far inconclusive10,11. While recent studies revealed examples of growing national econo-
mies where policies implemented in the last decade achieved reduc-
tions in energy demand and CO2 emissions12–14, neither those studies
nor a meta-analysis15 yielded evidence for reductions in energy use and
GHG emissions consistent with ambitious climate targets. Other potential determinants of energy demand and emissions
receive less attention than GDP, even though additional entry points to
accelerate decarbonization are urgently required. Population density
has been studied with varied outcomes. An econometric study
of OECD countries 1980–2011 suggested an inverse relation between
population density and CO2 emissions16; a regression analysis of
materials used in >100 countries in 2000 found a similar effect17. A global regression analysis18 found no effect of population density on
energy demand, whereas a panel analysis of 11 Asian countries from
1960–200419 to conditions that influence energy demand for heating
or cooling of buildings20, fuel prices affecting demand for transport
energy21, and the urbanization rate (urban population as percent of the Economic activity (called affluence in IPAT-style analyses, usually
measured as Gross Domestic Product or GDP) is acknowledged as
a major determinant of energy use and greenhouse gas (GHG)
emissions7–9. The discussion mainly focuses on whether GDP can be
decoupled from emissions, i.e., whether energy use and emissions can 1Institute of Social Ecology, University of Natural Resources and Life Sciences, Vienna, Austria. 2Economics Department and Eco-SOS, Universitat Rovira i
Virgili, Tarragona, Spain. 3Mercator Research Institute on Global Commons and Climate Change, EUREF 19, 10829 Berlin, Germany. 4Technical University
Berlin, Straße des 17 Junis 135, 10623 Berlin, Germany. 5These authors contributed equally: Helmut Haberl, Markus Löw, Alejandro Perez-Laborda. e-mail: helmut.haberl@boku.ac.at Nature Communications| (2023) 14:3898 1 https://doi.org/10.1038/s41467-023-39728-3 Article total population). A suitable proxy for the climate dependency of
heating energy demand are heating-degree days (abbreviated as
HDD)20. Built structures influence patterns of energy
demand and CO2 emissions across countries As conventional factors, we use
GDP/cap/yr (abbreviated as GDP), population density (DENS), UPOP as
a development indicator, HDD as a proxy of climate dependency of
energy demand, and PGAS as an energy price indicator. Extensive
variables are expressed as per-capita values to facilitate country
comparisons and remove countries’ population numbers from the
analysis. Multivariate lasso analysis The capability of the material stock pattern indicators to add insights
beyond conventional factors is further analyzed in Table 2. We use the
least absolute shrinkage and selection operator (lasso) approach to
select variables for multivariate statistical models capable of predict-
ing cross-country patterns of TFC and CO2. Lasso is a widely-
used procedure for automatically performing variable selection in
linear regression models39,40. It overcomes the drawbacks of overfitting
and multicollinearity associated with ordinary least square (OLS)
methods41. The lasso method penalizes complexity to derive the best
parsimonious model for any predefined value of λ (the factor deter-
mining how strongly model complexity is penalized; for detail, see
Methods section), thereby allowing to identify the factors that most
strongly affect TFC and CO2. The standard procedure to select λ is
cross-validation, where randomly chosen samples of countries are
used to develop a model used for the prediction of patterns in out-of-
sample countries. The procedure gradually reduces λ, which generally
results in more indicators being selected41. Alternative Lasso schemes
and other variable selection techniques, such as stepwise regression,
yield very similar results (SI). Nature Communications| (2023) 14:3898 tional-level indicators of material stock patterns National-level indicators of material stock patterns p
We test three hypotheses based on aggregated indicators of material
stock patterns (Fig. 1). Material stock patterns are represented by three
types of indicators: (1) The area of built-up land is represented by two
indicators, one as a fraction of a nation’s inhabited land, the other per
inhabitant. Other indicators describe patterns of built-up areas,
including their spatial clustering, form, and distribution, which reflect
geomorphological factors as well as historical contingencies. (2) Road
indicators that describe the density (length per unit area) of roads in
urban and rural regions and the relations between urban and rural road
lengths and densities. (3) Railway indicators are defined in the same
manner as those for roads (Table 1). The leftmost three columns of Table 2 show the knots in the lasso
paths, i.e., the λ values at which indicators are added (or removed due
to collinearity). Knots are arranged in decreasing order of λ, with
indicators being ranked in order of selection. In the first column, the λ
value marked with an asterisk (*) indicates the optimal model identified
by the cross-validation. GDP is always selected as the first indicator, but
various material stock pattern indicators appear very early on (i.e., at
high λ values), and remain active in the optimal model. BLcap is the
second-chosen indicator for both CO2 and TFC, and several other Nature Communications| (2023) 14:3898 2 Article https://doi.org/10.1038/s41467-023-39728-3 g. 1 | Workflow of this study. National-level indicators of the extent and spatial
tterns of settlements and infrastructures (material stock patterns) were derived
om global maps, here illustrated using Paraguay, the UK, Kenia, and Bangladesh as
amples. Results were statistically analyzed along with the conventional factors
assumed to co-determine energy use and CO2 emissions. The main aim was
the hypotheses formulated in the lower-right box. Copyright for administr
boundaries: © Eurogeographics. Fig. 1 | Workflow of this study. National-level indicators of the extent and spatial
patterns of settlements and infrastructures (material stock patterns) were derived
from global maps, here illustrated using Paraguay, the UK, Kenia, and Bangladesh as
examples. Results were statistically analyzed along with the conventional factors assumed to co-determine energy use and CO2 emissions. The main aim was to test
the hypotheses formulated in the lower-right box. Copyright for administrative
boundaries: © Eurogeographics. tional-level indicators of material stock patterns Table 1 | Indicators of the extent and patterns of built-up land and transport infrastructures material stock pattern indicators are selected much earlier than
widely-used conventional factors such as population density (DENS). Heating-degree days (HDD) and the price of gas (PGAS) are important
for both CO2 and TFC, whereas UPOP is important for predicting CO2
but not selected by lasso for TFC. type approaches, and will enable researchers to broaden their ana-
lysis and scenario modeling capabilities by including material stock
patterns as crucial factors for analyzing the possible decoupling of
energy use and emissions from GDP. Population density plays a smaller role than widely assumed, while
many material stock patterns strongly influence the cross-country
differences in energy demand and CO2 emissions. The material stock
pattern indicator with the most consistent predictive power across all
analyses is the area of built-up land per capita (BLcap), which emerges
as the second-most important variable (after GDP) in most of our
statistical analyses, even in analyses considering the GDP effect. This is
plausible because infrastructures and buildings require energy for
being built and used, which results in CO2 emissions in current fossil
fuel-dominated energy systems34,43. Higher BLcap also means larger
floor size and longer distances between destinations, which all raises
energy demand in buildings and transport. These findings corroborate
and expand on previous analyses (that used entirely different models,
did not capture spatial patterns, and mostly referred to temporal
trends), suggesting that challenges for climate-change mitigation
strongly depend on the past and future accumulation of material
stocks in buildings and infrastructures43–46. This is worrying because
material stocks are growing globally largely in unison with GDP32,46. The rightmost three columns of Table 2 report the estimated
coefficients of models selected by cross-validation as well as measures
of in-sample and out-of-sample fit (r2). Models A comprises only vari-
ables selected by lasso (path shown in the left part of the table) among
all indicators. For the benchmark Models B, only conventional factors
are selected. Including material stock pattern indicators in Model A
yields better prediction than Models B for both CO2 and TFC in terms
of in-sample and out-of-sample goodness of fit, and improves the
Bayesian Information Criterion (BIC) of model selection. Other criteria
to develop optimal models, and alternative variable selection proce-
dures yielded similar results (section 5 in the SI). tional-level indicators of material stock patterns Nature Communications| (2023) 14:3898 3 3 https://doi.org/10.1038/s41467-023-39728-3 Article Table 1 | Indicators of the extent and patterns of built-up land and transport infrastructures
Name
Abbreviation
Description and interpretation
Unit
(1) Indicators for the extent and pattern of a nation’s built-up land
Fraction of built-up land
BLfract
Built-up land (buildings & infrastructures) as % of the inhabited land area. m²/m²
Built-up land per capita
BLcap
Built-up land per capita. m²/cap
Dispersion of built-up land
BLdisp
Index based on the average distance of each patch of built-up land to the nearest adjacent patch. High
values indicate strong dispersion. –
Monocentricity of built-up land
BLmono
Area of the largest contiguous built-up patch as % of the sum of the areas of the ten largest patches. High values indicate dominance of one large center. m2/m2
Compactness of built-up land
BLcomp
Index describing how round or compact the shapes of a nation’s built-up land patches are on average. –
Urban population density
UPdens
Urban population per unit of urban built-up land
cap/m2
(2) Indicators of road density and distribution
Road density
RDtotal
Length of roads per unit area of inhabited land. m/m²
Urban road density
RDurban
Length of roads in urban areas per unit area of urban areas. m/m²
Rural road density
RDrural
Length of roads in rural areas per unit area of rural areas; proxy of rural accessibility and connectivity
between urban centers. m/m²
Ratio of urban-to-rural road lengths
RLurb-rur
Ratio of urban to rural road lengths, indicating the extent to which roads are concentrated in cities. –
Ratio of urban-to-rural road density
RDurb-rur
Ratio of RDurban and RDrural, indicating the difference between urban and rural road densities. –
(3) Indicators of railway density and distribution
Railway density
RWDtotal
Length of railways per unit area of inhabited land. m/m²
Urban railway density
RWDurban
Length of railways in urban areas per unit area of urban areas. m/m²
Rural railway density
RWDrural
As RDrural for railways. m/m²
Ratio of urban-to-rural railway
lengths
RWLurb-rur
As RLurb-rur for railways. –
Ratio of urban-to-rural railway
density
RWDurb-rur
As RDurb-rur for railways. –
These indicators condense spatially explicit information in maps to national-level indicator values assumed to co-determine a nation’s per-capita level of energy demand and CO2 emissions. Global
maps showing the indicator values are in Supplementary Figs. 5–7 (SI). tional-level indicators of material stock patterns Even models that
include only material stock pattern indicators and exclude all con-
ventional factors (including GDP) achieve good predictions (out-of-
sample r2 of 0.65 for TFC and 0.62 for CO2; see results for Model C
shown in section 5.2 of the SI). Nature Communications| (2023) 14:3898 Discussion Our indicators and results create options for analyzing which of
the characteristics of material stock patterns are most important in
determining and predicting energy demand and emissions in cross-
country analyses. Analyses presented in Fig. 2, Table 2 as well as the SI
clearly show that many specific aspects of material stock patterns play
a role in determining cross-country differences in energy demand and
emissions. The multivariate analysis also shows that these patterns
interact in many ways that are difficult to disentangle due to the col-
linearities of the material stock pattern indicators. Future research
could employ refined study designs addressing how changes over time
and space affect these relationships and elucidate the different causal
pathways involved. The analysis shows that extent and spatial patterns of built struc-
tures, here denoted as material stock patterns, play an important role
in co-determining and predicting the level of resource use, here TFC
and CO2, in a cross-country analysis. This implies that insights from
urban studies23–26,42 generally hold at the national scale. Despite the
unavoidable loss of information resulting from the aggregation of
maps to the national scale, the indicators in Table 1 maintain key
information representing important characteristics of material stock
patterns that strongly affect cross-country patterns of TFC and CO2. The indicators have substantial additional explanatory and pre-
dictive power over conventional factors. Discussion Correlations were not
significant for
between mate
for GDP and D
ficient of the s
correlation co
indicate insign UPOP
HDD
PGAS
BLfract
BLcap
UPdens
BLdisp
BLmono
BLcomp
RDtotal
RDurban
RDrural
RLurb-rur
RDurb-rur
RWDtotal
RWDurban
RWDrural
RWLurb-rur
RWLurb-rur
-0,8
-0,6
-0,4
-0,2
0,0
0,2
0,4
0,6
0,8
-0,4
-0,3
-0,2
-0,1
0,0
0,1
0,2
0,3
0,4
Zero correlation
Semi-partial correlation
Semi-partial correlations for TFC
UPOP
HDD
PGAS
BLfract
BLcap
UPdens
BLdisp
BLmono
BLcomp
RDtotal
RDurban
RDrural
RLurb-rur
RDurb-rur
RWDtotal
RWDurban
RWDrural
RWLurb-rur
RWDurb-rur
-0,8
-0,6
-0,4
-0,2
0,0
0,2
0,4
0,6
0,8
-0,4
-0,3
-0,2
-0,1
0,0
0,1
0,2
0,3
0,4
Zero correlation
Semi-partial correlation
Semi-partial correlations for CO2
Fig. 2 | Correlation analyses of total final energy demand per capita (TFC) and
per-capita CO2 emissions (CO2) with conventional factors and material stock
pattern indicators. a Pearson’s zero correlation coefficients of correlations
between TFC (left) and CO2 (right) and material stock pattern indicators as well as
conventional factors. Natural logarithms of the variables were analyzed. Squaring
the correlation coefficients gives the percentage of the cross-country variation of
CO2 or TFC explained by the respective indicator alone. Correlations were not
significant for variables marked with an asterisk (p < 0.1).b Semi-partial correlations
between material stock pattern indicators and TFC (left) and CO2 (right) controlling
for GDP and DENS. Distance from the vertical axis indicates the correlation coef-
ficient of the semi-partial correlation, and distance from the horizontal axis is the
correlation coefficient of the bivariate (uncontrolled) correlation. Red contours
indicate insignificant results (significance level p < 0.1). Semi-partial correlations for CO2 Fig. 2 | Correlation analyses of total final energy demand per capita (TFC) and
per-capita CO2 emissions (CO2) with conventional factors and material stock
pattern indicators. a Pearson’s zero correlation coefficients of correlations
between TFC (left) and CO2 (right) and material stock pattern indicators as well as
conventional factors. Natural logarithms of the variables were analyzed. Squaring
the correlation coefficients gives the percentage of the cross-country variation of
CO2 or TFC explained by the respective indicator alone. Correlations were not Fig. 2 | Correlation analyses of total final energy demand per capita (TFC) and
per-capita CO2 emissions (CO2) with conventional factors and material stock significant for variables marked with an asterisk (p < 0.1).b Semi-partial correlations
between material stock pattern indicators and TFC (left) and CO2 (right) controlling
for GDP and DENS. Discussion They can help develop
much stronger models of national-level TFC and CO2 than usual IPAT- Nature Communications| (2023) 14:3898 4 https://doi.org/10.1038/s41467-023-39728-3 Article (a)
(b)
0,88
0,72
0,67
0,67
0,65
0,59
0,53
0,47
0,46
0,44
0,37
0,27
0,12
0,08
-0,10
-0,11
-0,12
-0,31
-0,44
-0,45
-0,52
-1,00
-0,50
0,00
0,50
1,00
GDP
BLcap
UPOP
RWDtotal
RWDrural
RWDurban
HDD
RDrural
RDtotal
BLfract
RWLurb-rur
BLdisp
PGAS*
RLurb-rur*
DENS*
RDurban*
RWDurb-rur
BLmono
BLcomp
UPdens
RDurb-rur
Bivariate correlation with TFC
0,82
0,72
0,69
0,68
0,67
0,56
0,55
0,50
0,49
0,47
0,38
0,30
0,19
0,01
-0,05
-0,08
-0,19
-0,32
-0,36
-0,43
-0,50
-1,00
-0,50
0,00
0,50
1,00
GDP
UPOP
BLcap
RWDtotal
RWDrural
RWDurban
HDD
BLfract
RDtotal
RDrural
RWLurb-rural
BLdisp
RLurb-rur
PGAS*
RDurban*
DENS*
RWDurb-rur
BLmono
BLcomp
UPdens
RDurb-rur
Bivariate correlation with CO2
UPOP
HDD
PGAS
BLfract
BLcap
UPdens
BLdisp
BLmono
BLcomp
RDtotal
RDurban
RDrural
RLurb-rur
RDurb-rur
RWDtotal
RWDurban
RWDrural
RWLurb-rur
RWLurb-rur
-0,8
-0,6
-0,4
-0,2
0,0
0,2
0,4
0,6
0,8
-0,4
-0,3
-0,2
-0,1
0,0
0,1
0,2
0,3
0,4
Zero correlation
Semi-partial correlation
Semi-partial correlations for TFC
UPOP
HDD
PGAS
BLfract
BLcap
UPdens
BLdisp
BLmono
BLcomp
RDtotal
RDurban
RDrural
RLurb-rur
RDurb-rur
RWDtotal
RWDurban
RWDrural
RWLurb-rur
RWDurb-rur
-0,8
-0,6
-0,4
-0,2
0,0
0,2
0,4
0,6
0,8
-0,4
-0,3
-0,2
-0,1
0,0
0,1
0,2
0,3
0,4
Zero correlation
Semi-partial correlation
Semi-partial correlations for CO2
Fig. 2 | Correlation analyses of total final energy demand per capita (TFC) and
per-capita CO2 emissions (CO2) with conventional factors and material stock
pattern indicators. a Pearson’s zero correlation coefficients of correlations
between TFC (left) and CO2 (right) and material stock pattern indicators as well as
conventional factors. Natural logarithms of the variables were analyzed. Squaring
the correlation coefficients gives the percentage of the cross-country variation of
CO2 or TFC explained by the respective indicator alone. Correlations were not
significant for variables marked with an asterisk (p < 0.1).b Semi-partial correlations
between material stock pattern indicators and TFC (left) and CO2 (right) controlling
for GDP and DENS. Distance from the vertical axis indicates the correlation coef-
ficient of the semi-partial correlation, and distance from the horizontal axis is the
correlation coefficient of the bivariate (uncontrolled) correlation. Red contours
indicate insignificant results (significance level p < 0.1). Discussion Distance from the vertical axis indicates the correlation coef-
ficient of the semi-partial correlation, and distance from the horizontal axis is the
correlation coefficient of the bivariate (uncontrolled) correlation. Red contours
indicate insignificant results (significance level p < 0.1). significant for variables marked with an asterisk (p < 0.1).b Semi-partial correlations
between material stock pattern indicators and TFC (left) and CO2 (right) controlling
for GDP and DENS. Distance from the vertical axis indicates the correlation coef-
ficient of the semi-partial correlation, and distance from the horizontal axis is the
correlation coefficient of the bivariate (uncontrolled) correlation. Red contours
indicate insignificant results (significance level p < 0.1). pattern indicators. a Pearson’s zero correlation coefficients of correlations
between TFC (left) and CO2 (right) and material stock pattern indicators as well as
conventional factors. Natural logarithms of the variables were analyzed. Squaring
the correlation coefficients gives the percentage of the cross-country variation of
CO2 or TFC explained by the respective indicator alone. Correlations were not Our results have implications for countries pursuing ambitious
climate targets2,43. They demonstrate that insights from urban-level
studies translate to the national scale, which could so far not be
investigated at that level due to lacking data. This suggests that the
indicators presented above offer opportunities to investigate the
importance of built structures also for other aspects of national
economies’ resource use than those analyzed here. Empirical urban
studies have consistently shown that urban form and infrastructure
affect travel demand and, therefore, GHG emissions47. The relationship between built structures and GHG emissions has also been predicted
by theoretical analyses in urban economics21,48. This suggests that our
national-level observations—e.g., that large, sprawling material stocks
in built structures represent an important determinant of a nation’s
per-capita level of energy demand and emissions—are very likely
underpinned by similar causal relationships. Of course, more research,
in particular on how these relationships change over time, would be
desirable. Because high-resolution maps of decadal trends in built
structures are gradually becoming available49,50, such analyses may between built structures and GHG emissions has also been predicted
by theoretical analyses in urban economics21,48. This suggests that our
national-level observations—e.g., that large, sprawling material stocks
in built structures represent an important determinant of a nation’s
per-capita level of energy demand and emissions—are very likely
underpinned by similar causal relationships. Of course, more research,
in particular on how these relationships change over time, would be
desirable. Discussion (a) (a)
(b)
0,88
0,72
0,67
0,67
0,65
0,59
0,53
0,47
0,46
0,44
0,37
0,27
0,12
0,08
-0,10
-0,11
-0,12
-0,31
-0,44
-0,45
-0,52
-1,00
-0,50
0,00
0,50
1,00
GDP
BLcap
UPOP
RWDtotal
RWDrural
RWDurban
HDD
RDrural
RDtotal
BLfract
RWLurb-rur
BLdisp
PGAS*
RLurb-rur*
DENS*
RDurban*
RWDurb-rur
BLmono
BLcomp
UPdens
RDurb-rur
Bivariate correlation with TFC
0,82
0,72
0,69
0,68
0,67
0,56
0,55
0,50
0,49
0,47
0,38
0,30
0,19
0,01
-0,05
-0,08
-0,19
-0,32
-0,36
-0,43
-0,50
-1,00
-0,50
0,00
0,50
1,00
GDP
UPOP
BLcap
RWDtotal
RWDrural
RWDurban
HDD
BLfract
RDtotal
RDrural
RWLurb-rural
BLdisp
RLurb-rur
PGAS*
RDurban*
DENS*
RWDurb-rur
BLmono
BLcomp
UPdens
RDurb-rur
Bivariate correlation with CO2 Bivariate correlation with CO2 (b) UPOP
HDD
PGAS
BLfract
BLcap
UPdens
BLdisp
BLmono
BLcomp
RDtotal
RDurban
RDrural
RLurb-rur
RDurb-rur
RWDtotal
RWDurban
RWDrural
RWLurb-rur
RWLurb-rur
-0,8
-0,6
-0,4
-0,2
0,0
0,2
0,4
0,6
0,8
-0,4
-0,3
-0,2
-0,1
0,0
0,1
0,2
0,3
0,4
Zero correlation
Semi partial correlation
Semi-partial correlations for TFC
UPOP
HDD
PGAS
BLfract
BLcap
UPdens
BLdisp
BLmono
BLcomp
RDtotal
RDurban
RDrural
RLurb-rur
RDurb-rur
RWDtotal
RWDurban
RWDrural
RWLurb-rur
RWDurb-rur
-0,8
-0,6
-0,4
-0,2
0,0
0,2
0,4
0,6
0,8
-0,4
-0,3
-0,2
-0,1
0,0
0,1
0,2
0,3
0,4
Zero correlation
Semi partial correlation
Semi-partial correlations for CO2 UPOP
HDD
PGAS
BLfract
BLcap
UPdens
BLdisp
BLmono
BLcomp
RDtotal
RDurban
RDrural
RLurb-rur
RDurb-rur
RWDtotal
RWDurban
RWDrural
RWLurb-rur
RWLurb-rur
-0,8
-0,6
-0,4
-0,2
0,0
0,2
0,4
0,6
0,8
-0,4
-0,3
-0,2
-0,1
0,0
0,1
0,2
0,3
0,4
Zero correlation
Semi-partial correlation
Semi-partial correlations for TFC
P
-0,4
-0
Zero correlation
Fig. 2 | Correlation analyses of total final energy demand per capita (TFC) and
per-capita CO2 emissions (CO2) with conventional factors and material stock
pattern indicators. a Pearson’s zero correlation coefficients of correlations
between TFC (left) and CO2 (right) and material stock pattern indicators as well as
conventional factors. Natural logarithms of the variables were analyzed. Squaring
the correlation coefficients gives the percentage of the cross-country variation of
CO2 or TFC explained by the respective indicator alone. Discussion In the first column, the λ value marked with an asterisk
denotes the optimal model (Model A) emerging from the cross-validation. CV MSPE is the cross-validated mean-square prediction error evaluated with tenfolds (for detail, see Methods section). For
Model B, only conventional factors are selected. The same folds are used for the assessment of all models. BIC is the Bayesian Information Criterion for model selection. r2 is the goodness of fit within
the sample of countries, and oSr2 refers to the (cross-validated) out-of-sample goodness of fit. resolution land-cover data53. The indicators refer to the years 2015
(built-up land) and 2020 (roads and railroads). To reduce random
fluctuations for the energy and emission data as well as the conven-
tional factors, we calculated averages for as many years in the period
2015-2020 as were available in the statistical sources. Extensive vari-
ables were represented by per-capita values to facilitate comparisons
between different-sized countries, following the convention to regard
population as a scaling factor with an elasticity of one in STIRPAT
analyses5. soon be feasible. We conclude that the area and patterns of built-up
land emerge as an important entry point for efforts at reducing energy
demand and CO2 emissions at national levels51, suggesting that limiting
built-up area per capita could be a worthwhile policy goal, not only
from a land-use perspective, but also to limit future GHG emissions. Discussion Because high-resolution maps of decadal trends in built
structures are gradually becoming available49,50, such analyses may Nature Communications| (2023) 14:3898 5 https://doi.org/10.1038/s41467-023-39728-3 Article Table 2 | Multivariate analysis using cross-validation lasso |
y
g
Models predicting TFC
Lasso path for model A
Estimated coefficients
λ
(A)dded, (R)emoved
CV MPSE
Model A
Model B
0.711
GDP(A)
0.703
GDP
0.494
0.572
0.371
BLcap(A)
0.319
DENS
−0.017
−0.039
0.161
HDD(A), RWDtot(A)
0.179
UPOP
0.121
RDurb-rur(A)
0.166
HDD
0.021
0.051
0.111
PGAS(A)
0.161
PGAS
−0.352
−0.288
0.101
RDurb(A)
0.155
BLcap
0.184
0.076
RWDurb(A)
0.135
BLmono
−0.017
0.063
RWDtot(R)
0.125
RDurb
−0.392
0.048
DENS(A)
0.115
RLurb-rur
−0.024
0.030
BLmono(R)
0.108
RDurb-rur
−0.074
0.027
RLurb-rur(A)
0.107
RWDurb
0.045
0.017*
(Unchanged)
0.105
Intercept
3.457
2.542
0.016
RWDurb-rur(A)
0.105
0.013
BLcomp(A)
0.105
Measures of in-and-out-of-sample fit
0.011
(Unchanged)
0.106
BIC
84.17
100.32
r2
0.900
0.851
oSr2
0.865
0.833
Models predicting CO2
λ
(A)dded, (R)emoved
CV MPSE
variable
Model A
Model B
0.867
GDP(A)
1.278
GDP
0.449
0.582
0.544
BLcap(A)
0.801
DENS
−0.061
0.023
0.496
UPOP(A)
0.738
UPOP
0.466
0.558
0.452
RWDtot(A)
0.683
HDD
0.055
0.109
0.312
HDD(A)
0.530
PGAS
−0.680
−0.688
0.215
PGAS(A)
0.446
BLfract
0.201
0.135
RDurb-rur(A)
0.338
BLcap
0.176
0.123
BLfract(A)
0.323
BLcomp
0.489
0.085
RWLurb-rur(A)
0.289
RDurb
−0.201
0.070
BLfract(R)
0.280
RWDurb
0.130
0.048
BLcomp(A)
0.269
RWDrur
0.017
0.044
RDurb(A)
0.266
Intercept
−2.723
−4.294
0.037
RWDurb(A)
0.262
0.025
BLfract (A)
0.256
Measures of in-and-out-of-sample fit
0.023
DENS(A)
0.255
BIC
178.44
190.82
0.021
RWLurb-rur(R)
0.254
r2
0.873
0.812
0.016
RWDrur(A)
0.251
oSr2
0.817
0.785
0.016
RDurb-rur(R)
0.251
0.014
RWDtot(R)
0.250
0.012*
(Unchanged)
0.249
0.006
(Unchanged)
0.250
The leftmost three columns show the lasso path for predicting cross-country patterns of TFC (above) and CO2 (below) using all variables. In the first column, the λ value marked with an asterisk
denotes the optimal model (Model A) emerging from the cross-validation. CV MSPE is the cross-validated mean-square prediction error evaluated with tenfolds (for detail, see Methods section). For
Model B, only conventional factors are selected. The same folds are used for the assessment of all models. BIC is the Bayesian Information Criterion for model selection. r2 is the goodness of fit within
the sample of countries, and oSr2 refers to the (cross-validated) out-of-sample goodness of fit. The leftmost three columns show the lasso path for predicting cross-country patterns of TFC (above) and CO2 (below) using all variables. Deriving the built-up land layer To calculate IH, we first cut out high-altitude regions from the
country’s total territory. We calculated the elevational distribution of
BL features and excluded all areas above the area-weighted 99th per-
centile of BL-elevations. In a second step, we excluded the LC100 land
cover types bare/sparse vegetation (deserts and rocks), moss & lichen,
snow & ice, and permanent water bodies. Thirdly, we spatially inter-
sected the map resulting from the previous two steps with a synthetic
layer that represents the gapless inhabited land area. To derive this
layer, we applied an area-dependent buffer for all BL features. See Eq. (1) for the dynamic BL buffer width (wBL); ABL feature denotes the area of
an exemplary built-up land feature. g
p
y
The built-up land vector (BL) for every country is one primary data
product derived by the vectorized LC100 grid. This standard BL layer
comprised all national built-up features and was used to derive the BL-
related indicators shown in part (1) of Table 1. To map each country’s
urban agglomerations, and thereby distinguish urban from rural land
respectively infrastructures (see Supplementary Figs. 2–4, SI), we
clustered features of the BL vector layer using an empirical growing-
neighborhood approach: We started with the country’s largest BL
feature and created convex hulls, which were buffered with the fifth of
the area-equal radius. We then identified intersecting BL features
within this buffered hull and created a new hull and buffer. These two
steps were repeated as long as the BL area of all intersecting features
reached 33.3% of the area of the buffered hull (used to check the
intersections), and the BL area increased at least 0.5%, compared to the
BL area of the previous iteration. If these criteria were not given, the
growing procedure terminated and the collected BL features dissolved
to one BL agglomeration. The algorithm subsequently went on with
the next-biggest BL feature, which had not been assigned to an already-
created agglomeration, again starting the growing-neighborhood
procedure. The whole process terminated when the next BL feature
(to restart the growing-neighborhood procedure again) represented
less than 0.1% of the total BL area of the respective country. The maps ð1Þ maxfwBL : 100kmg Finally, we added cropland areas (from LC100) and the original BL
areas to re-include those BL areas excluded by the elevation threshold. The infrastructure layers The preprocessed OSM database comprises globally consistent road (R)
and railway vector data (RW), but the availability of OSM data varies
strongly between countries. While the OSM data in countries of the
global North (industrial or even postindustrial countries) also include
minor road and train track categories (e.g., cycleways, steps, or private
gauges), OSM data in countries of the global South only comprise the
main road and railway network. To reduce this inconsistency, we
excluded minor OSM classes in order to derive a more comparable
global database (Supplementary Table 3). The maps showing the road
network indicators are in Supplementary Fig. 6 (SI). Fractions of the area
refer to an inhabited land. The railway indicators were derived from
OSM in the same manner as those for roads and are shown in Supple-
mentary Fig. 7 (SI). The railway types considered are defined in Sup-
plementary Table 4. Fractions refer to an inhabited land. The planar
extent of road and railway networks was calculated using width data
reported in Supplementary Table 5. The distinction between urban and
rural infrastructures was based on a spatial intersection of OSM road
and railway data with the BL features, which resulted in layers of urban
and rural networks (example shown in Supplementary Fig. 4, SI). Reference layer for inhabited land Reference layer for inhabited land
The definition of some material stock pattern indicators requires a
reference area (AREF). The area of the total national territory (ANT) may
not be suitable, given that in some countries (almost), the entire area is
inhabitable, whereas other countries contain large tracts of land
unsuitable for human habitation and hence largely uninhabited. We,
therefore, developed the proxy layer inhabited land (IH) as a more
suitable area reference (AIH). In contrast to existing similar datasets69,
this IH mainly uses land cover information of the LC100 grid. This
guarantees thematic consistency in spatial intersections with BL data
that were derived using the same dataset. The IH includes not only area
that is covered with settlements or infrastructures, but also cropland
areas and areas with ambiguous land cover that fall within a zone of
influence around existing built-up areas, which is approximated by a
buffer that depends on the area of a built-up land feature (Eq. 1). The IH
is based on the current settlement and cropland extent, as well as the
spatial distribution, density, and elevation occurrence of built-up areas
according to the LC100 grid. Data used to derive spatial indicators The spatial indicators presented in this study rely on three spatially
explicit datasets; (1) built-up features and urban agglomerations, (2)
main infrastructure features (road and railway), and (3) an inhabited
land area layer used for reference. All variables that express quan-
tities as fractions of a country’s area, relate to inhabited land. We
used the LC100 grid of the Copernicus Global Land Cover Service53 to
derive relevant land cover information. The choice of input data and
data quality checks are discussed in the SI; see also Supplementary
Fig. 1 (SI). In contrast to other built-up land datasets54–56, the LC100
not only provides globally consistent information on built-up areas, it
also includes all other complementary land cover types at a global
scale. For data preparation, we vectorized the LC100 grid to allow
further spatial intersection procedures (e.g., to clip to national bor-
ders). The NOAA-DEM grid was used as a global digital elevation
model. We utilized the Geofabrik-download hub57 to obtain the entire
global OSM data. The GISCO archive from EUROSTAT58 provides the
applied country borders. Details on the data sources are given
in Supplementary Table 1 (SI); datasets59 and software code60 are
available online. Methods The material stock pattern indicators were derived from crowd-
sourced data on infrastructures (roads and railways)52 as well as high- Nature Communications| (2023) 14:3898 Nature Communications| (2023) 14:3898 6 Article https://doi.org/10.1038/s41467-023-39728-3 in Supplementary Fig. 5 (SI) show the built-up (BL) land indicators. Fractions of land areas refer to inhabited land. in Supplementary Fig. 5 (SI) show the built-up (BL) land indicators. Fractions of land areas refer to inhabited land. Sources of energy and emission data as well as conventional
factors Gross domestic product (GDP) in constant 2015 US$ was sourced from
UN Statistics Division National Accounts61. Population data used to
calculate population density (DENS) and urban population rates
(UPOP) were taken from official census data 2017 of the World
Bank62,63. The World Bank database was also used to source data on
pump prices for gasoline (PGAS)64. Heating-degree days (HDD) were
calculated as a population-weighted average of °C days above 18 °C
and sourced from the International Energy Agency (IEA) Weather for
Energy Tracker65. Territorial CO2 emissions from fossil fuel combus-
tion and cement production (CO2) were sourced from the Global
Carbon Project database66,67 and total final energy consumption (TFC)
from the IEA energy balances, which could only be accessed for the
latest year 201768. The record date for the download of these indicators
was 25 March 2021. We always used the latest available year for our
cross-sectional analysis, but also conducted robustness checks using
arithmetic averages over the latest five years for all indicators to
exclude potential bias from outstanding annual values. Details on the
data are available in Supplementary Table 2 in the SI. Data availability Datasets on spatial data on patterns of global infrastructure and set-
tlements, the inhabited land layer, as well as the indicator values of
dependent and independent variables used in the statistical analyses is
freely available here: https://doi.org/10.5281/zenodo.5876941. An
interim result that was too large to be uploaded as zenodo archive is
available from the authors for non-commercial research purposes
upon reasonable request (for detail, see ref. 59). The semi-partial correlation is calculated by first fitting a linear
regression of Y on X and computing the coefficient as: rY Xi∣Xi
ð
Þ = sign tð Þ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
t2 1 R2
n k
v
u
u
t
ð2Þ ð2Þ References 1. IPCC. Climate Change 2021, The Physical Science Basis. (Working
Group I contribution to the Sixth Assessment Report of the Inter-
governmental Panel on Climate Change, 2021). 1. IPCC. Climate Change 2021, The Physical Science Basis. (Working
Group I contribution to the Sixth Assessment Report of the Inter-
governmental Panel on Climate Change, 2021). 2. IPCC. Global Warming of 1.5 °C. An IPCC special report on the
impacts of global warming of 1.5 °C above pre-industrial levels and
related global greenhouse gas emission pathways, in the context of
strengthening the global response to the threat of climate change,
sustainable development, and efforts to eradicate poverty. (WMO,
UNEP, 2018). Pearson correlations The Pearson correlation is a measure of linear association between two
variables. The coefficient can be obtained from bivariate data
X1,Y 1
,:::, Xn,Y n
as rXY = SXY=SXSY , where SXY and Si denote the
sample covariance and standard deviation. The correlation coefficient
is between −1 and 1. Correlations equal to 1 (or −1) indicate a perfect
linear association, with data points lying exactly on a positive (nega-
tive) line. A value equal to zero indicates the absence of any linear
association. The squared correlation coefficient r2
xy is the coefficient of
determination (R-squared) of the linear regression of variable x on
variable y; it measures the fraction of the variance explained by the
regression line. The first part of the term (3) is the in-sample squared error mini-
mized by a classical least-squares approach. Lasso also includes the
absolute sum of coefficients in the objective function, which penalizes
complexity driving some of the estimated coefficients to zero. The value of λ is typically chosen so that the estimated model
satisfies a predetermined condition. Several criteria can be employed,
the most common of which is cross-validation. Cross-validation selects
the λ to minimize the out-of-sample prediction error. First, sample
observations are split into K random folds (validation sets). For each
validation set, the model is fitted using data from the other folds, and
the out-of-sample deviance for the observations in the validation set is
computed (i.e., using data not employed for estimation). Finally, the
overall out-of-sample performance of the model in all the validation
sets is assessed by the mean-square prediction error (MSPE), a statis-
tical parameter in squared (log) units required for model selection. Cross-validation selects the λ over a grid of possible values such that
the corresponding model has the minimum MSPE71. In Table 2, MSPE is
transformed into r2, i.e., the goodness of fit within the sample of
countries, and oSr2, i.e., the cross-validated out-of-sample goodness of
fit for the optimal models. If no variable is added or removed at λ*, this
is reported in the left columns of Table 2 as Unchanged. Note that
beyond λ*, more variables could be added but would not further
improve the out-of-sample prediction. Definitions of spatial pattern indicators The spatial pattern indicators were derived from the built-up, road,
and railway layers. Details on the definitions of the indicators (Table 1)
are given in the SI in section 3, Supplementary Tables 6–8. 1
2N Y XB
ð
Þ0 Y XB
ð
Þ + λ
XP
j = 1∣Bj∣
ð3Þ ð3Þ Code availability
h
l
l In (2) t is the t-statistic of variable X i in the previous regression, R2
is the R-squared, k is the number of independent variables plus the
constant, and n is the sample size. Finally, the significance level is given
by 2=Pr tnk > ∣t∣
, where Pr is the probability, t is as described above
and tnk follows a Student’s t distribution with n k degrees of free-
dom. Further details on the correlation techniques used are available
e.g. here70. The code used for calculations of maps is freely available here: https://
doi.org/10.5281/zenodo.5883652. The code used for calculations of maps is freely available here: https://
doi.org/10.5281/zenodo.5883652. Deriving the built-up land layer Please note that for spatial indicators that depend on AREF, the specific
spatial pattern (shape) of IH is not relevant: we just use the national
total area of IH instead of the area of the national territory as reference
value. The potential usefulness of this IH layer for other research Nature Communications| (2023) 14:3898 7 Article https://doi.org/10.1038/s41467-023-39728-3 questions, in particular where its spatial accuracy is of high impor-
tance, needs to be tested and is not in focus in this study. model with the maximum complexity. In general, the larger the λ, the
smaller the number of non-zero coefficients. Consider
a
linear
specification
Y = B0 + B1X1 + . . . + BPXP + ϵ,
where variables have been previously standardized to account for
differences in scales. Lasso finds estimates for model coefficient B
keeping the model sparse by minimizing the following term: Semi-partial correlations Suppose that Y is determined by X = X1,::::,Xk
. Then, the semi-partial
correlation between Y and Xi, controlled for the other predictors Xi,
attempts to measure the correlation between Y and Xi that would be
observed if the effect of Xi would be removed from Xi but not fromY. This means that the semi-partial regression measures the correlation
between Y and the part of Xi that is orthogonal to the other variables
Xi. It is calculated by constructing a new variable X 0
i that is orthogonal
(i.e., entirely uncorrelated) to all previously considered variables (i.e.,
those controlled for). The squared semi-partial correlation coefficient measures the
fraction of the variance of the dependent variable Y that is uniquely
explained by Xi. Thus, it can also be interpreted as the increase
(decrease) in the model R-squared value that results from including
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Investigation of relative risk estimates from studies of the same population with contrasting response rates and designs
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University of Wollongong
University of Wollongong
Research Online
Research Online University of Wollongong
University of Wollongong
Research Online
Research Online Faculty of Informatics - Papers (Archive)
Faculty of Engineering and Information
Sciences
2010
Investigation of relative risk estimates from studies of the same population
Investigation of relative risk estimates from studies of the same population
with contrasting response rates and designs
with contrasting response rates and designs
Nicole M. Mealing
University of Western Sydney
Emily Banks
Australian National University
Louisa R. Jorm
University of Western Sydney
David G. Steel
University of Wollongong, dsteel@uow.edu.au
Mark S. Clements
University of Western Sydney
See next page for additional authors
Follow this and additional works at: https://ro.uow.edu.au/infopapers
Part of the Physical Sciences and Mathematics Commons
Recommended Citation
Recommended Citation
Mealing, Nicole M.; Banks, Emily; Jorm, Louisa R.; Steel, David G.; Clements, Mark S.; and Rogers, Kris D.:
Investigation of relative risk estimates from studies of the same population with contrasting response
rates and designs 2010, 26-38. https://ro.uow.edu.au/infopapers/1470
Research Online is the open access institutional repository for the University of Wollongong. For further information
contact the UOW Library: research-pubs@uow.edu.au Faculty of Engineering and Information
Sciences Faculty of Informatics - Papers (Archive) y
p
(
)
2010
Investigation of relative risk estim
Investigation of relative risk estim
with contrasting response rates
with contrasting response rates
Nicole M. Mealing
University of Western Sydney
Emily Banks
Australian National University
Louisa R. Jorm
University of Western Sydney
David G. Steel
University of Wollongong, dsteel@uow.edu.au
Mark S. Clements
University of Western Sydney
See next page for additional authors
Follow this and additional works at: https://ro
Part of the Physical Sciences and Mathem
Recommended Citation
Recommended Citation
Mealing, Nicole M.; Banks, Emily; Jorm, Louis
Investigation of relative risk estimates from s
rates and designs 2010, 26-38. https://ro.uow.edu.au/infopapers/1470
Research Online is the open access institutional re
contact the UOW Library: research-pubs@uow.edu Investigation of relative risk estimates from studies of the same population
Investigation of relative risk estimates from studies of the same population
with contrasting response rates and designs
with contrasting response rates and designs Follow this and additional works at: https://ro.uow.edu.au/infopapers Follow this and additional works at: https://ro.uow.edu.au/infopapers Part of the Physical Sciences and Mathematics Commons Disciplines
Disciplines Physical Sciences and Mathematics Physical Sciences and Mathematics Recommended Citation
Recommended Citation Mealing, Nicole M.; Banks, Emily; Jorm, Louisa R.; Steel, David G.; Clements, Mark S.; and Rogers, Kris D.:
Investigation of relative risk estimates from studies of the same population with contrasting response
rates and designs 2010, 26-38. Research Online is the open access institutional repository for the University of Wollongong. For further information
contact the UOW Library: research-pubs@uow.edu.au Abstract
Abstract Background
Background: There is little empirical evidence regarding the generalisability of relative risk estimates from
studies which have relatively low response rates or are of limited representativeness. The aim of this
study was to investigate variation in exposure-outcome relationships in studies of the same population
with different response rates and designs by comparing estimates from the 45 and Up Study, a
population-based cohort study (self-administered postal questionnaire, response rate 18%), and the New
South Wales Population Health Survey (PHS) (computer-assisted telephone interview, response rate
~60%). Methods
Methods: Logistic regression analysis of questionnaire data from 45 and Up Study participants (n =
101,812) and 2006/ 2007 PHS participants (n = 14,796) was used to calculate prevalence estimates and
odds ratios (ORs) for comparable variables, adjusting for age, sex and remoteness. ORs were compared
using Wald tests modelling each study separately, with and without sampling weights. Results
Results: Prevalence of some outcomes (smoking, private health insurance, diabetes, hypertension,
asthma) varied between the two studies. For highly comparable questionnaire items, exposure-outcome
relationship patterns were almost identical between the studies and ORs for eight of the ten relationships
examined did not differ significantly. For questionnaire items that were only moderately comparable, the
nature of the observed relationships did not differ materially between the two studies, although many ORs
differed significantly. Conclusions
Conclusions: These findings show that for a broad range of risk factors, two studies of the same
population with varying response rate, sampling frame and mode of questionnaire administration yielded
consistent estimates of exposure-outcome relationships. However, ORs varied between the studies where
they did not use identical questionnaire items. Publication Details
Publication Details Mealing, N., Banks, E., Jorm, L., Steel, D., Clements, M. & Rogers, K. (2010). Investigation of relative risk
estimates from studies of the same population with contrasting response rates and designs. BMC
Medical Research Methodology, 10 26-38. Keywords
Keywords Investigation, relative, risk, estimates, from, studies, same, population, contrasting, response, rates,
designs Investigation, relative, risk, estimates, from, studies, same, population, contrasting, response, rates,
designs Disciplines
Disciplines
Physical Sciences and Mathematics BioMed Central
© 2010 Mealing et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. Abstract Background: There is little empirical evidence regarding the generalisability of relative risk estimates from studies
which have relatively low response rates or are of limited representativeness. The aim of this study was to investigate
variation in exposure-outcome relationships in studies of the same population with different response rates and
designs by comparing estimates from the 45 and Up Study, a population-based cohort study (self-administered postal
questionnaire, response rate 18%), and the New South Wales Population Health Survey (PHS) (computer-assisted
telephone interview, response rate ~60%). Methods: Logistic regression analysis of questionnaire data from 45 and Up Study participants (n = 101,812) and 2006/
2007 PHS participants (n = 14,796) was used to calculate prevalence estimates and odds ratios (ORs) for comparable
variables, adjusting for age, sex and remoteness. ORs were compared using Wald tests modelling each study separately,
with and without sampling weights. Results: Prevalence of some outcomes (smoking, private health insurance, diabetes, hypertension, asthma) varied
between the two studies. For highly comparable questionnaire items, exposure-outcome relationship patterns were
almost identical between the studies and ORs for eight of the ten relationships examined did not differ significantly. For
questionnaire items that were only moderately comparable, the nature of the observed relationships did not differ
materially between the two studies, although many ORs differed significantly. Conclusions: These findings show that for a broad range of risk factors, two studies of the same population with
varying response rate, sampling frame and mode of questionnaire administration yielded consistent estimates of
exposure-outcome relationships. However, ORs varied between the studies where they did not use identical
questionnaire items. Authors
Authors Nicole M. Mealing, Emily Banks, Louisa R. Jorm, David G. Steel, Mark S. Clements, and Kris D. Rogers Nicole M. Mealing, Emily Banks, Louisa R. Jorm, David G. Steel, Mark S. Clements, and Kris D. Rogers This journal article is available at Research Online: https://ro.uow.edu.au/infopapers/1470 Mealing et al. BMC Medical Research Methodology 2010, 10:26
http://www.biomedcentral.com/1471-2288/10/26 Open Access * Correspondence: Nicole.Mealing@saxinstitute.org.au
1 The Sax Institute, P.O. Box 123, Broadway NSW 2007, Australia
Full list of author information is available at the end of the article Mealing et al. BMC Medical Research Methodology 2010, 10:26
http://www.biomedcentral.com/1471-2288/10/26 (male or female), remoteness (major city, inner regional,
outer regional or remote) and age (five year age groups
from 45-85 years or ≥85 years). have relatively high response rates within these groups,
recent response rates to population-based cohort studies
are usually below 50% [8-12]. Furthermore, cohort study
participants are generally healthier and more health con-
scious than non-participants [3,13-16]. Concern is often
expressed at the low response rates for cohort studies, or
the selectiveness of the group under study, and the gener-
alisability of their results [17]. The 45 and Up Study The 45 and Up Study is a population-based cohort study
of more than 260,000 men and women aged 45 years and
over in New South Wales (NSW), Australia [10]. Partici-
pants were randomly selected from the database that is
used to administer the national universal health insur-
ance scheme (Medicare Australia), which has almost
complete coverage of the population. Equal numbers of
males and females were selected for participants less than
80 years old. Individuals aged 80 years or over and resi-
dents in rural areas were oversampled by a factor of two,
males aged 80 years or over were oversampled compared
to females and all residents in remote areas were com-
pletely enumerated. Participants entered the study by
completing a baseline postal questionnaire and providing
written consent to have their health followed over time. The
study
questionnaire
is
available
at
http://
www.45andUp.org.au. The survey was available only in
English. The current overall response rate to the baseline
questionnaire is estimated to be 17.9% [10]. The final ana-
lytic sample consisted of 44,851 men and 52,961 women
joining the study up to July 2008 after excluding 125
respondents who had a missing Accessibility Remoteness
Index of Australia (ARIA+) [19] score. The NSW Population Health Survey The NSW Population Health Survey (PHS) is an ongoing
survey on the health of people in NSW using computer
assisted telephone interviewing [21]. Independent sam-
ples of NSW households with private telephones are
drawn each year using random digit dialling, and one per-
son is randomly selected to participate in the survey. Informed consent was obtained from participants by
their willingness to complete the telephone interview. The survey questionnaire is available at http://
www.health.nsw.gov.au/publichealth/surveys/phs.asp. The survey is administered in 6 languages. In 2006 partic-
ipants were asked all survey questions and in 2007 they
were asked a random subset of the survey questions. We
report analyses of data for 5,766 men and 9,030 women
aged 45 years or over who responded to the 2006 (n =
5,480) or 2007 (n = 9,316) PHS, with response rates of
59.3% [22] and 63.6% [23] respectively. Direct empirical data to support the assumption that
internal comparisons remain reliable, despite low
response rates or highly selected study groups, is lacking. Furthermore, concerns are also expressed that elements
of study design, such as sampling methods and use of
postal questionnaires versus interviews, may influence
the observed relationships [18]. This paper investigates
whether or not cross-sectional estimates of exposure-
outcome relationships are affected by survey aspects
(response rate, sampling frame and mode of question-
naire administration) or the wording of questionnaire
items, by comparing estimates computed from two inde-
pendent studies of the same target population with diver-
gent response rates and different designs. Weights were assigned to each year of data to adjust for
the differences in the probability of selection within the
household, number of residential telephone connections
to the house and the varying sampling fraction between
each of the 8 NSW area health services to provide esti-
mates that were representative of the NSW population
[21]. These area health services can include several
remoteness categories. Post-stratification weights were
also assigned according to sex (male or female) and age
(five year age groups from 45-85 years or 85-110 years)
using 2005 and 2007 mid-year population statistics
released by the ABS for each area health service [22,23]. After weighting, Indigenous people are slightly under-
represented in the PHS sample, and Australian-born peo-
ple slightly over-represented, compared to the overall
NSW population [22,23]. Background sarily required for reliable estimates of relative risk based
on internal comparisons within study populations [3-5]. Indeed, having a greater proportion of respondents in
extreme categories compared to the population of inter-
est may often be necessary, in order to yield sufficient
information about specific exposure-outcome relation-
ships [5]. A key issue is whether there is any nonresponse
bias after conditioning on the covariates included in the
analysis. The aim of most epidemiological studies is to obtain esti-
mates that can be generalised to a population of interest. For surveys concerned with disease prevalence, the main
means to achieve this is to draw a sample that is suffi-
ciently representative of the target population. However,
few surveys have perfect response rates and any level of
nonresponse can potentially lead to biased estimates of
prevalence [1,2]. Cohort studies generally require more extensive data
collection than one-off surveys, as well as the provision of
identifying details and a long-term commitment to fol-
low-up. While cohort studies often focus on selected
population groups (e.g. occupational groups) [6,7] and In contrast, much epidemiological practice is based
around the principle that representativeness is not neces- * Correspondence: Nicole.Mealing@saxinstitute.org.au
1 The Sax Institute, P.O. Box 123, Broadway NSW 2007, Australia
Full list of author information is available at the end of the article Page 2 of 12 Mealing et al. BMC Medical Research Methodology 2010, 10:26
http://www.biomedcentral.com/1471-2288/10/26 Mealing et al. BMC Medical Research Methodology 2010, 10:26
http://www.biomedcentral.com/1471-2288/10/26 Analysis Before analyses commenced, twenty exposure-outcome
pairs were selected for inclusion in our analyses. These
were selected on the basis of demonstrating relationships
across a wide range of domains of research interest. This
consisted of i) ten pairs where both the exposure and the
outcome variables were highly comparable across the two
studies; and ii) ten pairs where the exposure and/or out-
come variables were only moderately comparable across
the two studies. Participants were classified as having hypertension,
diabetes and/or asthma if they reported that these condi-
tions had ever been diagnosed by a doctor (both studies)
or at a hospital (PHS only). Only participants who
answered version two of the 45 and Up baseline question-
naire were asked if they had ever been diagnosed with
asthma (n = 65,522). Unweighted and survey weighted prevalence estimates
with 95% confidence intervals (CI) were calculated for
each study for all highly and moderately comparable vari-
ables used in the exposure-outcome relationship analy-
ses. Odds ratios (ORs) were used to approximate relative
risks and logistic regression analyses were used to calcu-
late the 20 pre-determined exposure-outcome relation-
ships for each study; separated into highly and
moderately comparable ORs. In each case two sets of
ORs were calculated; namely the crude OR and the OR
adjusting for age, sex and remoteness since these were the
sampling variables common to both surveys. Unweighted
and weighted comparisons of these two types of ORs by
study are presented in Additional files 2 and 3 respec-
tively (45 and Up Study) and Additional files 4 and 5
respectively (PHS). Indicator variables were constructed for being born in
Australia, missing all natural teeth, speaking a language
other than English at home, having fallen in the past 12
months, having private health insurance (excluding
Medicare) and having a hysterectomy. Daily fruit consumption was grouped into participants
who don't eat fruit, participants who eat fruit but less
than two serves per day, and participants who eat two or
more serves per day. An indicator for females who were breast screened in
the past two years was ascertained from responses to ever
having a mammogram and the year of (45 and Up Study)
or time interval (PHS) since their last mammogram. Psychological distress was evaluated using the Kessler
(K10) measure [26] ascertained as the sum of responses
for 10 questions. Moderately comparable questionnaire items The wording of questions across the two studies differed
for household income before tax (45 and Up Study
included benefits, pensions and superannuation), bowel
screening (screening tests varied by study) and current
smoking status (45 and Up Study participants recorded
whether they were 'regular smokers' currently without a
definition for regular, whereas PHS participants recorded
if they 'smoke daily' where smoking was defined to
include cigarettes, cigars and pipes). Questions on mammography screening and hysterec-
tomy were only asked in the 2006 PHS and hypertension
and bowel screening in the 2007 PHS. Highly comparable questionnaire items The response categories varied across the two studies
for highest level of educational attainment (for these
analyses similar categories were constructed) and self-
rated health status (the PHS had an additional response
category - for these analyses the categories "poor" and
"very poor" on the PHS were combined). Remoteness was determined using the mean ARIA+
score for the postcode of the participant's residential
address and categorised as major city, inner regional,
outer regional or remote, according to the Australian
Institute of Health and Welfare [24]. Self reported height and weight were used to calculate
participants' body mass index (BMI) as weight in kilo-
grams divided by the square of height in meters. BMI was
categorised as underweight (BMI < 18.5 kg/m2), normal
range (BMI 18.5-24.9 kg/m2), overweight (BMI 25.0-29.9
kg/m2) or obese (BMI ≥ 30 kg/m2) according to the World
Health Organisation [25]. Mealing et al. BMC Medical Research Methodology 2010, 10:26
http://www.biomedcentral.com/1471-2288/10/26 Page 3 of 12 those with a score less than 22 as having low/moderate
levels of psychological distress [27]. of participants a question of interest then the same
restriction was applied to the other study. Data are
reported on falls in the past 12 months for participants
aged 60 years and over, hysterectomy operation in
females less than 70 years, mammography screening in
the past two years for females less than 80 years and
bowel screening in the past 5 years for all persons aged 50
years and over. Questionnaire items We obtained the original questionnaires from the 45 and
Up Study and the 2006 and 2007 PHS and compared the
wording of questions and response categories. We classi-
fied questionnaire items as highly comparable, moder-
ately comparable or not comparable, based on whether
the item was expected to yield identical, similar or non-
comparable responses, respectively, for a given individ-
ual. Analyses focused on items considered highly or mod-
erately comparable; items used in these analyses are
compared in Additional file 1. All variables used in these
analyses were derived from self-reported data except
postcode (45 and Up Study only). Post-stratification estimation weights were assigned to
the 45 and Up baseline survey to adjust the sample to
account for the differences in selection probabilities and
response rates and give consistency with 2006 population
estimates produced by the Australian Bureau of Statistics
(ABS) [20]. The post-strata were formed according to sex All analyses included all participants in both studies,
unless otherwise stated. If one study only asked a sub-set Mealing et al. BMC Medical Research Methodology 2010, 10:26
http://www.biomedcentral.com/1471-2288/10/26 Mealing et al. BMC Medical Research Methodology 2010, 10:26
http://www.biomedcentral.com/1471-2288/10/26 Analysis If a respondent answered nine of the 10
items then the missing item was imputed as the average
of the other nine responses. If a respondent answered less
than nine items their K10 score was set to missing. Partic-
ipants with a K10 score of 22 or greater were assigned as
having high/very high levels of psychological distress and In the figures, the squares and lines represent each OR
estimate and CI, with the area of each square being pro-
portional to the sample size used for each estimate. Wald chi-square statistics were computed to test for
differences in the log odds ratios between the two surveys
for each of the 20 exposure-outcome pairs and compared
to a chi-square distribution with degrees of freedom
equal to the number of categories minus one in the expo- Page 4 of 12 Mealing et al. BMC Medical Research Methodology 2010, 10:26
http://www.biomedcentral.com/1471-2288/10/26 Mealing et al. BMC Medical Research Methodology 2010, 10:26
http://www.biomedcentral.com/1471-2288/10/26 sure variable. Each study was modelled separately and
then the Wald statistics were calculated by combining the
two sets of estimated parameters, variances and covari-
ances. Analyses were conducted with and without using
sampling weights. With survey data the Wald test can be
unreliable if the degrees of freedom on the estimated
covariance matrix are small [28]. In this case the samples
were large and the designs relatively simple. distress was observed (Wald test P = 0.02). Similar obser-
vations were seen when the ORs from these ten relation-
ships were calculated using sampling weights (Additional
file 6). The ten exposure-outcome relationships where the
exposure and/or outcome variables were only moderately
comparable across the two studies are presented in Figure
2, with ORs adjusted for age, sex and remoteness. Each
exposure-outcome pair had a similar relationship pattern
for both studies and all OR estimates were in the same
direction and of similar magnitude, except when self-
rated health status was the exposure variable. The rela-
tionships did not differ significantly for 4 out of 10 of the
exposure-outcome associations. Significant but relatively
minor differences in ORs were observed for smoking and
educational attainment and pre-tax income in relation to
psychological distress, private health insurance and
remoteness of residence. Results The distributions of social and demographic characteris-
tics and of health risk factors and conditions in the two
studies are shown in Tables 1 and 2, respectively. Younger
persons and/or those living in major cities were under-
represented in both surveys as were males in the PHS
(the 45 and Up Study sample was stratified by sex). The
prevalence confidence intervals were narrower on the 45
and Up Study compared to the PHS, because of the larger
sample size. The weighted estimates of prevalence were similar
across the two studies for variables such as age, sex, and
remoteness (the variables used for weighting), country of
birth, educational attainment, fruit consumption, body-
mass-index and falls. However, the prevalence of speak-
ing a language other than English at home and of holding
private health insurance was higher in the 45 and Up
Study compared to the PHS, while the prevalence of
smoking, high/very high psychological distress, ever
diagnosed with hypertension, ever diagnosed with diabe-
tes and ever diagnosed with asthma was lower (Table 1;
Table 2). The PHS tended to have less missing data than
the 45 and Up Study, particularly for variables relating to
mammography screening, K10 score and household
income before tax. Prevalence estimates for self-rated
health status varied across the two studies with the pro-
portion who reported the lowest category of self-rated
health status on the 45 and Up baseline questionnaire (i.e. "poor") being similar to the proportion who reported the
lowest category on the PHS (i.e. "very poor")(Table 2). Following adjustment for age, sex and remoteness,
additional weighting of the OR for age, sex and remote-
ness did not change any of the ORs from the 45 and Up
Study materially (i.e. no changes were >10%) (Additional
files 2, 3). This is because the weighting is determined by
the variables used in the logistic regression. Weighting
the PHS resulted in some changes to the ORs because not
all variables used to determine the weighting (i.e. house-
hold size to account for the selection of a person from
each selected household and the 8 area health services)
were used in the logistic regression (Additional files 4, 5). Weighting did not change the general nature of the
observed relationships. Analysis In spite of the similarity in the
shape of the relationship, substantial heterogeneity and
large differences in ORs were observed for relationships
with self-rated health (where the PHS had an additional
response category, "very poor"). Similar observations
were seen when the ORs from these ten relationships
were calculated using sampling weights (Additional file
7). Analyses were carried out using SAS, version 9 [29]. This study has the approval of the University of New
South Wales Ethics Committee and the NSW Population
and Health Services Research Ethics Committee. Discussion Discussions around epidemiological methods often con-
clude that representativeness is not necessarily required
for reliable estimates of relative risk based on internal
comparisons within study populations [4]. By their
nature, cohort studies tend not to be directly representa-
tive of the general population, however over time, their
results have usually been shown to be both reproducible
and generalisable to the larger population [6,7]. Miettinen
explains that "an empirical relation is not distorted by any
manipulation of the distribution of the study base accord-
ing to the elements in the occurrence relation - the deter-
minant, the modifiers and/or confounders. For example,
the empirical relation of body weight to gender does not The ten exposure-outcome relationships where both
the exposure and the outcome variables were highly com-
parable across the two studies are presented in Figure 1,
with ORs adjusted for age, sex and remoteness. The
observed relationships were virtually identical between
the two studies. For 8 out of the 10 relationships there
was no significant difference between the results from the
different studies. There was borderline evidence of a dif-
ference in the risk of falling according to BMI across the
two studies (Wald test P = 0.04) and minor heterogeneity
in the relationship of age to high/very high psychological Mealing et al. depend on the gender distribution in the study base." [[5],
p. 56] depend on the gender distribution in the study base." [[5],
p. 56] pleted the survey in languages other than English. It was
not possible to definitively separate the individual effects
of sampling frame, response rate and mode of adminis-
tration, since response rates and aspects of study design
are closely linked [33]. It is generally accepted that in order to produce results
that are generalisable, studies should exhibit sufficient
variability in the determinant and modifiers to be studied
and a limited range for confounders [4,5]. Nevertheless
the possibility of bias cannot be excluded, and empirical
data on how exposure-outcome relationships might vary
according to the degree of nonresponse are lacking. We were unable to locate other empirical comparisons
of relative risk estimates in independent studies with
divergent response rates and different study designs that
were drawn from the same target population. Indirect
evidence supporting our findings comes from studies that
have observed consistent ORs in study respondents and
non-respondents using linked data [34] and in initial
cohort study participants and participants responding to
a subsequent questionnaire [35]. Two studies found only
small biases in relative risk estimates due to nonresponse,
in cross-sectional ORs from a cohort study relating to
cardiovascular disease [31], and in cohort analyses relat-
ing to reproductive outcomes [12]. One study found con-
sistent ORs related to smoking in respondents recruited
by postal survey and those recruited through postal and
telephone surveys and home visits [18]. Nonresponse is a form of self-selection. Selection solely
by the exposure or outcome variable does not bias the
estimates of ORs in logistic regression [14,30,31] and
selection solely on the basis of covariates in the logistic
regression also leads to unbiased ORs. Although evidence
from simulation supports the principle of generalisability
[12], specific scenarios may result in significant bias if
selection criteria and dependent variables are closely
related [32]. In particular, biases can occur if selection
depends on both the exposure and outcome [14,17]. We found that although some prevalence estimates var-
ied between the two studies of the same population inves-
tigated here, exposure-outcome relationships did not
differ materially, where the variables used were highly
comparable. Discussion BMC Medical Research Methodology 2010, 10:26
http://www.biomedcentral.com/1471-2288/10/26 Page 5 of 12 Table 1: Social and demographic characteristics of the 45 and Up Study and the NSW PHS populations
45 and Up Study
PHS
Social and Demographics
Characteristics a
N
Crude %
(95% CI)
Weighted b
% (95% CI)
N
Crude %
(95% CI)
Weighted c
% (95% CI)
Sex
Male
48851
48.0 (47.7, 48.3)
48.0 (47.6, 48.4)
5766
39.0 (38.2, 39.8)
48.4 (47.4, 49.5)
Female
52961
52.0 (51.7, 52.3)
52.0 (51.6, 52.4)
9030
61.0 (60.2, 61.8)
51.6 (50.5, 52.6)
Age
45-49 years
12399
12.2 (12.0, 12.4)
18.9 (18.6, 19.2)
1803
12.2 (11.7, 12.7)
19.3 (18.3, 20.3)
50-54 years
16120
15.8 (15.6, 16.1)
17.1 (16.8, 17.4)
2043
13.8 (13.3, 14.4)
17.5 (16.6, 18.4)
55-59 years
17350
17.0 (16.8, 17.3)
15.9 (15.7, 16.2)
2188
14.8 (14.2, 15.4)
16.2 (15.4, 17.0)
60-64 years
15342
15.1 (14.8, 15.3)
12.6 (12.4, 12.8)
2252
15.2 (14.6, 15.8)
12.5 (11.9, 13.1)
65-69 years
12800
12.6 (12.4, 12.8)
10.1 (9.9, 10.3)
1939
13.1 (12.6, 13.6)
10.2 (9.6, 10.8)
70-74 years
9564
9.4 (9.2, 9.6)
8.3 (8.1, 8.5)
1710
11.6 (11.0, 12.1)
8.5 (8.0, 8.9)
75-79 years
7235
7.1 (6.9, 7.3)
7.3 (7.2, 7.5)
1490
10.1 (9.6, 10.6)
7.3 (6.9, 7.8)
80-84 years
7545
7.4 (7.2, 7.6)
5.4 (5.3, 5.6)
922
6.2 (5.8, 6.6)
5.9 (5.4, 6.3)
≥ 85 years
3457
3.4 (3.3, 3.5)
4.3 (4.1, 4.4)
449
3.0 (2.8, 3.3)
2.6 (2.3, 2.9)
Remoteness (ARIA+)
Major City
44146
43.4 (43.1, 43.7)
69.1 (68.8, 69.4)
6718
45.4 (44.6, 46.2)
60.6 (59.6, 61.6)
Inner Regional
36640
36.0 (35.7, 36.3)
22.7 (22.5, 23.0)
4351
29.4 (28.7, 30.1)
24.4 (23.5, 25.2)
Outer Regional
18926
18.6 (18.4, 18.8)
7.6 (7.5, 7.7)
3086
20.9 (20.2, 21.5)
12.2 (11.6, 12.7)
Remote
2100
2.1 (2.0, 2.1)
0.6 (0.5, 0.6)
456
3.1 (2.8, 3.4)
1.3 (1.2, 1.5)
Missing
0
-
-
185
1.3 (1.1, 1.4)
1.5 (1.2, 1.8)
Language other than English spoken at home
No
92230
90.6 (90.4, 90.8)
87.4 (87.1, 87.7)
13379
93.0 (92.6, 93.4)
89.5 (88.7, 90.3)
Yes
9580
9.4 (9.2, 9.6)
12.6 (12.3, 12.9)
986
6.9 (6.4, 7.3)
10.4 (9.6, 11.2)
Missing
2
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
17
0.1 (0.0, 0.2)
0.1 (0.0, 0.2)
Country of Birth
Australia
75821
74.5 (74.2, 74.7)
70.8 (70.5, 71.1)
11332
76.6 (75.9, 77.3)
71.1 (70.1, 72.2)
Not Australia
24964
24.5 (24.3, 24.8)
28.2 (27.9, 28.6)
3433
23.2 (22.5, 23.9)
28.7 (27.7, 29.7)
Missing
1027
1.0 (0.9, 1.1)
1.0 (0.9, 1.0)
24
0.2 (0.1, 0.2)
0.2 (0.1, 0.2)
Private Health Insurance
No
38300
37.6 (37.3, 37.9)
33.9 (33.6, 34.3)
6766
45.7 (44.9, 46.5)
41.5 (40.4, 42.5)
Yes
63508
62.4 (62.1, 62.7)
66.1 (65.7, 66.4)
7973
53.9 (53.1, 54.7)
58.2 (57.1, 59.3)
Missing
4
0.0 (0.0, 0.0)
0.0 (0.0, 0.0)
57
0.4 (0.3, 0.5)
0.3 (0.2, 0.4) ble 1: Social and demographic characteristics of the 45 and Up Study and the NSW PHS populations Mealing et al. Discussion BMC Medical Research Methodology 2010, 10:26
http://www.biomedcentral.com/1471-2288/10/26 Page 6 of 12 Educational Attainment
No School Certificate
12385
12.2 (12.0, 12.4)
10.9 (10.7, 11.1)
2017
13.7 (13.1, 14.2)
11.7 (11.1, 12.3)
School Certificate
22608
22.2 (22.0, 22.5)
20.3 (20.0, 20.6)
3732
25.3 (24.6, 26.0)
20.6 (19.8, 21.4)
Trade/Certificate/Diploma
32289
31.7 (31.4, 32.0)
31.0 (30.6, 31.3)
1631
26.5 (25.8, 27.2)
29.5 (28.5, 30.5)
Higher School Certificate
9787
9.6 (9.4, 9.8)
10.1 (9.9, 10.3)
3902
11.1 (10.6, 11.6)
10.9 (10.2, 11.6)
Tertiary Qualification
22802
22.4 (22.1, 22.7)
25.9 (25.6, 26.3)
3096
21.0 (20.3, 21.7)
25.0 (24.0, 26.0)
Missing
1941
1.9 (1.8, 2.0)
1.8 (1.8, 1.9)
360
2.4 (2.2, 2.7)
2.3 (2.0, 2.6)
Household Income
<$20,000 p.a
20633
20.3 (20.0, 20.5)
17.9 (17.6, 18.1)
4505
30.4 (29.7, 31.2)
22.2 (21.4, 22.9)
$20,000-$39,999 p.a
18386
18.1 (17.8, 18.3)
15.3 (15.1, 15.6)
3145
21.3 (20.6, 21.9)
20.3 (19.5, 21.1)
≥$40,000 p.a
39792
39.1 (38.8, 39.4)
44.6 (44.2, 44.9)
4768
32.2 (31.5, 33.0)
41.4 (40.3, 42.5)
Missing
23001
22.6 (22.3, 22.8)
22.2 (21.9, 22.5)
2378
16.1 (15.5, 16.7)
16.2 (15.4, 17.0)
ARIA+, Accessibility Remoteness Index of Australia; CI, Confidence Interval; p.a, per annum
a Characteristics above the line-break are highly comparable across the two surveys. Characteristics below the line-break are moderately
comparable across the two surveys. Weighted prevalence estimates for age, sex and remoteness differ across the two studies since the
weighting schemes vary. b Weighted by age, sex and remoteness
c Weighted by area health service, probability of selection in the household and the number of residential connections to the house
Table 1: Social and demographic characteristics of the 45 and Up Study and the NSW PHS populations (Continued) nd demographic characteristics of the 45 and Up Study and the NSW PHS populations (Continued) Table 1: Social and demographic characteristics of the 45 and Up Study and the NSW PHS popula depend on the gender distribution in the study base." [[5],
p. 56] This was despite major differences between
the studies, including varying response rates, sampling
frames and modes of administration; the PHS had a
smaller proportion of missing and invalid responses due
to the nature of the computer assisted telephone inter-
viewing system and it included respondents who com- Having established the lack of any major differences
attributable to response rate and study design (including
sampling frame and mode of questionnaire administra-
tion), the comparison of exposure-outcome relationships
containing moderately comparable variables across the
two studies can be seen as illustrating the additional
effect of the specific questionnaire items used. Our find-
ings demonstrate that an apparently minor difference in Mealing et al. depend on the gender distribution in the study base." [[5],
p. 56] BMC Medical Research Methodology 2010, 10:26
http://www.biomedcentral.com/1471-2288/10/26 Page 8 of 12 Last Mammography Screening
Not within past 2 years
12622
26.2 (25.9, 26.6)
28.6 (28.1, 29.1)
1018
34.4 (32.7, 36.1)
36.7 (34.5, 39.0)
Within past 2 years
30056
62.5 (62.1, 62.9)
60.1 (59.6, 60.6)
1925
65.1 (63.4, 66.8)
62.8 (60.6, 65.1)
Missing
5413
11.3 (11.0, 11.5)
11.3 (11.0, 11.7)
15
0.5 (0.3, 0.8)
0.4 (0.2, 0.7)
Hysterectomy
No
29487
73.9 (73.4, 74.3)
76.6 (76.1, 77.0)
1583
71.1 (69.2, 73.0)
74.2 (72.0, 76.4)
Yes
10431
26.1 (25.7, 26.6)
23.4 (23.0, 23.9)
630
28.3 (26.4, 30.2)
25.0 (22.8, 27.2)
Missing
0
-
-
13
0.6 (0.3, 0.9)
0.8 (0.3, 1.3)
Psychological Distress
Low\Moderate
81542
80.1 (79.8, 80.3)
80.7 (80.4, 81.0)
9503
88.8 (88.2, 89.4)
88.9 (88.1, 89.7)
High\Very high
6663
6.5 (6.4, 6.7)
6.8 (6.6, 7.0)
1101
10.3 (9.7, 10.9)
10.2 (9.4, 11.0)
Missing
13607
13.4 (13.2, 13.6)
12.5 (12.2, 12.7)
100
0.9 (0.8, 1.1)
0.9 (0.7, 1.2)
Current Smoker
No
93685
92.0 (91.9, 92.2)
91.9 (91.7, 92.1)
9468
87.9 (87.3, 88.6)
88.1 (87.3, 89.0)
Yes
7575
7.4 (7.3, 7.6)
7.6 (7.4, 7.8)
1292
12.0 (11.4, 12.6)
11.8 (11.0, 12.7)
Missing
552
0.5 (0.5, 0.6)
0.5 (0.5, 0.6)
7
0.1 (0.0, 0.1)
0.1 (0.0, 0.1)
Last Bowel Screening
Not within past 5 years
49036
54.8 (54.5, 55.2)
55.9 (55.6, 56.3)
2497
53.5 (52.1, 54.9)
54.6 (52.7, 56.4)
Within past 5 years
35990
40.3 (39.9, 40.6)
39.2 (38.8, 39.6)
2098
45.0 (43.5, 46.4)
43.3 (41.5, 56.4)
Missing
4387
4.9 (4.8, 5.0)
4.9 (4.7, 5.0)
72
1.5 (1.2, 1.9)
2.2 (1.4, 2.9)
Self-reported Health Status
Excellent
14920
14.7 (14.4, 14.9)
15.5 (15.2, 15.8)
2433
17.8 (17.2, 18.5)
18.1 (17.2, 18.9)
Very Good
36046
35.4 (35.1, 35.7)
35.4 (35.0, 35.7)
3815
28.0 (27.2, 28.7)
28.5 (27.5, 29.5)
Good
33178
32.6 (32.3, 32.9)
32.1 (31.7, 32.4)
3990
29.2 (28.5, 30.0)
29.8 (28.7, 30.8)
Fair
11878
11.7 (11.5, 11.9)
11.4 (11.2, 11.6)
2265
16.6 (16.0, 17.2)
16.0 (15.2, 16.8)
Poor
2090
2.1 (2.0, 2.1)
2.0 (1.9, 2.1)
863
6.3 (5.9, 6.7)
5.8 (5.3, 6.3)
Very Poor d
N/A
239
1.8 (1.5, 2.0)
1.6 (1.3, 1.9)
Missing
3700
3.6 (3.5, 3.7)
3.7 (3.5, 3.8)
44
0.3 (0.2, 0.4)
0.3 (0.2, 0.4)
CI, Confidence Interval; NSW, New South Wales;
a Variables above the line-break are highly comparable across the two surveys. depend on the gender distribution in the study base." [[5],
p. 56] BMC Medical Research Methodology 2010, 10:26
http://www.biomedcentral.com/1471-2288/10/26 Page 7 of 12 Table 2: Health risk factors and conditions of the 45 and Up Study and the NSW PHS populations
45 and Up Study
PHS
Variable a
N
Crude %
(95% CI)
Weighted b
% (95% CI)
N
Crude %
(95% CI)
Weighted c
% (95% CI)
Fruit Consumption
Don't eat fruit
6620
6.5 (6.4, 6.7)
6.5 (6.4, 6.7)
321
6.1 (5.5, 6.8)
6.1 (5.2, 6.9)
< 2 serves per day
31707
31.1 (30.9, 31.4)
31.0 (30.7, 31.4)
1719
32.8 (31.6, 34.1)
33.4 (31.8, 35.1)
≥ 2 serves per day
57620
56.6 (56.3, 56.9)
56.7 (56.3, 57.0)
3135
59.9 (58.5, 61.2)
59.4 (57.7, 61.2)
Missing
5865
5.8 (5.6, 5.9)
5.7 (5.6, 5.9)
61
1.2 (0.9, 1.5)
1.0 (0.7, 1.3)
Teeth
Some/all natural teeth
88501
86.9 (86.7, 87.1)
88.0 (87.8, 88.2)
9321
86.7 (86.0, 87.3)
90.3 (89.7, 90.9)
No natural teeth
9953
9.8 (9.6, 10.0)
8.7 (8.5, 8.9)
1427
13.3 (12.6, 13.9)
9.7 (9.1, 10.3)
Missing
3358
3.3 (3.2, 3.4)
3.3 (3.2, 3.5)
6
0.1 (0.0, 0.1)
0.0 (0.0, 0.1)
Body Mass Index
Underweight
1461
1.4 (1.4, 1.5)
1.5 (1.4, 1.6)
230
2.1 (1.9, 2.4)
1.9 (1.6, 2.3)
Normal Range
35219
34.6 (34.3, 34.9)
35.6 (35.2, 35.9)
4214
39.0 (38.1, 40.0)
38.4 (37.2, 39.6)
Overweight
37373
36.7 (36.4, 37.0)
36.6 (36.2, 36.9)
3774
35.0 (34.1, 35.9)
36.0 (34.8, 37.2)
Obese
20271
19.9 (19.7, 20.2)
19.2 (18.9, 19.5)
2205
20.4 (19.7, 21.2)
20.5 (19.4, 21.5)
Missing
7488
7.4 (7.2, 7.5)
7.1 (6.9, 7.3)
373
3.5 (3.1, 3.8)
3.2 (2.8, 3.7)
Ever diagnosed with hypertension
No
72023
70.7 (70.5, 71.0)
72.1 (71.7, 72.4)
1189
52.1 (50.1, 54.2)
55.2 (52.6, 57.8)
Yes
25144
24.7 (24.4, 25.0)
22.9 (22.6, 23.2)
1086
47.6 (45.6, 49.7)
44.6 (42.0, 47.2)
Missing
4645
4.6 (4.4, 4.7)
5.0 (4.9, 5.2)
6
0.3 (0.1, 0.5)
0.2 (0.0, 0.4)
Ever diagnosed with diabetes
No
88056
86.5 (86.3, 86.7)
86.6 (86.3, 86.8)
9450
88.4 (87.8, 89.0)
89.2 (88.4, 90.0)
Yes
9111
8.9 (8.8, 9.1)
8.4 (8.2, 8.6)
1202
11.2 (10.6, 11.8)
10.5 (9.7, 11.2)
Missing
4645
4.6 (4.4, 4.7)
5.0 (4.9, 5.2)
36
0.3 (0.2, 0.4)
0.3 (0.2, 0.4)
Ever diagnosed with asthma
No
55034
84.0 (83.7, 84.3)
83.8 (83.5, 84.1)
8753
82.0 (81.3, 82.8)
83.2 (82.2, 84.1)
Yes
7653
11.7 (11.4, 11.9)
11.4 (11.1, 11.7)
1894
17.8 (17.0, 18.5)
16.7 (15.8, 17.6)
Missing
2835
4.3 (4.2, 4.5)
4.8 (4.6, 5.0)
22
0.2 (0.1, 0.3)
0.1 (0.1, 0.2)
Fallen in the past 12 months
No
42038
75.1 (74.8, 75.5)
74.1 (73.7, 74.6)
2957
77.3 (76.0, 78.7)
77.5 (75.9, 79.1)
Yes
10757
19.2 (18.9, 19.6)
20.0 (19.6, 20.4)
856
22.4 (21.1, 23.7)
22.1 (20.6, 23.7)
Missing
49017
5.6 (5.4, 5.8)
5.8 (5.6, 6.1)
11
0.3 (0.1, 0.5)
0.4 (0.1, 0.6) Table 2: Health risk factors and conditions of the 45 and Up Study and the NSW PHS populations Mealing et al. b Weighted by age, sex and remoteness
c Weighted by area health service, probability of selection in the household and the number of residential connections to the house
d The category 'Very Poor' was only available on the NSW Population Health Survey bove the line-break are highly comparable across the two surveys. Variables below the line-break are moderately compara
ss the two surveys
b
d depend on the gender distribution in the study base." [[5],
p. 56] Variables below the line-break are moderately comparable but
similar across the two surveys
b Weighted by age, sex and remoteness
c W i ht d b
h
lth
i
b bilit
f
l
ti
i th h
h ld
d th
b
f
id
ti l
ti
t th h
Table 2: Health risk factors and conditions of the 45 and Up Study and the NSW PHS populations (Continued) lth risk factors and conditions of the 45 and Up Study and the NSW PHS populations (Continued) Table 2: Health risk factors and conditions of the 45 and Up Study and the NSW PHS popu Mealing et al. BMC Medical Research Methodology 2010, 10:26
http://www.biomedcentral.com/1471-2288/10/26 Mealing et al. BMC Medical Research Methodology 2010, 10:26
http://www.biomedcentral.com/1471-2288/10/26 Page 9 of 12 Figure 1 Odds ratios a by study where the exposure and outcome variables were highly comparable across the 45 and Up Study and the
NSW PHS b. ARIA+, Accessibility Remoteness Index of Australia; CI, Confidence Interval; NSW, New South Wales; PHS, Population Health Survey. a Ad-
justing for age, sex and remoteness.b Black squares represent ORs with area inversely proportional to the sample size contributing to the OR and the
corresponding line represents the 95% confidence interval. c P-value from Wald chi-square test: testing for a difference between the two studies for
the specific exposure-outcome pair and outcome variables were highly comparable across the 45 and Up Study and the
Australia; CI, Confidence Interval; NSW, New South Wales; PHS, Population Health Survey. a Ad-
esent ORs with area inversely proportional to the sample size contributing to the OR and the
rval. c P-value from Wald chi-square test: testing for a difference between the two studies for Figure 1 Odds ratios a by study where the exposure and outcome variables were highly comparable across the 45 and Up Study and the
NSW PHS b. ARIA+, Accessibility Remoteness Index of Australia; CI, Confidence Interval; NSW, New South Wales; PHS, Population Health Survey. a Ad-
justing for age, sex and remoteness.b Black squares represent ORs with area inversely proportional to the sample size contributing to the OR and the
corresponding line represents the 95% confidence interval. c P-value from Wald chi-square test: testing for a difference between the two studies for
the specific exposure-outcome pair the wording of questions can significantly influence mea-
sures of prevalence and estimates of risks. depend on the gender distribution in the study base." [[5],
p. 56] This empha-
sizes the critical importance of maintaining the
consistency of survey questions if valid comparisons are
to be made and is consistent with previous studies [36-
38]. Although most differences attributable to question
wording resulted in minor heterogeneity, highly signifi-
cant heterogeneity was evident for the question on self-
rated health status, where the response categories varied
across the two studies. However, despite differences
between questionnaire items, the observed ORs would
lead to similar conclusions regarding the nature of the
exposure-outcome relationships. bias. Under ideal circumstances we could use census
data; however the Australian Census includes only very
limited health data. Additionally, these findings relate to
two large studies with considerable variability in the fac-
tors included in our analyses. This ensured that there
were substantial numbers of participants from each study
in each exposure-outcome category, and allowed for
adjustment for multiple factors. Although these findings
support the principle of generalisability of findings from a
relatively select group of participants, it remains possible
that they are less applicable to smaller, less heterogeneous
studies. These findings relate to cross-sectional analyses;
prospective, longitudinal analyses are less prone to the
potential biases investigated here, since baseline selection
cannot be influenced by outcome status. One shortcoming of our study is the lack of strict gold-
standard measures for the study variables. The PHS has a
40% nonresponse rate and may be subject to nonresponse Mealing et al. BMC Medical Research Methodology 2010, 10:26
http://www.biomedcentral.com/1471-2288/10/26 Page 10 of 12 Figure 2 Odds Ratios a by study where either the exposure or outcome or both variables were only moderately comparable across the 45
and Up Study and the NSW PHS b. ARIA+, Accessibility Remoteness Index of Australia; CI, Confidence Interval; NSW, New South Wales; p.a, per an-
num; PHS, Population Health Survey. a Adjusting for age, sex and remoteness. b Black squares represent ORs with area inversely proportional to the
sample size contributing to the OR and the corresponding line represents the 95% confidence interval. c P-value from Wald chi-square test: testing for
a difference between the two studies for the specific exposure-outcome pair Figure 2 Odds Ratios a by study where either the exposure or outcome or both variables were only moderately comparable across the 45
and Up Study and the NSW PHS b. depend on the gender distribution in the study base." [[5],
p. 56] ARIA+, Accessibility Remoteness Index of Australia; CI, Confidence Interval; NSW, New South Wales; p.a, per an-
num; PHS, Population Health Survey. a Adjusting for age, sex and remoteness. b Black squares represent ORs with area inversely proportional to the
sample size contributing to the OR and the corresponding line represents the 95% confidence interval. c P-value from Wald chi-square test: testing for
a difference between the two studies for the specific exposure-outcome pair Applying weights to survey data to calculate prevalence
estimates that account for the differences in probability of
selection is standard practice. However, use of sampling
weights is less common when calculating relative risks
from cohort study data; instead adjustments are usually
made to account for potential confounders. The relative
risk estimates adjusting for age, sex and remoteness from
the 45 and Up Study were not altered materially by fur-
ther weighting. Hence weighting did not appear to be
necessary when the variables used in calculating the
weights were used as covariates in the analysis. Weighting
is potentially important in the PHS because of the role of
household size and area health service in the weighting. Conclusions These findings show that broad ranges of exposure-out-
come relationships estimated from two studies of the
same population remained consistent regardless of the
underlying response rate or mode of questionnaire
administration. They provide empirical support for the
basic epidemiological principle that results based on
internal comparisons remain generalisable even when
study subjects are drawn from a relatively select group. They emphasize the crucial importance of maintaining
the consistency of question wording in order to permit
comparisons between studies. Page 11 of 12 Mealing et al. BMC Medical Research Methodology 2010, 10:26
http://www.biomedcentral.com/1471-2288/10/26 The authors declare that they have no competing interests. ,
(
)
12. Nohr EA, Frydenberg M, Henriksen TB, Olsen J: Does low participation in
cohort studies induce bias? Epidemiology 2006, 17(4):413-8. cohort studies induce bias? Epidemiology 2006, 17(4):413-8 Authors' contributions 13. Eastwood BJ, Gregor RD, MacLean DR, Wolf HK: Effects of recruitment
strategy on response rates and risk factor profile in two cardiovascular
surveys. Int J Epidemiol 1996, 25(4):763-9. NM, EB, LJ and KR participated in the study concept and design and were
involved in the acquisition of data. NM and EB carried out the literature review
and drafted the manuscript. All authors were involved in the analysis and inter-
pretation of data. NM performed the statistical analysis and DS, MC and KR pro-
vided statistical advice. All authors critically revised the manuscript for
important intellectual content. EB and LJ obtained funding for this study and
provided study supervision. All authors read and approved the final manu-
script. 14. Criqui MH: Response bias and risk ratios in epidemiologic studies. Am J
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(unweighted and weighted), where either the exposure or outcome or
both variables were only moderately comparable across the 45 and Up
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(unweighted and weighted), where either the exposure or outcome or
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outcome variables were highly comparable across the 45 and Up Study and
the NSW PHS. Additional file 6 Weighted odds ratios by study where the exposure and
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the NSW PHS. Additional file 6 Weighted odds ratios by study where the exposure and
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1):95-103. Additional file 7 Weighted odds Ratios by study where either the expo-
sure or outcome or both variables were only moderately comparable
across the 45 and Up Study and the NSW PHS. Additional file 7 Weighted odds Ratios by study where either the expo-
sure or outcome or both variables were only moderately comparable
across the 45 and Up Study and the NSW PHS. Additional file 7 Weighted odds Ratios by study where either the expo-
sure or outcome or both variables were only moderately comparable
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Epidemiol 2008, 37(5):941-7. 10. 45 and Up Study Collaborators: Cohort profile: the 45 and up study. Int J
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expert assistance and advice on the NSW Population Health Survey and use of
the NSW Population Health Survey data. We thank the men and women partic-
ipating in the 45 and Up Study, and the NSW Department of Health for permit-
ting use of the 2006 and 2007 NSW Population Health Survey Datasets. The 45 and Up Study is run by The Sax Institute in collaboration with the Can-
cer Council of New South Wales; the New South Wales Division of the National
Heart Foundation of Australia; the New South Wales Department of Health;
beyondblue: the national depression initiative and the New South Wales Depart-
ment of Ageing, Disability and Home Care. This work was also supported by
the National Health and Medical Research Council [402810]. NM was employed
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Environmental Factors in health (SEEF) project. The SEEF investigators are:
Adrian Bauman, Sally Redman, Emily Banks, Mark Harris, Deborah Schofield,
Tony McMichael, Hilary Bambrick, John Beard, Dorothy Broom, Julie Byles,
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parable on the 45 and Up Study and the NSW PHS and used in the analyses. Additional file 1 Questionnaire items rated as highly or moderately com-
parable on the 45 and Up Study and the NSW PHS and used in the analyses. Additional file 1 Questionnaire items rated as highly or moderately com-
parable on the 45 and Up Study and the NSW PHS and used in the analyses. Additional file 2 Ten crude and adjusted ORs from the 45 and Up Study
(unweighted and weighted), where the exposure and outcome variables
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© 2010 Mealing 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 re
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1797, Penrith South DC NSW 1797, Australia, 3National Centre for
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© 2010 Mealing 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 rep
BMC Medical Research Methodology 2010, 10:26 Received: 14 December 2009 Accepted: 1 April 2010
Published: 1 April 2010
This article is available from: http://www.biomedcentral.com/1471-2288/10/26
© 2010 Mealing 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 rep
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http://www.biomedcentral.com/1471-2288/10/26/prepub The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2288/10/26/prepub The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2288/10/26/prepub doi: 10.1186/1471-2288-10-26
Cite this article as: Mealing et al., Investigation of relative risk estimates from
studies of the same population with contrasting response rates and designs
BMC Medical Research Methodology 2010, 10:26
|
https://openalex.org/W2905254169
|
https://www.redalyc.org/journal/5396/539658227014/539658227014.pdf
|
English
| null |
Variáveis associadas à ansiedade gestacional em primigestas e multigestas
|
Temas em Psicologia
| 2,018
|
cc-by
| 8,546
|
Abstract This study aimed to describe and compare anxiety on primigravidae and multiparous in the third quarter,
as well as to identify, among sociodemographic variables and of pregnancy, which ones were associated
with high anxiety. There were 479 pregnant women participants who responded to an anxiety instru-
ment (STAI) and a questionnaire on sociodemographic variables and about pregnancy. The groups were
statistically compared e a logistic regression model was composed to evaluate associations. The results
indicated that 36% of pregnant women presented symptoms of high anxiety on third gestational trimes-
ter, with a predominance of the multiparous in relation to the primigravidae. For the multiparous, the
highest chance for high anxiety was associated with low income and for the primigravidae to the threat
of miscarriage in early pregnancy. For both primigravidae and multiparous, wanting the baby appeared
as a protective factor for high anxiety. The expressive percentage of pregnant women with anxiety
symptoms justifi es the offering of prevention services and promotion of mental health for pregnant
women, with a differentiated attention to primigravidae and multiparous. Keywords: Anxiety, pregnant, primigravidae, multiparous. * Mailing address: Rua Quintino Bocaiúva, s/n, Aparecida de São Manuel, São Manuel, SP, Brazil 18650-000
Phone (fax) 2(14) 3841-3766. E-mail: professora.schiavo@gmail.com, olgarolim@fc.unesp.br and gimol@
fmb.unesp.br
Agradecemos a Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pelo auxílio fi nanceiro para execução desse estudo. Article Article Variables Associated with Gestational Anxiety
in Primigravidas and Multigravidas Rafaela de Almeida Schiavo*, 1, 2
Orcid.org/0000-0001-7290-3610
Olga Maria Piazentin Rolim Rodrigues3
Orcid.org/0000-0002-5332-256X
Gimol Benzaquen Perosa4
Orcid.org/0000-0002-7825-2537 Rafaela de Almeida Schiavo*, 1, 2
Orcid.org/0000-0001-7290-3610
Olga Maria Piazentin Rolim Rodrigues3
Orcid.org/0000-0002-5332-256X
Gimol Benzaquen Perosa4
Orcid.org/0000-0002-7825-2537 1Universidade Paulista, Bauru, SP, Brasil
2Instituto Municipal de Ensino Superior de São Manuel, São Manuel, SP, Brasil
3Universidade Estadual Paulista “Júlio de Mesquita Filho”, Bauru, SP, Brasil
4Universidade Estadual Paulista “Júlio de Mesquita Filho”, Botucatu, SP, Brasil 1Universidade Paulista, Bauru, SP, Brasil
2Instituto Municipal de Ensino Superior de São Manuel, São Manuel, SP, Brasil
3Universidade Estadual Paulista “Júlio de Mesquita Filho”, Bauru, SP, Brasil
4Universidade Estadual Paulista “Júlio de Mesquita Filho”, Botucatu, SP, Brasil Trends in Psychology/Temas em Psicologia
ISSN: 2358-1883
Sociedade Brasileira de Psicologia Trends in Psychology/Temas em Psicologia
ISSN: 2358-1883
Sociedade Brasileira de Psicologia Trends in Psychology/Temas em Psicologia
ISSN: 2358-1883 Schiavo, Rafaela de Almeida; Rodrigues, Olga Maria Piazentin Rolim; Perosa, Gimol Benzaquen
Variáveis Associadas à Ansiedade Gestacional em Primigestas e Multigestas
Trends in Psychology/Temas em Psicologia, vol. 26,
núm. 4, Outubro-Dezembro, 2018, pp. 2091-2104
Sociedade Brasileira de Psicologia
DOI: 10.9788/TP2018.4-14Pt
Disponível em: http://www.redalyc.org/articulo.oa?id=539658227014 Disponível em: http://www.redalyc.org/articulo.oa?id=539658227014 Como citar este artigo
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Sem fins lucrativos acadêmica projeto, desenvolvido no âmbito da iniciativa acesso aberto Sistema de Informação Científica Redalyc
Rede de Revistas Científicas da América Latina e do Caribe, Espanha e Portugal
Sem fins lucrativos acadêmica projeto, desenvolvido no âmbito da iniciativa acesso aberto Trends in Psychology / Temas em Psicologia
ISSN 2358-1883 (online edition) DOI: 10.9788/TP2018.4-14En ISSN 2358-1883 (online edition) 1Universidade Paulista, Bauru, SP, Brasil
2Instituto Municipal de Ensino Superior de São Manuel, São Manuel, SP, Brasil
3Universidade Estadual Paulista “Júlio de Mesquita Filho”, Bauru, SP, Brasil
4Universidade Estadual Paulista “Júlio de Mesquita Filho”, Botucatu, SP, Brasil Resumo Este estudo teve por objetivo descrever e comparar a ansiedade de primigestas e multigestas, no terceiro
trimestre gestacional, bem como identifi car, dentre as variáveis sociodemográfi cas e de gestação, as que p
gradecemos a Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Coordenação d
perfeiçoamento de Pessoal de Nível Superior (CAPES) pelo auxílio fi nanceiro para execução desse estudo Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Resumen Este estudio tuvo como objetivo describir y comparar la ansiedad en el primigestas y multigestas en
tercer trimestre, y para identifi car, entre las variables sociodemográfi cas y el embarazo, que se asocian
a mayor ansiedad. Participaron en 479 mujeres embarazadas que respondieron a un instrumento de an-
siedad (STAI) y un cuestionario sobre variables sociodemográfi cas y el embarazo. Los investigadores
compararon los grupos estadísticamente y montaron un modelo de regresión logística para evaluar las
asociaciones. Los resultados indicaron que el 36% de las mujeres embarazadas tenían síntomas altos
de ansiedad en tercer trimestre, con un predominio de multigestas en relación con el primigestas. Para
multigestas la mayor oportunidad para alta ansiedad se asoció con bajas ingresos y para el primigestas
con un aborto involuntario en el embarazo temprano. Ambos primigestas y multigestas quieren el bebé
apareció como un factor protector para alta ansiedad. Un porcentaje signifi cativo de mujeres embaraza-
das con síntomas de ansiedad justifi ca los servicios dirigidos a la prevención y promoción de la salud
mental de las mujeres embarazadas, con una especial atención a las primigestas y multigestas. Palabras clave: Ansiedad, embarazo, primigestas, multigestas. Anxiety is part of the process of human
development, and can be present in all periods
of the life cycle. It is a characteristic that occurs
in times of fear, danger or tension. However, in
some moments of life, anxiety is more intense
than in others. Depending on the period, it
is considered to be an expected emotional
state during a change of employment, faced
with the result of a job application or medical
examination, marriage, pregnancy and the birth
of a child, among others. being higher than in the puerperium (Agrati et
al., 2015; Baptista et al., 2006) for both fathers
and mothers (Figueiredo & Conde, 2011). Due to
the negative regulation of placental 11β-HSD2,
which increases fetal exposure to maternal
cortisol (O’Donnell et al., 2012), high levels of
anxiety in pregnancy can result in complications
and intercurrences such as premature birth
and low birth weight (Bener, 2013; Schetter &
Tanner, 2012). Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. 2106 se associaram para alta ansiedade. Participaram 479 gestantes que responderam a um instrumento de
ansiedade (IDATE) e um questionário sobre variáveis sociodemográfi cas e da gestação. Compararam-
se os grupos estatisticamente e montou-se um modelo de regressão logística para avaliar o peso das
associações (p<0,05). Os resultados indicaram que 36% das gestantes apresentaram sintomas de alta
ansiedade no terceiro trimestre gestacional, com um predomínio das multigestas em relação às primi-
gestas. Para as multigestas, a maior chance para alta ansiedade esteve associada à baixa renda e, para as
primigestas, à ameaça de aborto no início da gestação. Tanto para primigestas como para multigestas,
desejar o bebê apareceu como fator de proteção para alta ansiedade. A expressiva porcentagem de ges-
tantes com sintomas de ansiedade justifi ca o oferecimento de serviços voltados à prevenção e promoção
de saúde mental das gestantes, com atenção diferenciada às primigestas e multigestas. Palavras-chave: Ansiedade, gravidez, primigesta, multigesta. Variables Associated with Gestational Anxiety in Primigravidas and Multigravidas. 2107 Melchiori, Salgado, & Tavano, 2015). It may
also affect growth and cause delays in childhood
development (Beltrami, Moraes, & Souza, 2013;
O’Donnell, Glover, Barker, & O’Connor, 2014;
Schetter & Tanner, 2012). Melchiori, Salgado, & Tavano, 2015). It may
also affect growth and cause delays in childhood
development (Beltrami, Moraes, & Souza, 2013;
O’Donnell, Glover, Barker, & O’Connor, 2014;
Schetter & Tanner, 2012). of being a mother, which can increase anxiety. Due to cultural issues, this new responsibility is
often accompanied by guilt and anxiety, with the
woman being insecure about whether she will
fully correspond to the ideal of mothering spread
in Western society, expecting perfect mothers,
housewives and wives (Pitilin, Haracemiw,
Marcon, & Pelloso, 2013). Regarding the maternal mental health,
gestational anxiety has been associated with
gestational depression (Schetter & Tanner,
2012) and is indicative of puerperal anxiety
and depression (Agrati et al., 2015; Coelho,
Murray, Royal-Lawson, & Cooper, 2011;
Faisal-Cury et al., 2010) coinciding with the
studies of Airosa and Silva (2013) and Nardi et
al. (2015) who observed that gestational anxiety
left women vulnerable to the emergence of
emotional disturbances. However, in the study
by Saviani-Zeoti and Petean (2015), elevated
levels of gestational anxiety did not present any
impairment in the attachment relationship with
the fetus. For Simas et al. (2013), primigravidas and
multigravidas are equally anxious, however,
they present anxiety for different reasons: the
primigravidas are more distressed by their
inexperience in the task of being a mother while
the multigravidas are more ambivalent with the
unplanned pregnancy. On the other hand, for
Lopes et al. (2012), it is a myth to believe that
the experience of motherhood with the fi rst-born
would facilitate the relationship with the second
child. According to the authors, multigravidas,
especially those who are economically vulnerable
and have to assume countless costs, experience a
silenced social violence, where there is no room
for error, anger and sadness, which may justify
the high anxiety rates. According to the literature, some of the
risk factors for high gestational anxiety are low
maternal age, low educational level (Araújo,
Pacheco, Pimenta, & Kac, 2008), low income
(Simas, Souza, & Scorsolini-Comin, 2013)
and threat of abortion (Alves, Alves, Ibiapina,
Busgaib, & Costa, 2010). Fonseca-Machado,
Alves, Freitas, Monteiro, and Gomes-Sponholz
(2014), in an integrative literature review,
identifi ed that domestic violence during the
pregnancy had negative repercussions on the
mental health of pregnant women. Resumen Gestational anxiety is also associated with
shorter exclusive breastfeeding (Adedinsewo,
Fleming, Steiner, Meaney, & Girard, 2014;
Ystrom, 2012), which may have negative
consequences for the mother/baby relationship
(Airosa & Silva, 2013; Nardi, Rodrigues, Anxiety in pregnancy is one of the most
common
psychological
changes
(Baptista,
Baptista, & Torre, 2006; Faisal-Cury, Araya,
Zugaib, & Menezes, 2010), varying in intensity
in different societies and cultures, with its indices Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Participants Between 2010 and 2015, 485 pregnant
women in the third trimester that were waiting
for prenatal care at the Primary Health Units
(PHUs) were invited to participate in the study. Of these, 479 agreed to participate in the study
and, while waiting, completed the instruments
individually, in an appropriate room. There were
222 primigravidas and 257 multigravidas. Method This was a cross-sectional study that
evaluated the sociodemographic characteristics,
the perception of motherhood and the anxiety
of pregnant women. To compose the sample,
locations were selected that concentrated a large
number of consultations for the pregnant woman
through the Brazilian Nation Health System
(Sistema Único de Saúde - SUS), in three
medium-sized cities in the state of São Paulo. Data Collection Procedure The recruitment of the participants took
place in the PHUs of the three cities of São Pau-
lo state. The researcher, the fi rst author of this
study, approached all the pregnant women who
were in the third trimester and who waited in the
waiting room to be attended by the obstetrician
and invited them to participate in the study, stat- Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. 2108 women breastfed in the fi rst hour after birth than
primiparous women. cerned the medical history (health and mental
health prior to gestation, health during gestation)
and two were related to the pregnancy (threat of
abortion of the current pregnancy and previous
abortion). The pregnant women were also asked
if they and their partners had wanted the baby
(or not). As can be seen, studies comparing anxiety
levels between primigravidas and multigravidas,
as well as its associated factors, are still scarce
with differing results, possibly due to the
samples, evaluation instruments and diverse
contexts of investigation. These fi ndings suggest
the need for further investigation in order to assist
in offering differentiated support services to
primiparous and multiparous pregnant women,
focused on the control of maternal anxiety in
this period of the life cycle. Based on the above
considerations, this study had the objective
of describing and comparing the anxiety of
primigravidas and multigravidas in the third
gestational trimester, as well as identifying the
sociodemographic and gestational variables
associated with high anxiety. For the assessment of anxiety, the State-
Trait Anxiety Inventory (STAI), developed by
Spielberger, Gorsuch, and Lushene (1970) and
translated and adapted into Portuguese by Bi-
aggio and Natalício (1979/2003), was used. It
is an self-application instrument, for youths
and adults, composed of two scales that evalu-
ate State anxiety (S-anxiety) and Trait anxiety
(T-anxiety). From 20 items, individuals have to
evaluate themselves, according to a four-point
Likert-type scale. Regarding its psychometric qualities, its
reliability in the test-retest was 0.74 for State
anxiety and 0.83 for Trait anxiety. The internal
consistency, assessed through Cronbach’s Alpha,
was 0.88 in relation to female State anxiety
and 0.87 for female Trait anxiety (Biaggio &
Natalício, 1979/2003). For the present study,
internal consistency was calculated through
Cronbach’s Alpha for state and trait anxiety
in the third trimester. Alphas of 0.67 for State
anxiety and 0.63 for Trait anxiety were obtained,
indicating that the consistency indices for the
Portuguese version of the STAI applied with
pregnant women were moderate to good. As
the indication of symptoms of high anxiety, the
cut-off point used was a score equal to or above
the 75th percentile, as previously used in other
studies (Perosa, Canavez, Silveira, Padovani,
& Peraçou, 2009; Perosa, Silveira, & Canavez,
2008). Instruments The authors used a single form with 11
questions, fi ve of which were related to sociode-
mographic data (age, marital status, schooling,
family income, number of children), three con- Variables Associated with Gestational Anxiety in Primigravidas and Multigravidas. In the review
carried out for the present article, no references
were found for studies that emphasized the
protective factors for high anxiety. In the puerperium, the results are also
controversial. In Brazil, Faisal-Cury and
Menezes (2006) identifi ed that multiparous
women presented higher indices of anxiety than
primiparous women. In the study by Verissimo
(2010) in Portugal, primiparas were found to
have greater anxiety. In addition to the incipient literature and
the inconclusive data regarding the possible
mental health problems in primigravidas and
multigravidas, some research has been interested
in the repercussion that the number of children
can have on the quality of life of the couple
as well as their relationship with the children
(Camarneiro & Justo, 2012; Pereira, Fonseca,
Oliveira, Souza, & Mello, 2013; Saviani-Zeoti
& Petean, 2015). There are data indicating that
primigravidas have higher marital satisfaction,
greater preoccupation with the fetus and
better perception of their quality of life than
multigravidas (Camarneiro & Justo, 2012). On the other hand, in the study by Pereira et
al. (2013), a higher percentage of multiparous Few studies have addressed gestational
anxiety in multigravidas and primigravidas and
the results are still inconclusive, sometimes
associating high anxiety with primigravidas
(Alves et al., 2007), or with multigravidas
(Lopes, Vivian, Oliveira, Pereira, & Piccinini,
2012), or presenting no differences between the
two groups (Simas et al., 2013). According to
Alves et al. (2007), primiparity, even though it is
a period of adaptive psychic normality, it is also
a critical moment marked by anguish due to the
prominence of having to assume a new role, that Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. Data Analysis Procedure The sociodemographic, clinical and gesta-
tional, numerical and categorical variables were
binarized and organized in the Statistical Pack-
age for the Social Sciences (SPSS, version 21.0,
Chicago INc II, USA) database for further data
analysis. The criterion to binarize the age was
the one proposed by the World Health Organiza-
tion (WHO, 1986) that defi nes the ages between
10 and 19 years as adolescence. The STAI was
corrected according to the specifi c norms of the
manual and the binarized results, considering the
75th percentile as a marker of high anxiety. The
numerical variables (maternal age and number of
children) and categorical variables (all dummy
type) were analyzed using descriptive statistics
(frequency and percentage). Before proceeding
to the inferential statistical analysis, the Shap-
iro-Wilk test was applied to test the normality
of the variables. The chi-square test was used to
compare the primigravidas and multigravidas re-
garding the sociodemographic variables related
to the clinical and gestational history and high
anxiety. To identify possible variables that were
predictors of risk or protection for high anxiety,
a logistic regression model with all dichotomous
variables in a single block was applied for the
total sample and, separately, for the primigravi-
das and multigravidas. The signifi cance level ad-
opted was p<.05. Variables Associated with Gestational Anxiety in Primigravidas and Multigravidas. 2109 Ethical Procedures ing that they had to respond to two instruments,
and that one would assess their level of anxiety. The pregnant women who agreed to participate,
after signing the consent form, completed the
Form and the STAI in a room of the health unit
reserved for this purpose. For the non-literate
pregnant women and those who, for some rea-
son, preferred not to read, the researcher read
the inventory questions and fi lled out the answer
sheet of the instrument. All the pregnant women
were informed about the result of the STAI soon
after its application, the ones that scored for high
anxiety were advised to seek specialized clini-
cal psychological care, the researcher gave these
women the telephone number and address of the
place where they could fi nd this care. The project was approved by the Research
Ethics Committee of the Botucatu Medical
School Universidade Estadual Paulista “Júlio de
Mesquita Filho” – UNESP (process No. 4166-
2012) and by the Faculty of Sciences of UNESP
Bauru campus (process No. 614/46/01/09). The
data collection was authorized by the heads
of the three centers that attended the pregnant
women in primary care. Data collection only
took place after the signing of the consent
form that was given to the participants. All 479
participants, after responding to the instruments,
awaited correction by the researcher at the
application site, which immediately provided
the result. Those who scored for high anxiety
were referred to specialized professional care
locations in their city. Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Variables Associated with Gestational Anxiety in Primigravidas and Multigravidas. Results Primarily, sociodemographic and maternity
data will be presented. This will be followed by
the trait and state anxiety characteristics among
the primigravidas and multigravidas in the third
gestational trimester, the factors associated with
high anxiety in the third trimester and the factors
associated with high anxiety in primigravidas
and multigravidas. From Table 1 it is possible to verify that
the multigravidas were signifi cantly older
(p<.001), with a mean of 27 years (SD=5.4),
while the primigravidas had a mean age of
20 years (SD=5.5). Regarding the number of
children, 36.6% of the multigravidas already
had one child, 34.4% had two and 42.2%,
three or more children. The multigravidas also
presented lower levels of education (p=.009) and
a higher percentage of the mothers (p=.004) and
fathers (p=.049) had not wanted the baby when
compared to the primigravidas. Considering the total sample, trait or state,
around 36% of the pregnant women presented
high anxiety. In Table 2 it can also be observed
that, signifi cantly, a higher percentage of mul-
tigravidas presented high anxiety, both trait
(61.8%) and state (60.9%) in relation to the pri-
migravidas. Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Results 2110 Table 1
Comparison between Primigravidas and Multigravidas according to Sociodemographic and Maternity
Characteristics (N = 479)
Associated Variables
Parity
p – value*
Primigravidas
n = 222
Multigravidas
n = 257
X2
F
%
F
%
Total
Age
107.395
< .001
< 19 years old
101
88.6
13
11.4
114 (24%)
> 20 years old
121
33.2
244
66.8
365 (76%)
Marital Status
2.674
.102
Lives with partner
164
44.3
206
55.7
370 (77%)
Don´t live with partner
58
53.2
51
46.8
109 (23%)
Education
6.479
.011
< nine years old
84
39.8
127
60.2
211 (44%)
> nine years old
138
51.5
130
48.5
268 (56%)
Paid Occupation
0.018
.893
Yes
73
45.9
86
54.1
159 (33%)
No
149
46.6
171
53.4
320 (67%)
Family Income
0.628
.428
< 2 minimum wages
206
45.9
243
54.1
449 (94%)
> 2 minimum wages
16
53.3
14
46.7
30 (6.3%)
Pre-gestational health
2.737
.098
Normal
206
47.6
227
52.4
433 (90%)
A problem
16
34.8
30
65.2
46 (10%)
Gestational health
1.482
.223
Normal
189
47.6
208
52.4
397 (83%)
A problem
33
40.2
49
59.8
82 (17%)
Threat of abortion
0.267
.605
Yes
21
42.9
28
57.1
49 (10%)
No
201
46.7
229
53.3
430 (90%)
Wanted the baby
8.223
.004
Yes
147
51.8
137
48.2
284 (59%)
No
75
38.5
120
61.5
195 (41%)
Partner wanted the baby
3.892
.049
Yes
158
49.5
161
50.5
319 (67%)
No
64
40.0
96
60.0
160 (33%)
Note. F – frequency; % - percentage; X2 - Chi-Square. *p-value for the Chi-Square test. Table 1
Comparison between Primigravidas and Multigravidas according to Sociodemographic and Maternity
Characteristics (N = 479) een Primigravidas and Multigravidas according to Sociodemographic and Maternity
= 479) son between Primigravidas and Multigravidas according to Sociodemographic and Maternity
eristics (N = 479) Marital Status Family Income Note. F – frequency; % - percentage; X2 - Chi-Square. q
y
p
*p-value for the Chi-Square test. Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Variables Associated with Gestational Anxiety in Primigravidas and Multigravidas. Results 2111
Table 2
Difference between Primigravidas and Multigravidas in High Trait/State Anxiety (N = 479)
Parity
p – value*
Primigravidas
Multigravidas
Total
X2
F
%
F
%
F
%
Trait anxiety
7.316
.007
No high anxiety
156
51.0
150
49.0
306
63.9
With high anxiety
66
38.2
107
61.8
173
36.1
Total
222
46.3
257
53.7
479
100
State anxiety
5.802
.016
No high anxiety
154
50.5
151
49.5
305
63.7
With high anxiety
68
39.1
106
60.9
174
36.3
Total
222
46.3
257
53.7
479
100
Note. F – frequency; % - percentage; X2 - Chi-Square. *p-value for the Chi-Square teste. Variables Associated with Gestational Anxiety in Primigravidas and Multigravidas. 2111 able 2
fference between Primigravidas and Multigravidas in High Trait/State Anxiety (N = 479) *p-value for the Chi-Square teste. Table 3
Associated Factors for Greater and Lesser Chance of High Trait/State Anxiety in the Third Trimester of
Gestation (N = 479)
Anxiety trait
Anxiety state
p-value*
OR
IC 95%
p-value*
OR
IC 95%
Age
.490
1.185
0.732 1.921
.068
1.593
0.966 2.628
Marital Status
.777
1.071
0.667 1.719
.766
1.079
0.665 1.742
Education
.109
1.395
0.929 2.093
.317
1.234
0.818 1.863
Paid Occupation
.220
0.757
0.485 1.181
.629
0.896
0.574 1.398
Family Income
.247
1.736
0.682 4.419
.082
2.335
0.898 6.069
Pre-gestational health
.266
1.464
0.748 2.865
.135
1.672
0.852 3.280
Gestational health
.520
1.187
0.704 2.001
.679
0.893
0.521 1.529
Threat of abortion
.920
1.034
0.539 1.983
.021
2.126
1.122 4.027
History of abortion
.066
1.589
0.970 2.600
.029
1.734
1.057 2.845
Wanted the baby
.031
0.571
0.343 0.949
.003
0.455
0.273 0.758
Partner wanted the baby
.037
0.568
0.334 0.967
.122
0.655
0.384 1.120
Note. OR – Odds Ratio; IC – Confi dence Interval. *p-value for the Logistic Regression. Factors for Greater and Lesser Chance of High Trait/State Anxiety in the Third Trimester of
N = 479) Table 3
Associated Factors for Greater and Lesser Chance of High Trait/State Anxiety in the Third Trimester of
Gestation (N = 479) able 3
ssociated Factors for Greater and Lesser Chance of High Trait/State Anxiety in the Third Trimest
estation (N = 479) Note. OR – Odds Ratio; IC – Confi dence Interval. *p-value for the Logistic Regression. tion with high trait or state anxiety. Table 4 Table 4
Associated Factors for Greater and Lesser Chance of High Trait/State Anxiety on Primigravidas in in the
Third Trimester of Gestation (n = 222)
Primigravidas
Anxiety Trait
Anxiety State
p-value*
OR
IC 95%
p-value*
OR
IC 95%
Age
.933
0.970
0.481 1.958
.514
1.273
0.617 2.626
Marital Status
.504
0.779
0.374 1.622
.567
1.235
0.600 2.543
Education
.734
1.129
0.569 2.226
.789
1.100
0.549 2.204
Paid Occupation
.192
0.602
0.281 1.290
.942
0.973
0.462 2.047
Family Income
.355
0.554
0.158 1.938
.757
0.816
0.226 2.947
Pre-gestational health
.923
1.061
0.323 3.480
.703
1.253
0.393 3.996
Gestational health
.412
0.678
0.268 1.716
.381
0.662
0.263 1.666
Threat of abortion
.136
2.164
0.785 5.964
.017
3.464
1.245 9.640
History of abortion
.196
1.842
0.730 4.651
.086
2.263
0.891 5.749
Wanted the baby
.839
0.910
0.365 2.267
.033
0.390
0.164 0.928
Partner wanted the baby
.020
0.322
0.124 0.837
.358
0.653
0.263 1.620
Note. OR – Odds Ratio; IC – Confi dence Interval. *p-value for the Logistic Regression. Table 4
Associated Factors for Greater and Lesser Chance of High Trait/State Anxiety on Primigravidas in in the
Third Trimester of Gestation (n = 222) ors for Greater and Lesser Chance of High Trait/State Anxiety on Primigravidas in in the
r of Gestation (n = 222) Note. OR – Odds Ratio; IC – Confi dence Interval. *p-value for the Logistic Regression. er Brazilian studies evaluating pregnant and
puerpera women (Faisal-Cury & Menezes,
2006; Morais, Lucci, & Otta, 2013; Beltra-
mi et al., 2013), which permits comparisons
between the present study and others. As in
other studies (Borges, Santos, Nascimento,
Chofakian, & Gomes-Sponholz, 2016; Con-
ceição & Fernandes, 2015), a large number
of the pregnant women reported not having
wanted the pregnancy (41%), especially the With respect to multigravidas, low income
increased the chance of having high trait and
state anxiety while having wanted the baby de-
creased the chance, for both types of anxiety
(Table 5). Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. 2112 in multigravidas (Table 5) in the third trimester
of gestation are presented below. hibiting high trait anxiety in the third trimester
of the gestation. Similarly, the mother having
wanted the baby decreased the chance of scoring
for high state anxiety, in the third trimester of the
gestation. The pregnant women who had a clini-
cal history of a threat of abortion in early preg-
nancy and previous abortion had a higher chance
of presenting high state anxiety (Table 3). In Table 4, it can be seen that for the
primigravidas, having wanted the baby appeared
as a protective factor, while the threat of abortion
increased the chance for high state anxiety
by 3.464. Regarding trait anxiety, the partner
having wanted the baby reduced the chance of
the pregnant woman presenting high anxiety. The factors associated with high trait and
state anxiety in the primigravidas (Table 4) and Results However, it
was found that the mother and her partner hav-
ing wanted the baby decreased the chance of ex- In the prediction analysis for the total of the
sample, it can be observed that no sociodemo-
graphic variable showed a signifi cant associa- Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Discussion The sociodemographic characteristics of
the participants were similar to those of oth- Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Variables Associated with Gestational Anxiety in Primigravidas and Multigravidas. 2113 Table 5
Associated Factors for Greater and Lesser Chance of High Trait/State Anxiety on Multigravidas in in the
Third Trimester of Gestation (n = 257)
Multigravidas
Anxiety Trait
Anxiety State
p-value*
OR
IC 95%
p-value*
OR
IC 95%
Age
.802
0.856
0.253 2.892
.137
2.641
0.735 9.489
Marital Status
.328
1.405
0.711 2.773
.758
0.898
0.455 1.775
Education
.119
1.553
0.894 2.698
.276
1.355
0.785 2.340
Paid Occupation
.761
0.914
0.512 1.632
.655
0.878
0.495 1.557
Family Income
.043
5.094
1.050 24.704
.021
6.622
1.337 32.793
Pre-gestational health
.236
1.702
0.706 4.104
.167
1.836
0.775 4.352
Gestational health
.129
1.727
0.853 3.496
.793
1.098
0.545 2.215
Threat of abortion
.146
0.503
0.199 1.270
.360
1.493
0.633 3.525
History of abortion
.368
1.327
0.717 2.453
.162
1.538
0.841 2.813
Wanted the baby
.007
0.397
0.203 0.776
.015
0.439
0.226 0.850
Partner wanted the baby
.641
0.848
0.425 1.693
.303
0.698
0.352 1.384
Note. OR – Odds Ratio; IC – Confi dence Interval. *p-value for the Logistic Regression. *p-value for the Logistic Regression. er number of multigravidas presented high
anxiety, perhaps associated with their low
income and number of children, as will be
discussed later. multigravidas. This suggests that, despite
the law guaranteeing family planning as a
State responsibility, the aim of providing
conditions for men and women to access in-
formation about contraception (Prates, Abib
& Oliveira, 2008) may not be occurring ef-
fectively. For some authors, this is due to the
limitation and defi ciency in the care infra-
structure, the lack of information regarding
contraception among women or the resis-
tance of the spouse to seek family planning
services (Caetano, 2014; Penaforte et al.,
2010; Pitilin et al., 2013). This study allowed the identifi cation of
approximately 36% of the participants, in
the last trimester of gestation, with high trait
and state anxiety. This proportion is smaller
than that of other national studies, probably
due to the cut-off point adopted for high anx-
iety in this study being higher than in other
studies (Baptista et al., 2006; Faisal-Cury &
Menezes, 2006). The multigravidas were older than the
primigravidas. Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. 2114 In both the primigravidas and multigrav-
idas, the history of abortion signifi cantly in-
creased the chance of high anxiety (state),
possibly because this experience causes
pregnant women to experience the new preg-
nancy with ambivalent feelings of attach-
ment to the baby, as well as insecurity and
fear (Freire & Chatelard, 2009). In the study
by Fernandes et al. (2012), both women who
spontaneously aborted and those who had
provoked an abortion presented anxiety with
a new pregnancy associated with fear of an-
other abortion. Menezes, 2006; Morais et al., 2013). Studies
have highlighted that multiparity is associ-
ated with a lower degree of maternal edu-
cation, which is, in turn, refl ected in lower
wages, lower family income and greater eco-
nomic vulnerability (Moura, Silva, Gomes,
Almeida, & Evangelista, 2010; Prates et al.,
2008; Severinski, Mamula, Severinski, &
Mamula, 2009). Confi rming the positions of Simas et al. (2013), the percentage of pregnant women
with high anxiety, as well as sociodemo-
graphic, clinical and gestational risk factors,
differed in both groups: for the multigravi-
das low-income appeared to be a risk factor
for high anxiety (trait and state), while for
the primigravidas it was the threat of abor-
tion in early pregnancy (state). While the possibility of losing the child
appeared as a risk factor, having wanted the
child was associated with less chance of
primigravidas and multigravidas present-
ing symptoms of high anxiety (trace and
state). For Pitilin et al. (2013), most of the
time, even if the pregnancy was unplanned
and women believe they have become preg-
nant at the wrong time, the initial anguish
and anxiety become feelings of resigna-
tion, adaptation and, during the pregnancy,
they start to want the baby. In some stud-
ies, women who did not plan the pregnancy
due to fi nancial and marital diffi culties had a
belief that the baby came to restore the fam-
ily relationship, including the conjugal one,
and the desire for the child became a satis-
factory, even saving experience, increasing
their self-esteem and protecting them from
emotional problems (Moura et al., 2010; Pri-
etsch, González-Chica, Cesar, & Mendoza-
Sassi, 2011; Simas et al., 2013). Discussion It was hoped that because the
multigravidas were older, indicating greater
experience, knowledge and the ability to an-
ticipate occurrences acquired in raising other
children, they would present lower anxiety
compared to the primigravidas. However, in
this study, as in Lopes et al. (2012) a high- The multigravidas also had lower educa-
tion levels than the primigravidas, possibly
due to the multigravidas not being favored
by the new educational policies of continu-
ous progression, which started in 1996, re-
sponsible for higher levels of education of
pregnant women and puerperas in recent
studies (Beltrami et al., 2013; Faisal-Cury & Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. As 42.2% of the multigravidas had more
than three children, the arrival of one more
child, especially for the low-income mul-
tigravidas, may have led to a new recon-
fi guration of the family system, with more
responsibilities and possible losses in their
perspective for the future, fi nancial diffi cul-
ties and reduced quality of life, generating
uncertainties and anxiety (Borges, Ferreira,
Mariutti, & Almeida, 2011; Prates et al.,
2008; Simas et al., 2013). In the study by
Pitilin et al. (2013), with multiparas of four
or more children, SUS users, it was observed
that economic vulnerability was not limited
to income deprivation, but also interfered
with the possibility of obtaining quality
work with adequate remuneration, with ac-
cess to public services and with the dynam-
ics of family life. The partner wanting the baby decreased
the odds for high trait anxiety in the primi-
gravidas, showing that the desire for a child
and possible paternal involvement during
the pregnancy may have attenuated the un-
certainties regarding the care, especially for
the primiparas. According to Simas et al. (2013), when the partner accepts the preg-
nancy and wants to be a father, he becomes
more involved in the process, takes respon-
sibility for the preparations for the arrival
of the baby with the pregnant woman, of-
fers emotional support, tries to make contact Thus, fi nancial diffi culties may have
increased the tension in the multigravidas
faced with the birth of a new child, while the
possibility of fetal loss increased the chance
of high state anxiety in the primigravidas. Often, the threat of abortion is a condition
with unknown pathophysiology that requires
several types of intervention that, on one
hand, seek to prevent fetal loss and, on the
other, increase the sensation of unpredict-
ability and anxiety (Alves et al., 2010). Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Variables Associated with Gestational Anxiety in Primigravidas and Multigravidas. 2115 limits the generalization of the results. It is rec-
ommended that, in new investigations, in addi-
tion to not binarizing the variables, the subjects
should be randomized, increasing the generality
of the results. with the baby and listens to the concerns and
desires of the women, constituting an impor-
tant social support. Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. The present study contributed with ad-
vances in this area of research, since it com-
pared the anxiety of primigravidas and mul-
tigravidas in the third gestational trimester,
and identifi ed, among the sociodemographic
and gestational variables, those that were
associated with high anxiety in each group. This can allow socio-educative measures to
be developed from the data identifi ed with
greater attention to the emotional health of
pregnant women, mainly low-income multi-
gravidas and primigravidas with the threat of
abortion in early pregnancy. Despite the limitations, the present study
identifi ed high anxiety in 36% of the pregnant
women using the SUS, in the third trimester of
pregnancy, mainly among the multigravidas. The
already proven association of gestational anxiety
with obstetric complications and puerperal de-
pression, as well as with damage that can affect
childhood growth and development, indicate the
need for professionals that work in prenatal care
to pay special attention to the emotional health
of the pregnant women. The study also reinforces that the risk fac-
tors for high anxiety are different in primigravi-
das and multigravidas, justifying the provision
of differentiated care for women pregnant for the
fi rst time and for those that have been through
previous pregnancies. In the case of the primi-
gravidas, experiencing a threat of abortion in
early pregnancy appeared as the main variable
predicting high anxiety in the third trimester,
which indicates the importance of providing
maximum information, along with psychologi-
cal support, at that moment. Often, there are no
reasons to justify the threat of abortion (Alves et
al., 2010), which leads many women to perceive
it as an individual fault, associated with feelings
of guilt and fantasies of “a deserved punish-
ment”, relevant aspects to be considered in the
actions of psychological health care. Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. 2116 materno. Cogitare Enfermagem, 12(4), 416-427. Retrieved from revistas.ufpr.br/cogitare/article/
download/10063/6918 structural variable in which health profession-
als cannot act, with public policies in the care of
these women, therefore, being relevant, in order
to favor social-assistance, education and voca-
tional resources. In addition, it is possible to act
preventively by investing in programs that pro-
mote more effi cient and effective family plan-
ning for families using the public health system. Advances in contraception have enabled greater
decision-making power for couples and the
woman herself, with the possibility of choosing
when to have a child becoming a decisive factor
for a healthy pregnancy and maternity/paternity. Alves, J. A. G., Alves, N. G., Ibiapina, F. L. P., Bus-
gaib, R. P. S., & Costa, F. S. (2010). Ameaça de
aborto: Conduta baseada em evidências. Femi-
na, 38(2), 111-115. Retrieved from http://fi les. bvs.br/upload/S/0100-7254/2010/v38n2/a010. pdf Araújo, D. M. R., Pacheco, A. H. R. N., Pimenta, A. M., & Kac, G. (2008). Prevalência e fatores as-
sociados a sintomas de ansiedade em uma coorte
de gestantes atendidas em um centro de saúde do
município do Rio de Janeiro. Revista Brasileira
de Saúde Materno Infantil, 8(3), 333-340. doi:
10.1590/S1519-38292008000300013 It is also hoped that these results can help in
the development of new studies and provide sup-
port for policies aimed at pregnant users of pub-
lic services. The intention is to provide health-
care that goes beyond the biomedical scope and
takes into account emotional and social vulner-
abilities from the development of approaches
that ultimately improve the quality of life of this
population. Baptista, M. N., Baptista, A. S. D., & Torres, E. C. R. (2006). Associação entre suporte social, de-
pressão e ansiedade em gestantes. PSIC. Revista
de Psicologia da Vetor Editora, 7(1), 39-48. Re-
trieved from http://pepsic.bvsalud.org/pdf/psic/
v7n1/v7n1a06.pdf Beltrami, L., Moraes, A. G., & Souza, A. P. R. A. (2013). Ansiedade materna puerperal e risco
para o desenvolvimento infantil. Distúrbios da
Comunicação, 25(2), 229-239. Retrieved from
http://revistas.pucsp.br/index.php/dic/article/
view/16476/12373 Final Considerations The results obtained bring advances to the
knowledge in the area, however, it is important
to consider some limitations of this study that
could help in the planning of new investigations. The fi rst limitation refers to the instrument used
to measure anxiety, the STAI, which was also
used in other studies to identify levels of anxiety
at the end of gestation (Agrati et al., 2015; Araú-
jo et al., 2008; Baptista et al., 2006; O’Donnell et
al., 2012; Roos, Faure, Louchner, Vythilingum,
& Stein, 2013). Although the reliability calcu-
lated for this study through Cronbach’s alpha
showed moderate to good indices, the STAI is
not a specifi c instrument for pregnant women,
since some symptoms evaluated as anxiety could
be characteristics expected during pregnancy. It
is recommended that further investigations be
conducted to identify the best instrument to mea-
sure anxiety in pregnant women, or to develop
an instrument specifi c for this population. The feelings and fantasies of primigravidas
need to be addressed from the moment the threat
of abortion occurred to prevent the chronifi ca-
tion of the pregnant woman’s emotional state,
which may ultimately negatively infl uence both
the maternal mental health and the birth, as well
as the physical and emotional health of the baby. However, studies are still needed to verify what
type of care, individual or group, can more ef-
fectively prevent the aggravation of anxiety at
the end of gestation. Another possible limitation of the study
may have been the fact that all the variables, in-
cluding the continuous ones, such as age and in-
come, were binarized and, if considered in their
continuous form, they would possibly allow a
more accurate analysis of anxiety in the different
age and socioeconomic groups, in the logistic re-
gression. Also, the fact that the sample was not
probabilistic, but constituted by convenience, With regard to the multigravidas, a high
percentage had not wanted the baby (60%) and
high anxiety was associated with low income, a Trends Psychol., Ribeirão Preto, vol. 26, nº 4, p. 2105-2118 - December/2018 Schiavo, R. A., Rodrigues, O. M. P. R., Perosa, G. B. Variables Associated with Gestational Anxiety in Primigravidas and Multigravidas. 2117 Conceição, S. P., & Fernandes, R. A. Q. (2015). In-
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β-ketoenole dyes: Synthesis and study as fluorescent sensors for protein amyloid aggregates
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Dyes and pigments
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Svitlana Chernii1, Vladyslava Kovalska1, Mykhaylo Losytskyy1, Yan Dovbii2, Iryna Tretyakova2, Rafal Czerwieniec3,
Victor Chernii2, Sergiy Yarmoluk1, Sergiy Volkov2 Svitlana Chernii1, Vladyslava Kovalska1, Mykhaylo Losytskyy1, Yan Dovbii2, Iryna Tretyakova2, Rafal Czerwieniec3
Victor Chernii2, Sergiy Yarmoluk1, Sergiy Volkov2 1Institute of Molecular Biology and Genetics NASU, 150 Zabolotnogo St., 03143 Kyiv, Ukraine
2V.I. Vernadskii Institute of General and Inorganic Chemistry NASU, 32/34 Palladin Av., 03080 Kyiv, Ukraine
3Institut für Physikalische und Theoretische Chemie, Universitätsstr. 31, 93053, Regensburg, Germany Dyes and Pigments 132 (2016) 274-281
DOI: 10.1016/j.dyepig.2016.04.053 aggregates Svitlana Chernii1, Vladyslava Kovalska1, Mykhaylo Losytskyy1, Yan Dovbii2, Iryna
Tretyakova2, Rafal Czerwieniec3, Victor Chernii2, Sergiy Yarmoluk1, Sergiy
Volkov2
1Institute of Molecular Biology and Genetics NASU, 150 Zabolotnogo St., 03143
Kyiv, Ukraine
2V.I. Vernadskii Institute of General and Inorganic Chemistry NASU, 32/34 Palladin
Av., 03080 Kyiv, Ukraine
3Institut für Physikalische und Theoretische Chemie, Universitätsstr. 31, 93053,
Regensburg, Germany Regensburg, Germany This work was supported in the framework of Marie Skłodowska-Curie Research and Innovation
Staff Exchange (RISE) program
Project Number: 645628
Project Acronym: METCOPH
Project title: Metallocomplexes of macrocyclic compounds for photonic devices Project title: Metallocomplexes of macrocyclic compounds for photonic devices ABSTRACT The series of the new β-ketoenole dyes ((2E,5Z,7E,9E)-6-hydroxy-2-
(alkylamino)-10-phenyldeca-2,5,7,9-tetraen-4-ones) with variation of alkylamino tail
groups was synthesized and studied as potential probes for the sensing of protein
aggregates amyloid fibrils. The dyes are low fluorescent when free but able to
increase their emission intensity in dozens of times in the presence of fibrillar insulin. The fluorescent response of the dye on fibrillar insulin strongly depends on
the nature of the alkylamino tail group. For compounds with propylamino (dye 13)
and 2-hydroxyethylamino (dye 14) fragments the fluorescence intensity in the
presence of fibrillar insulin exceeds that for the native one in 22 and 66 times
correspondingly. However dyes demonstrate from low or moderate exceed of the fluorescence
intensity in the presence of aggregated lysozyme compared to native one (up to 8.7
times for the dye 53 bearing methyl ester tail group), due to their pronounced
sensitivity to native lysozyme. The dyes in complexes with insulin have rather height quantum yield up to
0.15, the large Stokes shifts values (about 100 nm and more), their binding constant
values are about 105 M-1. The dye 14 allows fluorescent detection of the insulin
amyloid fibrils in the concentration range 1-50 μg/ml. This causes an interest in the
future study of the β-ketoenole as prospective fluorescent amyloid-sensitive
molecules. KEYWORDS: β-ketoenole dyes, fluorescent sensing, probes, amyloid fibrils, insulin, lysozime, proteins 1. INTRODUCTION One of the most convenient methods for the analysis of biomolecules is the use
of extrinsic fluorescent probes that noncovalently bind to them by electrostatic, van
der Waals and hydrophobic interactions. The wide range of fluorescent molecules is developed for high efficient sensing, quantification and visualization of proteins and
nucleic acids in in vitro and in vivo assays [1-4]. The spontaneous aggregation of proteins leading to formation of insoluble beta-
pleated aggregates (amyloid fibrils) is among the actual targets in the biomedical
researches, since these aggregates are connected with the range of harmful human
diseases including neurodegenerative ones. This causes an interest in the
development of new appropriate analytical tools to be used in the study of this
process. The extrinsic fluorescent probes are used for the detection and quantification of
amyloid fibrils, monitoring of the kinetics of their formation and study of the factors
and agents affecting these processes. For these purposes, amyloid sensitive
fluorescent probes Thioflavin T and it`s derivatives are commonly applied. The
histological dyes Congo Red and Chrysamine G are used for the staining and study
of amyloid formations on tissues [5-7]. Recently we discovered and developed mono
and polymethine cyanines dyes as efficient fluorescent probes for the detection of
protein β-pleated aggregates [8, 9]. Cyanines with high sensitivity to the amyloid
fibrils and wide detection range (1.5-120 µg/ml for trimethine cyanine 7519 [10])
surpassing that of Thioflavine T were proposed. On the base of these cyanine dyes
the inhibitory assay for the search of the compounds with anti-fibrillogenic activity
was developed and applied. At the same time further search for the amyloid-
sensitive probes with high fluorescent response to the fibrillar protein presence is
still urgent. One of the necessary requirements for the molecule to be applicable as the
fluorescent probe for the amyloid formations detection is high affinity of the
complex formation between this molecule and the amyloid fibril. As the most
probable mode of such complex formation, the insertion of the dye molecule into the
groove of the amyloid fibril is suggested [11]. As a result of such binding, the
fluorescent response is observed due to the quite rigid fixation of the dye molecule,
and the polarization of the absorbed light is caused by the same orientation the
bound dye molecules. 1. INTRODUCTION Thus, in order to obtain an efficient response to the presence
of amyloid fibril, the molecule should have shape complimentary to that of the fibril
groove and the size fitting the fibril groove (about 6,5-7 Å) [12]. (Fig 1) Figure 1. Scheme of the layout of the dye molecule in the fibrilar groove (left) [11]
and AFM image of the insulin amyloid fibrils (right) [13]. Figure 1. Scheme of the layout of the dye molecule in the fibrilar groove (left) [11]
and AFM image of the insulin amyloid fibrils (right) [13]. In the present work we firstly studied the β-ketoenole dyes as potential
fluorescent probes for the sensing of amyloid aggregates of proteins (Table 1). The
β-ketoenoles are the molecules of the elongated shape that is suggested as preferable
for fitting to the groove of the amyloid fibril; besides they have rather flexible aliphatic chromophore chain providing the low intrinsic fluorescence intensity to the
unbound dye. Unlikely to the majority of the amyloid-sensitive dyes bearing either
positive (Thioflavin T, cyanine dyes) or negative (Congo Red) charge, the molecules
of β-ketoenoles are uncharged. With this aim the series of ((2E,5Z,7E,9E)-6-hydroxy-2-(alkylamino)-10-
phenyldeca-2,5,7,9-tetraen-4-ones) dyes with variation of alkylamyno substituents
was synthesized and the fluorescent properties were characterized for the free dyes
as well as in the presence of amyloidogenic proteins lysozyme and insulin in the
native and aggregated form. The range of detection of the amyloid aggregates with
the most efficient dye was determined. Besides, the fluorescent sensitivity of β-
ketoenole dye to the serum albumin able to bind the variety of the small molecules
was studied for the comparison. Table 1. Dye structures. 13
14
47
50 14 13 13 14 50 47 47 50 47 51
52
53
54
88 51 52 52 51 54 53 53 54 88 88 2.1. Synthesis and characterization of the dyes 2.1. Synthesis and characterization of the dyes The general procedure of ((2E,5Z,7E,9E)-6-hydroxy-2-(alkylamino)-10-
phenyldeca-2,5,7,9-tetraen-4-ones) synthesis is as follows. The dyes were prepared by mixing the 5mM 4-hydroxy-6-methyl-3-((2E,4E)-5-
phenylpenta-2,4-dienoyl)-2H-pyran-2-one in 10 ml of DMF (described in [14]) with
an excess of 10% amine. The mixture was heated at 100 ºC for 30 minutes; during
the reaction path it changed the color to red. The heating was stopped after the finishing of the carbon dioxide elimination. The cooled reaction mixture was
precipitated by water. The formed product was washed twice with water and
crystallized from DMF-ethanol mixture, filtered off, washed twice with ethanol on
the filter and dried. All obtained compounds are yellow-orange fine-crystalline
substances. Structures of investigated compounds were confirmed with 1H NMR and Anal. Calcd. (%). Calcd. (%). (2E,5Z,7E,9E)-6-hydroxy-10-phenyl-2-(propylamino)deca-2,5,7,9-tetraen-4-
one. 13. Yield 67%. Mp 255-259°C. 1H NMR (CDCl3, 400 MHz) δ 14.99 (1H, s),
10.43 (1H, s), 7.44 (2H, d, J = 7.4 Hz), 7.34 (2H, dd, J = 13.4, 6.2 Hz), 7.26 (1H, dd,
J = 9.8, 4.7 Hz,), 7.17 (1H, dd, J = 15.0, 10.8 Hz), 7.01 – 6.71 (2H, m), 6.01 (1H, d,
J = 15.1 Hz), 5.20 (1H, s), 4.78 (1H, s), 3.24 (2H, dd, J = 13.3, 6.7 Hz), 1.98 (3H, s),
1.65 (2H, dd, J = 14.4, 7.2 Hz), 1.09 – 0.96 (3H, m). Found (%): C, 76.79; H, 7.74;
N, 4.64; molecular formula C19H23NO2 requires C, 76.73; H, 7.80; N, 4.71. (2E,5Z,7E,9E)-6-hydroxy-2-((2-hydroxyethyl)amino)-10-phenyldeca-2,5,7,9-
tetraen-4-one. 14. Yield 69%. Mp 273-275°C. 1H NMR (CDCl3, 400 MHz) δ 14.84
(1H, s), 10.44 (1H, s ), 7.46 (2H, dd, J = 12.9, 7.2 Hz), 7.40 – 7.30 (2H, m ), 7.30 –
7.22 (1H, m ), 7.18 (1H, dd, J = 15.0, 10.8 Hz), 6.91 (1H, ddd, J = 23.8, 20.0, 13.2
Hz), 6.76 (1H, d, J = 15.5 Hz), 6.00 (1H, d, J = 15.0 Hz), 5.21 (1H, s ), 4.83 (1H, s),
3.79 (2H, dt, J = 10.7, 5.2 Hz), 3.45 (2H, dt, J = 11.4, 5.6 Hz), 2.29 (1H, s ), 2.01
(3H, s). Found (%): C, 72.14; H, 7.02; N, 4.63; molecular formula C18H21NO3
requires C, 72.22; H, 7.07; N, 4.68. (2E,5Z,7E,9E)-2-(allylamino)-6-hydroxy-10-phenyldeca-2,5,7,9-tetraen-4-one. 47. Yield 44%. M143-144°C. 1H NMR (CDCl3, 400 MHz) δ 15.13 (1H, s ), 10.30
(1H, t, J = 6.0 Hz), 7.52 (2H, d, J = 7.7 Hz), 7.36 (3H, t, J = 7.5 Hz), 7.28 (1H, t, J =
7.3 Hz), 7.06 (2H, dd, J = 9.2, 4.8 Hz), 6.89 (1H, d, J = 14.2 Hz), 6.15 (1H, dd, J =
14.0, 6.4 Hz), 6.02 – 5.86 (1H, m ), 5.34 (2H, s), 4.89 (1H, s ), 3.97 (2H, t, J = 5.6
Hz), 1.98 (3H, s). Found (%): C, 77.11; H, 7.12; N, 4.70; molecular formula
C19H21NO2 requires C, 77.26; H, 7.17; N, 4.74. (2E,5Z,7E,9E)-2-((3-(dimethylamino)propyl)amino)-6-hydroxy-10-
phenyldeca-2,5,7,9-tetraen-4-one. 50. Yield 40%. Mp 121-123°C. Calcd. (%). 1H NMR (DMSO,
400 MHz) δ 15.19 (1H, s ), 10.32 (1H, s ), 7.52 (2H, d, J = 7.5 Hz), 7.36 (2H, t, J =
7.5 Hz), 7.28 (1H, t, J = 7.3 Hz), 7.11 – 6.98 (2H, m ), 6.88 (1H, dd, J = 15.4, 6.4
Hz), 6.15 (1H, d, J = 14.0 Hz), 5.31 (1H, s ), 4.82 (1H, s ), 3.32 (2H, dd, J = 13.0,
6.3 Hz), 2.25 (2H, t, J = 6.8 Hz), 2.13 (6H, s), 1.99 (3H, s ), 1.75 – 1.57 (2H, m ). Found (%): C, 74.02; H, 8.24; N, 8.19; molecular formula C21H28N2O2 requires C,
74.08; H, 8.29; N, 8.23. (2E,5Z,7E,9E)-2-(sec-butylamino)-6-hydroxy-10-phenyldeca-2,5,7,9-tetraen-4-
one. 51. Yield 36%. Mp 159-162°C. 1H NMR (DMSO, 400 MHz) δ 15.05 (1H, s ),
10.30 (1H, d, J = 9.4 Hz), 7.52 (2H, d, J = 7.5 Hz), 7.36 (2H, t, J = 7.5 Hz), 7.28
(1H, t, J = 7.2 Hz), 7.04 (2H, dt, J = 14.3, 7.4 Hz), 6.88 (1H, d, J = 14.7 Hz), 6.23 –
6.08 (1H, m ), 5.31 (1H, s ), 4.81 (1H, s ), 3.68 – 3.53 (1H, m ), 2.01 (3H, s ), 1.50
(2H, dt, J = 14.0, 6.9 Hz), 1.16 (3H, d, J = 6.4 Hz), 0.89 (3H, t, J = 7.3 Hz). Found (%): C, 77.22; H, 7.97; N, 4.46; molecular formula C20H25NO2 requires C, 77.14; H,
8.09; N, 4.50. (2E,5Z,7E,9E)-6-hydroxy-2-(isobutylamino)-10-phenyldeca-2,5,7,9-tetraen-4-
one. 52. Yield 45%. Mp 170-173°C. 1H NMR (DMSO, 400 MHz) δ 15.06 (1H, s ),
10.44 (1H, t, J = 5.9 Hz), 7.52 (2H, d, J = 7.4 Hz), 7.37 (2H, dd, J = 14.1, 6.9 Hz),
7.28 (1H, t, J = 7.3 Hz), 7.12 – 6.98 (2H, m ), 6.89 (1H, t, J = 11.3 Hz), 6.27 – 6.03
(1H, m ), 5.32 (1H, s ), 4.85 (1H, s ), 3.15 (2H, t, J = 6.4 Hz), 1.99 (3H, s ), 1.78
(1H, d, J = 6.4 Hz), 0.94 (6H, d, J = 6.7 Hz). Found (%): C, 76.96; H, 8.01; N, 4.39;
molecular formula C20H25NO2 requires C, 77.14; H, 8.09; N, 4.50. (2E,5Z,7E,9E)-6-hydroxy-2-((2-methoxyethyl)amino)-10-phenyldeca-2,5,7,9-
tetraen-4-one. 53. Yield 65%. Mp 153-155°C. 2.2. Insulin and lysozyme aggregates formation Human insulin (Private Joint Stock Company «On the production of insulin
«Indar», Ukraine) was dissolved at 340 µM concentration in 0,1 mM HCl solution
(pH 2). Fibrils were formed by incubating the protein solution in a thermomixer
incubator at 65 °C for about 5 h. To prepare lysozyme aggregates, lysozyme of hen
egg white (Sigma-Aldrich Co. USA) was dissolved at 1 mM concentration in 0,1
mM HCl solution (pH 2) and incubated in a thermomixer incubator at 65 °C for 24
hours. Calcd. (%). 1H NMR (DMSO, 400 MHz) δ 15.15
(1H, s ), 10.30 (1H, s ), 7.52 (2H, d, J = 7.4 Hz), 7.37 (2H, t, J = 7.5 Hz), 7.28 (1H, t,
J = 7.3 Hz), 7.14 – 6.96 (2H, m ), 6.98 – 6.80 (1H, m ), 6.16 (1H, dd, J = 13.4, 6.8
Hz), 5.32 (1H, s ), 4.85 (1H, s ), 3.49 (4H, d, J = 6.6 Hz), 3.30 (3H, s ), 2.00 (3H, s ). Found (%): C, 72.89; H, 7.34; N, 4.49; molecular formula C19H23NO3 requires C,
72.82; H, 7.40; N, 4.47. (2E,5Z,7E,9E)-6-hydroxy-2-((3-methoxypropyl)amino)-10-phenyldeca-2,5,7,9-
tetraen-4-one. 54. Yield 57%. Mp 106-107°C. 1H NMR (DMSO, 400 MHz) δ 15.18
(1H, s ), 10.30 (1H, t, J = 5.8 Hz), 7.59 – 7.47 (2H, m ), 7.36 (2H, t, J = 7.5 Hz),
7.28 (1H, t, J = 7.3 Hz), 7.15 – 6.97 (2H, m ), 6.89 (1H, dd, J = 14.3, 7.6 Hz), 6.21 –
6.01 (1H, m ), 5.31 (1H, s ), 4.85 (1H, d, J = 9.5 Hz), 3.38 (4H, dd, J = 10.8, 4.7
Hz), 3.25 (3H, d, J = 2.4 Hz), 1.99 (3H, s ), 1.78 (2H, dd, J = 12.7, 6.2 Hz).. Found (%): C, 73.45; H, 7.65; N, 4.25; molecular formula C20H25NO3 requires C, 73.37; H, (%): C, 73.45; H, 7.65; N, 4.25; molecular formula C20H25NO3 requires C, 73.37; H, 7.70; N, 4.28 7.70; N, 4.28 7.70; N, 4.28 (2E,5Z,7E,9E)-2-(heptylamino)-6-hydroxy-10-phenyldeca-2,5,7,9-tetraen-4-
one. 88. Yield 62%. Mp 132-134°C. 1H NMR (DMSO, 300 MHz) δ 15.16 (1H, s),
10.35 (1H, s), 7.56 (2H, d, J = 7.3 Hz), 7.40 (3H, t, J = 7.4 Hz), 7.31 (1H, t, J = 7.3
Hz), 7.09 (2H, dt, J = 16.4, 11.3 Hz), 6.91 (1H, d, J = 14.4 Hz), 6.27 – 6.07 (1H, m),
5.34 (1H, s), 4.87 (1H, s), 3.36 – 3.24 (2H, m), 2.02 (2H, s), 1.56 (2H, m), 1.31 (8H,
m, J = 4.6 Hz), 0.89 (3H, m, J = 7.1 Hz). Found (%):C, 77.95; H, 8.68; N, 3.92;
molecular formula C23H31NO2 requires C, 78.15; H, 8.84; N, 3.96. 2.3. Preparation of dye solutions and protein solutions β-ketoenole stock solutions were prepared by dissolving the weighted amount
of the dyes at 2 mM concentration in DMSO or DMF. Working solutions of free
dyes were prepared by dilution of the dye stock solutions in 50 mM Tris-HCl buffer
(pH 7.9) to the concentration 2 µM. The working solutions of dye-proteins complexes were prepared by adding to the dye 2 µM solution the aliquot of
monomer or fibrillar protein stock solution, the protein concentration in the working
solution was thus 3,4 µM for insulin and 4 µM for lysozyme. Bovine serum albumin
(BSA) was obtained from Sigma-Aldrich Co. USA. To prepare the dye-BSA
working solution, to the 0.2mg/ml (3 µM) BSA solution in 50 mM Tris-HCl buffer
(pH 7.9) the aliquot of the dye stock solution was added, the dye concentration in the
working solution was thus 2 µM. 2.4. Spectral measurements Fluorescence excitation and emission spectra were registered using the
fluorescent spectrophotometer Cary Eclipse (Varian, Austria). Fluorescence
emission was excited at the maximum wavelength of excitation spectrum of
corresponding dye solution. The quantum yield value of the dyes 14 and 50 (2 μM)
in the presence of fibrillar insulin (13.6 μM) was determined using Rhodamine 6G
solution in ethanol as the reference (quantum yield value 0.95) [15]. All the spectral-
luminescent characteristics of unbound dyes in aqueous buffer were studied at room
temperature. 2.5. Estimation of equilibrium constants of the dye-to-fibril binding To estimate the equilibrium constant of the dyes 14 and 50 binding to fibrillar
insulin, fluorescent titration of the dyes (2 μM) upon addition of 0-45 μM of fibrillar
insulin was performed. Taking into account only the points for the concentrations 2
M of protein molecules and higher, we could consider the concentration of the
binding sites to be much higher than this of the dyes and thus the concentration of the free protein to be roughly equal to its total concentration. Under this assumption,
the equation for the equilibrium constant K of dye-fibril binding could be written as: Cd/Cbd = 1 + (K×CF)–1 (1) (1) where Cd, Cbd and CF are total dye, fibril-bound dye and fibrillar protein
concentrations respectively. Further, let us consider the totally unbound and totally bound with fibrils dye
solution to have the fluorescence intensity I0 and Imax respectively. In this case the
measured dye fluorescence intensity I at the fibrillar protein concentration CF could
be written as I = I0×(Cd – Cbd)/Cd + Imax×Cbd/Cd, that can be transformed into: Cd/Cbd = (Imax – I0)/(I – I0) (2) (2) Together with (1), (2) gives Together with (1), (2) gives Together with (1), (2) gives Together with (1), (2) gives I – I0 = A×K×CF/(1+K×CF) (3) (3) I – I0 = A×K×CF/(1+K×CF) (3)
A being the denotation for (Imax – I0) difference. Thus the experimental dependence
of I – I0 on CF was approximated with the equation (3), A and K being obtained as
approximation parameters. Accounting for several assumptions made, the obtained
K value could be regarded as a rough estimation of the binding constant value rather
than its precise value. Besides it should be reminded that the estimated binding
constant is only an apparent value calculated with respect to protein globule
concentration and not this of the binding sites that is unknown; actually the
estimation of binding constant with respect to protein globule concentration is
common for the ligand-fibril binding studies. 2.6. Computer simulations of the dye 14 dimensions 2.6. Computer simulations of the dye 14 dimensions To estimate the dimensions of the dye 14, geometry optimization of the dye
structure was first performed using the PM3 method from the HyperChem 6.03
program package. Further the isosurface with the total charge density 0.002 that
characterizes the molecular dimensions was built; linear dimensions i.e. length,
height and width of the obtained isosurface were then estimated. 3.1. Synthesis of the β-ketoenole dyes In present work we obtained series of new compounds by the pyran ring
opening reaction of 4-hydroxy-6-methyl-3 - ((2E, 4E) -5-phenylpenta-2,4-dienoyl) -
2H-pyran-2-one with primary aliphatic amines. The mechanism of this reaction was
studied in [14, 16], it occurs through the nucleophilic attack of the 6-carbon atom of
the pyran cycle leading to its next opening and decarboxilation. We have found that
on the first step of this reaction the interaction of 4-hydroxy-6-methyl-3 - ((2E, 4E) -
5-phenylpenta-2,4-dienoyl) -2H-pyran-2-one with amines (Scheme 1) leads to the
formation of the corresponding salt. When heated, it is dissociated and at the same
time the amino group attacks a carbon atom of the methyl group in the pyran ring,
causing its opening and subsequent decarboxylation [14, 16]. In the case of n-alkyl
amines, formation of the corresponding salts, ring opening and decarboxylation
occurs quite easily, but iso-amines react considerably worse and require higher
reaction temperature. Scheme. 1. Synthesis of the studied β-ketoenole dyes. Scheme. 1. Synthesis of the studied β-ketoenole dyes. In the case of the tert-butylamine, the reaction stopped at the stage of the salt
formation, opening of the pyran ring does not occur (Fig. 1), the behavior of -
phenyl ethyl amine is similar. This observation indirectly confirms the reaction
mechanism proposed in [14, 16]. Such molecules could be divided into two parts: hydrophobic ones containing
phenyl moiety and polymethine chain, and hydrophilic molecules containing keto-
enol fragment and tail alkylamino group. For related compounds the existence of the
set of tautomeric forms of the ketoenol fragment was established by NMR [16]. The
compounds described here are also able to the formation of the tautomers. In their
NMR spectra in addition to the main groups of signals there are the minor signals
with similar morphology corresponding to the presence of 3-6% of admixtures. According to data of LC/MS the molecular ions of these admixtures have the same
molecular weight as that of the base compound that also confirms the presence of the
tautomeric forms of compounds (data not presented). 3.2. Spectral properties of dyes in buffer and in the presence of native
roteins 3.2. Spectral properties of dyes in buffer and in the presence of native
proteins proteins The fluorescent characteristics of the 9 β-ketoenole compounds in buffer
solution and in the presence of insulin, lysozyme and BSA in their native form are
presented in Table 2. The excitation maxima for the studied dyes in the free state are located in the
range 414-428 nm; emission maxima are in the range 523–560 nm, except for the
dye 88 (emission maximum at 604 nm) and the dye 52 possessing long-wavelength
emission maximum (at 606 nm) together with the short-wavelength one (545 nm). The addition of insulin, lysozyme and BSA could cause the shift of the excitation
and emission spectrum maxima both to longer or shorter wavelengths for up to 35
nm. Excitation maxima in the presence of proteins are places in the range 408-435
nm respectively, while positions of the emission maxima are at 522-571 nm. Exceptions are the dye 88 with long alkyl tail that has emission bands with maxima
in the range 604-611 nm respectively. For the dyes 52 (free) and the dye 47 (in the
presence of insulin) emission spectra contain two bands, the long-wavelength one
for the both dyes has the maximum at 606-607 nm. The large values of the shift
between the excitation and emission maxima for free dyes and the dyes in the
presence of proteins (98-190 nm) should be mentioned. Table 2. Spectral-luminescent properties of β-ketoenole dyes in the presence of the native proteins. Name
free dye
with insulin
with lysozyme
with BSA
λex,
nm
λem, nm
I0, a.u. λex,
nm
λem, nm
IM, a.u. IM/I0
λex,
nm
λem,
nm
IM,
a.u. IM/I0
λex,
nm
λem,
nm
IM,
a.u. * – I0 was this at the wavelength 545 nm 3.2. Spectral properties of dyes in buffer and in the presence of native
roteins IM/I0
13
427
542
1.7
413
542
3.7
2.2
427
525 17.7
10.4
427
536
1
0.6
14
426
539
1.3
426
539
1.6
1.2
426
540
10
7.7
410
540
7
5.4
47
415
545
2.6
415 433/ 607 4.7/ 7.3
1.8 / 2.8
410
541
1.9
7.3
415
530
5
1.9
50
414
523
0.8
427
526
2
2.5
427
526
7.7
9.6
418
523
0.8
1
51
417
554
5.5
415
539
6.8
1.2
415
543
10
1.8
417
550
3.8
0.7 52
414
545 / 606
7.5 / 15 415
553
6.2
0.8*
415
571
9.3
1.2*
408
555
5
0.7*
53
415
535
0.7
427
522
4.3
6.1
423
525
2.6
3.7
415
535
4
5.7
54
428
560
3.6
420
536
4.9
1.4
427
525 17.7
4.9
428
531
2
0.6
88
414
604
0.7
416
604
0.7
1
410
611
3.2
4.6
435
611
1.5
2.1 λex, λem – maximum wavelength of fluorescence excitation and emission spectra;
I0 (IM) - fluorescence emission intensity of the dye in free state (in the presence of native protein);
a.u. - arbitrary units. λex, λem – maximum wavelength of fluorescence excitation and emission spectra; λex, λem – maximum wavelength of fluorescence excitation and emission spectra; I0 (IM) - fluorescence emission intensity of the dye in free state (in the presence of native protein);
a.u. - arbitrary units. I0 (IM) - fluorescence emission intensity of the dye in free state (in the presence of native protein); * – I0 was this at the wavelength 545 nm * – I0 was this at the wavelength 545 nm Fig.2. The excitation and emission spectra of the dye 50(left) and dye 14(right)
in the presence of the monomeric or fibrillar insulin. Fig.2. The excitation and emission spectra of the dye 50(left) and dye 14(right)
in the presence of the monomeric or fibrillar insulin. Fig.2. The excitation and emission spectra of the dye 50(left) and dye 14(right)
in the presence of the monomeric or fibrillar insulin. As it was suggested, the fluorescence intensity of the free β-ketoenole dyes is
low 0.7-7.5 a.u. The enhancement of the dyes fluorescence intensity in the presence
of the native proteins does not exceed 10 times, and formed protein-dye complexes
are of the quite low intensity. The dyes 51 and 52 with the branched alkyl substituent
slightly change their emission upon the addition of proteins. 3.2. Spectral properties of dyes in buffer and in the presence of native
roteins Other dyes demonstrate
the quite pronounced emission increase (in 3.7-10 times) in the presence of
lysozyme. The dye 14 having the tail alkyl hyrdoxy group gives fluorescent response
to the presence of both lysozyme the albumin. At the same time, the compound 53
bearing the methyl ester as tail group possesses better sensitivity to insulin and BSA
than to lysozyme. 3.3. Spectral properties of dyes in the presence of fibrillar proteins Insertion of the dye molecule into the groove of the amyloid fibril is suggested
to be the most possible model of the dye-fibril binding. With the help of the
computer simulation, the dimensions of the dye 14 were estimated to be about
23Å×7.8Å×4.8Å, that is characteristic for the dyes of the studied series. Thus the
studied dyes fit to the fibrillar groove formed by the β-pleated structure of the fibril,
the width of which is believed to be equal to 6.5-7Å [12]. Thus, we can expect the
formation of the dye-fibril fluorescent complex due to the fixation of the dye
molecules in the fibril groove. The fluorescent characteristics of the 9 β-ketoenole compounds in the presence
of fibrillar aggregates of insulin and lysozyme are presented in Table 3. For the majority of the studied β-ketoenole dyes, the addition of the fibrillar
insulin results in the long-wavelength shift of the excitation maximum wavelength
for up to 22 nm (except of the dye 88 with the 3 nm short-wavelength shift). The
short-wavelength shifts of the emission maximum wavelength for up to 26 nm were
also observed for the majority of the dyes except 50 (almost no change), 88 (7-nm
long-wavelength shift) and 54 (two maxima shifted to the short- and long-
wavelength region with respect to the free dye were observed). Table 3. Table 3. Table 3. Spectral-luminescent properties of β-ketoenole dyes in the presence of the
aggregated proteins
Name with fibrilar insulin
with fibrilar lysozyme Spectral-luminescent properties of β-ketoenole dyes in the presence of the
aggregated proteins
Name with fibrilar insulin
with fibrilar lysozyme Spectral-luminescent properties of β-ketoenole dyes in the presence of the λex, nm λem, nm
IF, a.u. IF/IM
λex, nm λem, nm IF, a.u. IF/IM
13
441
528
80
21.6
441
517
10
0.6
14
430
531
97
60.6
427
540
37
3.7
47
436
528
49.7
6.8*
426
532
5.9
0.3
50
436
524
25
12.5
426
522
27.7
3.6
51
437
528
20.9
3.1
415
543
8.5
0.8
52
435
535
17
2.7
408
555
9.2
1.0
53
434
525 / 591
22
5.1
440
526
22.7
8.7
54
436
545 / 588
33.7 / 39.6 6.9 / 8.1
441
517
10
0.6
88
411
611
1.9
2.7
410
611
3.5
1.1 λex, λem - maxima of fluorescence excitation and emission spectra; λex, λem - maxima of fluorescence excitation and emission spectra; IF - emission intensity of dye in the presence of fibrillar form of the insulin and
lysozyme; IF/ IM - emission increasing of the dye in the presence of corresponding proteins;
a.u. - arbitrary units; * – IM was this at the wavelength 607 nm The presence of fibrillar insulin leads to the enhancement of the dye
fluorescence intensity (IF) as compared to that of both free dye (I0) and the dye in the presence of native insulin (IM); the highest values of the IF/IM ratio were observed for
the dyes 13 and 14 containing “short” unbranched propylamino and
hydroxyethylamino groups as substituents (22 and 61 times respectively). The rather
high fluorescent response with IF/IM between 7 and 13 times was also observed for
the dyes 47, 50 and 54 containing allylamino, dimethylaminopropylamino and
methoxypropylamino substituents. The other dyes did not demonstrate significant
IF/IM values. It should be also mentioned that the fluorescence quantum yield value
for the dyes 14 and 50 in the presence if fibrillar insulin is rather high and equals to
0.12 and 0.15 respectively. For the dyes 14 and 50 the titration with fibrillar insulin was performed; the
dependence of the dye fluorescence intensity on the concentration of the added
fibrils allowed us to estimate the equilibrium constant of the dye to fibril binding. This estimation gave the values of the binding constant K = (2.70.9)×105 M-1 for
the dye 14 and (2.51.4)×105 M-1 for the dye 50, being thus of the same order of
magnitude. Spectral-luminescent properties of β-ketoenole dyes in the presence of the As for the fibrillar lysozyme, its presence leads to the long-wavelength shift of
the excitation spectrum maximum wavelength up to 25 nm, except for the dyes 51,
52 and 88 that slightly shift their maxima to the short-wavelength shift region. At the
same time, for the emission maximum the wavelength shift was predominantly to
the short-wavelength region (up to 43 nm for the dye 54), exceptions being the dyes
14, 52 and 88 (long-wavelength shifts for up to 10 nm). As for the emission intensity, only for three dyes 14, 50 and 53 the fluorescence
sensitivity to the fibrillar lysozyme overpasses that to native protein. The IF/IM
values for these three dyes are not significant: the highest value is for the dye 53 (8.7
times), while for the dyes 14 and 50 it is about 3.7. Other compounds possess the
fluorescent sensitivity to lysozyme aggregates equal to or lower than this for the
native protein. It should be mentioned that the insignificant IF/IM value observed for the most
insulin-sensitive dye 14, as well as for several other dyes in the presence of fibrillar
lysozyme is partially connected with the fluorescent sensitivity of these dyes to the
native lysozyme (IM value). The binding to native lysozyme leading to the fixation of the fluorescent
conformation of the molecule and thus to the fluorescent response occurs for the
majority of the studied dyes. That could be explained by the high content of the
charges amino acids in the structure of this protein (8 negatively charged residues
and 18 positively charged ones) and thus attributed to the electrostatic interaction. Generally the β-ketoenole molecules are uncharged, so its affinity to the charged
amino acid residues would be higher in the case of the molecule’s transition to the
zwitterionic tautomer form (Fig. 3). Fig. 3. Zwitterionic tautomer form of β-ketoenole dye Fig. 3. Zwitterionic tautomer form of β-ketoenole dye It was observed that some of β-ketoenoles are even more sensitive to the native
lysozyme than to its well-structured beta-pleated aggregates that formed the binding
places in the fibrillar grooves. Thus we could suggest that electrostatic interaction
with charged groups mainly drive the interaction of the dye molecules to the
lysozyme. The reorganization of the protein structure to beta-pleated aggregates
does not lead to the essential increase in the number of tightly bound and thus
fluorescent molecules, and can even decrease their number. linear detection range To examine the applicability of the dye 14 bearing 2-hydroxyethyl group as
amyloid-sensitive probe for the quantification of amyloid insulin, we performed
titration of the 2 µM dyes solutions with increasing amounts of the aggregated
protein (Fig. 4). The lower limit for the fibrillar insulin detection by the dye 14 was
determined as equal to the insulin concentration leading to the two times dye
fluorescence intensity increase. The upper limit of the detection range was
determined as the maximum concentration where the dependence of dye
fluorescence intensity on the fibrils concentration is still linear. Thus the 2μM
concentration of the dye 14 allows fluorescent detection of the insulin amyloid
fibrils in the concentration range 0.2-9 μM (1-50 μg/ml) (R = 0,97703; Fig. 4); the
upper limit of the fibrillar insulin detection by the dye 14 thus exceeds that for the
amyloid-specific dye Thioflavin T (0.5-25 μg/ml) [17]. Fig. 4. Titration of 14 dye by fibrillar insulin. The linear concentration range 0.2-9
μM (1-50 μg/ml) (linear, R = 0,977). The 2 µM dye solution was used. Fig. 4. Titration of 14 dye by fibrillar insulin. The linear concentration range 0.2-9
μM (1-50 μg/ml) (linear, R = 0,977). The 2 µM dye solution was used. μM (1-50 μg/ml) (linear, R = 0,977). The 2 µM dye solution was used. It should be mentioned also that the large shifts between the excitation and emission
maxima for the fibril-bound dye (about 100 nm), good fluorescence intensity
increase, rather high quantum yield and sufficient detection range make the dye 14
promising as probe for fluorescent detection of the fibrillar aggregates. 4. CONCLUSION The series of β-ketoenole dyes was firstly synthesized and characterized, their
fluorescent properties as potential probes for the sensing of amyloid aggregates of
proteins were studied. These dyes are low fluorescent when free, but have the large shift between
excitation and emission maxima reaching 190 nm. The majority of dyes possess the
insignificant sensitivity to the native proteins insulin and bovine serum albumin, while their fluorescent response on the presence of native lysozyme is more
pronounced (emission intensity increase up to 10 times was observed). This could be
explained by the high content of the charged aminoacids in the protein and allows
suggesting about high impact of the electrostatic interaction into this binding. The sensitivity of these dyes to fibrillar proteins depends strongly of the
nature of their functional substitutens. The most intensive fluorescent response on
the presence of amyloid fibrils of insulin is observed for dyes 13 and 14 that carry
“short” propylamino and or 2-hydroxyethylamino groups (fluorescence intensity in
the presence of fibrils exceeds that for the native proteins in 22 and 60 times
respectively). For
the
dyes
containing
2-hydroxyethylamino
(14)
and
dimethylaminopropylamino groups (50), the values of equilibrium constant of
binding to fibrillar insulin were found to be (2.70.9)×105 M-1 and (2.51.4)×105
M-1 respectively. At the same time, the studied dyes demonstrate significantly lower
fluorescent sensitivity to fibrillar lysozyme as compared to fibrillar insulin. fluorescent sensitivity to fibrillar lysozyme as compared to fibrillar insulin. The dye 14 demonstrates the good fluorescent response and linear range (1-50
μg/ml) of amyloid fibril detection, large value of the shift between excitation and
emission maxima (about 100 nm), its quantum yield in the complex with fibrillar
insulin reaches 0.15. We suggest this dye as prospective fluorescent molecule for the
sensing of β-pleated protein aggregates and investigation of the aggregation reaction. Thus we suggest the β-ketoenole dyes as prospective fluorescent molecules
for the design on their base of the probes for the detection of the β-pleated protein
aggregates and investigation of the aggregation reaction. This work was supported by the H2020-MSCA-RISE-2014-RISE-645628 -
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A Two-Stage Evolutionary Game Model for Collaborative Emergency Management Between Local Governments and Enterprises
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International Journal of Disaster Risk Science/International journal of disaster risk science
| 2,023
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cc-by
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ARTICLE ARTICLE ARTICLE A Two‑Stage Evolutionary Game Model for Collaborative Emergency
Management Between Local Governments and Enterprises Yanqing Wang1 · Hong Chen2,3 · Xiao Gu1 Accepted: 17 December 2023 / Published online: 4 January 2024
© The Author(s) 2023, corrected publication 2024 Accepted: 17 December 2023 / Published online: 4 January 2024
© The Author(s) 2023, corrected publication 2024 Abstract Enterprises play a vital role in emergency management, but few studies have considered the strategy choices behind such
participation or the collaborative relationship with the government. This study contended that enterprises have at least three
strategies regarding emergency management: non-participation, short-term participation, and long-term participation. We
constructed a two-stage evolutionary game model to explore the behavioral evolution rules and evolutionary stability strate-
gies of the government and enterprises, and employed numerical simulation to analyze how various factors influence the
strategy selection of the government and enterprises. The results show that if and only if the utility value of participation is
greater than 0, an enterprise will participate in emergency management. The evolutionary game then enters the second stage,
during which system stability is affected by a synergistic relationship between participation cost, reputation benefit, and gov-
ernment subsidies, and by an incremental relationship between emergency management benefit, government subsidies, and
emergency training cost. This study provides a new theoretical perspective for research on collaborative emergency manage-
ment, and the results provide important references for promoting the performance of collaborative emergency management. Keywords Collaborative relationship · Emergency management · Enterprise participation · Numerical simulation · Two-
stage evolutionary game International Journal of Disaster Risk Science (2023) 14:1029–1043
https://doi.org/10.1007/s13753-023-00531-9 International Journal of Disaster Risk Science (2023) 14:1029–1043
https://doi.org/10.1007/s13753-023-00531-9 www.ijdrs.com
www.springer.com/13753 * Hong Chen
hongchenxz@163.com * Hong Chen
hongchenxz@163.com
1
School of Economics and Management, China University
of Mining and Technology, Xuzhou 221116, China
2
School of Business, Jiangnan University, Wuxi 214122,
China
3
Research Institute of National Security and Green
Development, Jiangnan University, Wuxi 214122, China 1 Introduction governments, social organizations, enterprises, and the resi-
dents (Kong and Sun 2021; Fan et al. 2022). Collaborative emergency management (CEM) has been
defined as joint activity of two or more stakeholders that
work together in pursuit of greater public good. It aims to
eliminate wasted resources and efforts via communication,
coordination, and interoperability (Kapucu et al. 2010). With
the complexity of emergencies continues to increase, the
success of emergency management becomes increasingly
depending on effective collaboration (Oh et al. 2014). The
September 11 attacks in 2001 and Hurricane Katrina in 2005
have been followed by a significant increase in the role of
social forces in emergency management (Lv 2017). In par-
ticular, private enterprises have made great contributions
by enhancing their advantages in capital, technology, and
equipment. For example, Walmart and Home Depot imple-
mented effective relief efforts in responding to Hurricane
Katrina, combining proactivity with logistic expertise (Wang
et al. 2014). During the Covid-19 pandemic, Meituan pro-
vided people with many kinds of food, necessities, medi-
cines, epidemic prevention and disinfection supplies, and
other errand services, thereby deploying its professional The mounting frequency and intensity of various emergen-
cies are placing increased pressure on emergency manage-
ment (Bosomworth et al. 2017), and the traditional gov-
ernment-centered system struggles to respond effectively. To improve the market mechanism and give full play to
its efficiency, professionalism, and competitiveness, mar-
ket participation in emergency management is imperative
(Nan et al. 2022). In many countries, enterprises, non-profit
organizations, and residents have played an important role
in such work. The general trend of emergency management
worldwide has been to advocate collaboration between * Hong Chen
hongchenxz@163.com 1
School of Economics and Management, China University
of Mining and Technology, Xuzhou 221116, China 3
Research Institute of National Security and Green
Development, Jiangnan University, Wuxi 214122, China (012 3456789) Wang et al. A Two-Stage Evolutionary Game Model for Collaborative Emergency Management 1030 and in different regions (vertical and horizontal) (Li 2017);
and collaboration between different functional agencies at
the same government level (Bennett 2018). Previous stud-
ies focused mainly on the synergistic relationship between
government emergency management departments and social
organizations. For example, Tau et al. (2017) analyzed the
CEM between different countries in the Southern African
Development Community (SADC). 2 Literature Review This section introduces related works about CEM and evo-
lutionary game theory, discusses gaps in the literature, and
explains why we used evolutionary game method and system
dynamics (SD) simulation in this study. 1 Introduction Olszewski and Siebe-
neck (2021) discussed the nature of CEM between different
levels of government in the United States, and proposed a
framework visualizing collaboration as a trust-building and
outcome cycle. Chen et al. (2019b) revealed the functions
of social organizations in natural hazard-related disasters’
emergency relief, and analyzed their role orientation in the
emergency management network. Elsewhere, the role of
emerging technologies in promoting CEM capabilities has
also attracted widespread scholarly attention. For example,
Wang and Chen (2022) clarified the possibility of using
blockchain technology to improve the efficiency of multi-
agent CEM with respect to information sharing, supervision,
and reward/punishment. Xiong and Xue (2023) analyzed the
feasibility of using blockchain to improve the performance
of emergency material collaborative transportation. Gupta
et al. (2022) put forward an evidence-based framework that
emphasizes the role of artificial intelligence and cloud-based
collaboration platforms in emergency management. capabilities to provide necessary guarantees for people’s
live (Zhang 2021). According to the 2020 Meituan Deliv-
ery Action Report against COVID-19, Meituan’s order vol-
ume reached 3.96 million during the lockdown of Wuhan,
with 54.1% of Meituan riders delivering orders to hospitals. Meituan took the lead in launching a “contactless delivery”
service, which played an important role in avoiding contact
infection. Each enterprise has unique advantages, and the
strong “joint force” they can form is increasingly important
when dealing with emergencies, effectively mitigating inade-
quacies of government provision in emergency management. The above illustrates that enterprises have become an
important actor in emergency management. Government
failures in emergency response and the greater efficiency
of the private sector further reflect enterprises’ important
functions (Linnenluecke and McKnight 2017). However,
enterprises participating in emergency management con-
tinue to self-organize, rather than being included in the gov-
ernment response system (Zhang and Tong 2015). Previous
studies have focused mostly on the emergency management
capability of public organizations, such as the government
and schools (Shi 2012; Shah et al. 2018), while giving little
attention to private organizations. In particular, the greater
efficiency of enterprises’ participation in emergency man-
agement through the market mechanism has scarcely been
explored. Therefore, this study aimed to analyze the strategy
choices and game relationship between local governments
and private enterprises, and to explore what incentives pro-
mote CEM performance. Our findings will have important
theoretical and practical significance for promoting the
establishment of a sustainable, effective CEM system and
improving capabilities. 2.1 Related Works on Collaborative Emergency
Management (CEM) Due to the increasing frequency and severity of large-scale
disasters worldwide, since the beginning of the twenty-first
century, particularly following the September 11 attacks in
2001, multi-agent CEM for cross-border and major crises has
become a hot topic among scholars (Ansell et al. 2010; Hart
2013). Multi-agent collaboration in emergency management
has three main aspects: collaboration between the govern-
ment, the market, and social organizations (Diehlmann et al. 2021); collaboration between governments at different levels 1 Introduction capabilities to provide necessary guarantees for people’s
live (Zhang 2021). According to the 2020 Meituan Deliv-
ery Action Report against COVID-19, Meituan’s order vol-
ume reached 3.96 million during the lockdown of Wuhan,
with 54.1% of Meituan riders delivering orders to hospitals. Meituan took the lead in launching a “contactless delivery”
service, which played an important role in avoiding contact
infection. Each enterprise has unique advantages, and the
strong “joint force” they can form is increasingly important
when dealing with emergencies, effectively mitigating inade-
quacies of government provision in emergency management. q
g
p
g
y
g
The above illustrates that enterprises have become an
important actor in emergency management. Government
failures in emergency response and the greater efficiency
of the private sector further reflect enterprises’ important
functions (Linnenluecke and McKnight 2017). However,
enterprises participating in emergency management con-
tinue to self-organize, rather than being included in the gov-
ernment response system (Zhang and Tong 2015). Previous
studies have focused mostly on the emergency management
capability of public organizations, such as the government
and schools (Shi 2012; Shah et al. 2018), while giving little
attention to private organizations. In particular, the greater
efficiency of enterprises’ participation in emergency man-
agement through the market mechanism has scarcely been
explored. Therefore, this study aimed to analyze the strategy
choices and game relationship between local governments
and private enterprises, and to explore what incentives pro-
mote CEM performance. Our findings will have important
theoretical and practical significance for promoting the
establishment of a sustainable, effective CEM system and
improving capabilities. In terms of enterprises’ participation in emergency man-
agement, previous studies have mainly focused on enter-
prises’ own emergency management capability and role. For example, Li et al. (2021) explored the effect of personal
initiative on enterprise emergency management capability,
seeking an evidential basis for proposing effective measures
to improve emergency management ability. Ai and Zhang
(2019) studied the storage and distribution problem of mar-
itime emergency supplies under the CEM of the govern-
ment and enterprises using a two-stage optimization loca-
tion model. Xie et al. (2022) studied the decision-making
and cooperation problems for emergency medical supplies
considering corporate social responsibility with the govern-
ment, manufacturers, and retailers. However, few scholars
have examined how enterprises can participate more effec-
tively, or the CEM relationship between enterprises, govern-
ments, and other participants such as social organizations
and residents. 2.2 Evolutionary Game Theory Evolutionary games have occupied an increasing share of
game theory literature in recent years. Evolutionary game
is a combination of game theory and dynamic evolution-
ary process analysis, originated from the study of biological
evolutionary process in theory of evolution. In an evolution-
ary game involving any form of strategic interaction, higher 1031 International Journal of Disaster Risk Science payoff strategies tend to replace lower payoff strategies over
time, but there is some inertia, and players do not system-
atically attempt to influence other players’ future decisions. Friedman (1998) indicated that like the traditional game,
an evolutionary game model must have a game framework,
including hypothesis, structure, and rules of the game, and
the evolutionary game process is always carried out under
such a framework. Grafica, Campari Group, Caracol, Ellamp, Distillerie Silvio
Carta, and Mares) to the Covid-19 emergency; their find-
ings show that with the ability to rapidly reconfigure pro-
cess and innovation, these enterprises have made important
contributions to early recovery from Covid-19 by providing
needed supplies. Therefore, this study explored the strat-
egy choices and influencing factors of enterprises and gov-
ernments in CEM using the combination of evolutionary
game theory and SD simulation. This method should better
explain the interaction between collaborating governments
and enterprises. Evolutionary game has become a classical method for
studying the multi-agent collaborative governance mecha-
nism, and is widely used in research on the formation and
evolution process of social habits, rules, and institutions
(Liu et al. 2015; Semasinghe et al. 2015). In the field of
organizational cooperation, evolutionary game theory has
been widely used to various problems, such as industry-
university-research cooperation (Cao and Li 2020), market-
supervision cooperation (Lu et al. 2018), environmental
governance cooperation (Chen et al. 2019a), public goods
game (Wang et al. 2013), and sustainable humanitarian sup-
ply chains (Li et al. 2019). It is also widely applied in the
research of CEM involving multiple stakeholders. For exam-
ple, Du and Qian (2016) used evolutionary game theory to
explore the interactions between government and non-profit
organizations in emergency mobilization within China. Shao
et al. (2022) established an evolutionary game model for
the collaborative governance of construction waste with
multi-agent participation, and integrated system dynamics
(SD) to simulate and analyze the system’s strategy selection. Fan et al. 3 Methods Evolutionary game theory posits that players are subject to
bounded rationality, and uses the concept of natural selection
(rather than profit maximization) for economic analysis. At
the beginning of the game, both players of the game can-
not determine their optimal strategies, but find the optimal
strategy combination through continuous imitation, trial,
and error and learning, finally reach a stable state (Levine
and Pesendorfer 2007). One of the key concepts in evolu-
tionary game theory is evolutionary stable strategy (ESS),
which is when the vast majority of individuals in the popu-
lation choose a certain strategy; the group that chooses the
mutation strategy cannot invade the group that contains the
vast majority of individuals as it includes fewer individuals
(Wang et al. 2022). When analyzing multi-variable nonlinear complex
systems, SD can not only solve the modeling problem of
complex systems, but also perform quantitative coordina-
tion and optimization of the relationships among system
elements under the overall framework. System dynamics
model describes the causality and feedback relationship
between system elements through a casual flow diagram
(Fan et al. 2021), and carries out simulation experiments by
establishing computer-based models. Specifically, based on
evolutionary game modeling, this study used a SD model
to simulate the evolutionary game process and the dynamic
equilibrium results of CEM between governments and enter-
prises, and analyze the effect of various factors on the ESS. Although the importance of enterprise participation in
emergency management has been widely recognized, there
is little research on how they effectively collaborate with
the government. Most game studies on CEM have focused
on the cooperation between local governments, or between
governments and social organizations, while few have inves-
tigated collaboration between governments and enterprises. Research on collaborative governance of emergencies
between the government and enterprises mainly analyzed
the challenges of enterprise participation in emergency
management in a theoretical framework, or illustrated the
importance of their participation through case studies. For
example, Puliga and Ponta (2021) investigated the fast inno-
vation reactions of different enterprises (including Isinnova, f
Nearly all previous studies are based on a binary oppo-
sition perspective, such as “(not) governance,” “(not) par-
ticipation,” “(not) incentive,” and “(not) coordination.” In
practice, however, enterprises have at least three strate-
gies of non-participation, short-term participation, and
long-term participation with respect to emergency man-
agement, while governments also have at least three strat-
egies of non-incentive, short-term incentive, and long-
term incentive. 2.2 Evolutionary Game Theory (2021) analyzed interactions among the behavio-
ral strategies of government, community, and citizens amid
public health emergencies by combining evolutionary game
method and SD. Liu et al. (2021) examined the cooperation
relationship between government and social organizations
in emergency management by adopting evolutionary game
theory and simulation analysis. Evidently, as an effective
method to study the dynamic problems of complex systems,
SD simulation is often combined with evolutionary game
theory to better explore the strategy evolution mechanisms
of multiple stakeholders in the CEM system. 3 Methods In other words, the classical evolutionary
game method in existing studies cannot appropriately
describe enterprise strategy choice regarding emergency 1032 Wang et al. A Two-Stage Evolutionary Game Model for Collaborative Emergency Management 4 Construction of a Two‑Stage Evolutionary
Game Model management. Furthermore, in a traditional evolutionary
game, the matrix is usually static, and all players select
their respective strategies concurrently, making it difficult
to explain some social and economic phenomena with
successive decision making in real society. The two-stage
game model is an effective method to analyze multi-agent
interaction, multi-element collaboration, and multi-stage
progression. In the emergency management process, the
enterprises have priority in choosing whether or not they
will participate; accordingly, the choice behavior of both
players has the order of priority, but the game players can
see one another’s choices before choosing their own strat-
egies. Therefore, in the game of emergency management
between the government and enterprises, it is more appro-
priate to use a two-stage game method. Based on previous
studies involving two-stage games (Petrosyan et al. 2016;
Gao et al. 2017; Tang et al. 2021), we constructed a two-
stage evolutionary game framework for emergency man-
agement (Fig. 1), seeking to provide explanatory ideas for
the evolution of government-enterprise CEM. This section details the construction of a two-stage evolu-
tionary game model for CEM between the government and
enterprises, calculates the system’s ESS, and tests the model
stability. Fig. 1 Two-stage game matrix
with two players 4.1 Game Scenario From the rational choice perspective, collaboration between
the government and enterprises is premised on both sides
recognizing the possibility of benefit. On the one hand, the
government plays a key role in effectively promoting enter-
prises’ participation in emergency management, whether by
formulating preferential tax policies or providing subsidies
(Luo et al. 2022). Its aims in this endeavor are to improve
emergency management capability, quickly and effectively
respond to various emergencies, reduce disaster losses,
maximize the protection of people’s lives and property, and
maintain social security and stability. On the other hand,
enterprises’ participation in emergency management can
provide enormous social benefit by reducing pressure on
the government, while also enhancing their corporate image
(Hamann et al. 2020). For example, Lüttenberg et al. (2022)
indicated that enterprises’ involvement in emergency man-
agement is general highly valued by consumers, leading to
increase willingness to choose their products and services. In addition, active participation in emergency management
is also an important part of corporate social responsibil-
ity (Johnson et al. 2011). Studies have shown that fulfilling
social responsibility obligations can also lead to growth and
sustainability of an enterprise’s business. Li et al. (2023)
indicated that during Covid-19, enterprises involved in
emergency hospital construction and donations had higher
excess return rates.f As shown in Fig. 1, during the process of two-stage
evolutionary game, player 1 makes decisions first, i and
j refer to two strategies of player 1 in the first stage. If
player 1 adopts strategy i, the original benefit of player
1 and player 2 remains unchanged, which are Π1 and Π2
respectively, and then the game ends. If player 1 selects
strategy j, the game enters the second stage. In this stage,
player 1 has two strategies, p and q, player 2 has two strat-
egies, m and n, and the evolutionary game system has four
strategy combinations: (p, m), (p, n), (q, m), and (q, n);
and the benefit of player 1 and player 2 in each situation is
(Πp, Πm), (Πp, Πn), (Πq, Πm), and (Πq, Πn), respectively. In
addition, the benefit of both sides is affected by the strat-
egies of the other side, and they find their own optimal
strategies in the process of constant gaming, and finally
reach a stable state. 4.1 Game Scenario Enterprises’ participation strategies are also affected by
participation cost and stakeholder behaviors (Zhang and Enterprises’ participation strategies are also affected by
participation cost and stakeholder behaviors (Zhang and Fig. 1 Two-stage game matrix
with two players
The first stage
The second stage
Player 1
Player 2
(Π , Π )
(Π , Π )
(Π , Π )
(Π , Π )
Player 2
(Π1, Π2) The first stage 1033 International Journal of Disaster Risk Science Kong 2022).First, participating in emergency management
inevitably involves extra cost, and as a limited rational sub-
ject, if the cost of participating is greater than the benefit, the
enterprise will not choose to participate. Second, the gov-
ernment plays an important role in promoting enterprises’
participation in emergency management (Luo et al. 2022). Government subsidies can reduce the cost of participation,
and thereby improve the willingness of enterprises to par-
ticipate in emergency management. However, as the subject
of bounded rationality, the government will choose differ-
ent incentive strategies according to different participation
strategies of enterprises to maximize the performance of
emergency management. The differences in motivation and
preference of the government and enterprises make it diffi-
cult for them to construct long-term collaborative relation-
ships in emergency management. the emergency rescue stage (Yang and Zhang 2018). Long-term participation first refers to the enterprises’
participation in the whole process of emergency man-
agement, including prevention, preparation, response,
and recovery. In addition, long-term participation
means that enterprises formally become partners of the
government and effectively integrate with the govern-
ment’s emergency forces. Long-term participation of
enterprises will contribute more to emergency manage-
ment, but it also means more cost. Correspondingly, the
government also has two strategies to encourage enter-
prise participation in emergency management: “short-
term incentive” and “long-term incentive.” Compared
with short-term incentive, long-term incentive means
that the government provides emergency training to
enterprises participating in emergency management to
improve their participation efficiency. In addition, long-
term incentive aims to reduce the participation cost of
enterprises through government subsidies, thereby
enhance their willingness and capabilities to continue
to participate. The parameters of this evolutionary
game model are defined and described in Table 1. Furthermore, in the CEM of the government and enter-
prises, the latter retain the right to choose whether to par-
ticipate, and only if they do so can the CEM mechanism be
produced. 4.1 Game Scenario In the process of collaboration, the evolution of
the government and enterprise strategies has a mutually pro-
moting mechanism, and both sides seek the optimal strategy
combination through continuous evolution, imitation, and
learning, eventually reaching a stable state. Therefore, in this
study the collaborative relationships were explored based
on the perspective of evolutionary game theory, aiming at
seeking effective measures to improve the performance of
CEM between the government and enterprises. 4.3 Construction and Analysis of the Two‑Stage
Evolutionary Game Model According to the two-stage evolutionary game framework
presented in Fig. 1 and the special nature of emergency man-
agement, this study took the government and enterprises as
the players in dynamic and static games between the two
sides and constructed a pay-off matrix of two-stage evolu-
tionary game with collaborative relationships in emergency
management (Fig. 2). 4.2 Assumptions and Parameters Our proposed model is based on the following assumptions: (1) There are only two players in the evolutionary game
system—local government and private enterprises
(referred to as “government” and “enterprises”). (2) Both the government and enterprises are bounded
rational. Due to information asymmetry, they have lim-
ited abilities in rational cognition, analytical reasoning,
and decision making. 4.3.1 Analysis of the First Stage 2 Pay-off matrix of the government and enterprises 1034 Table 1 Parameters and description
Player
Notation
Description
Enterprises (E)
퓍
The probability that the enterprise chooses “participation” in the first stage ( 0 ≤퓍≤1)
퓏
The probability that the enterprise chooses “long-term participation” in the second stage ( 0 ≤퓏≤1)
CE1
The cost of the enterprise when choosing “short-term participation” in emergency management
CE2
The cost of the enterprise when choosing “long-term participation” in emergency management ( CE2 > CE1)
CE
The fixed cost of the enterprise when choosing “non-participation” strategy
RE
The original benefit of the enterprise when choosing “non-participation” strategy
퓇
The reputation benefit of the enterprise when choosing “participation” strategy
θ퓇
The incremental benefit of reputation for the enterprise when choosing “long-term participation” strategy ( θ > 0)
ρ
The benefit of emergency management when enterprises choose “short-term participation” strategy
αρ
The incremental benefit of emergency management when enterprises choose “long-term participation” strategy
( α > 0)
Government (G)
퓎
The probability that the government chooses “long-term incentive” strategy ( 0 ≤퓎≤1)
S
The subsidies provided by the government for enterprises that choose “long-term participation” strategy; enter-
prises that choose “short-term participation” strategy cannot receive these subsidies
CG1
The collaboration cost input by the government when enterprises choose “short-term participation” strategy
CG2
The collaboration cost input by the government when enterprises choose “long-term participation” strategy
(CG2 > CG1)
풹
The emergency training cost paid for enterprises when the government chooses “long-term incentive” strategy
κρ
The incremental benefit of emergency management after the enterprises have been trained ( κ > 0)
RG
The benefit of emergency management when enterprises choose “not participation” strategy
CG
The cost of emergency management of the government when enterprises choose “not participation” strategy Fig. 2 Pay-off matrix of the government and enterprises Fig. 2 Pay-off matrix of the government and enterprises of limited rationality, enterprises do not inevitably partici-
pate in emergency management even if their willingness to
do so improves. The interaction mechanism of influencing
factors on enterprises’ participation strategies is represented
by the dotted line in Fig. 3. Zhang and Kong 2022). First, according to the theory of
planned behavior (Luiza et al. 2020), some influencing fac-
tors enhance enterprises’ willingness to participate, thus
indirectly affecting their strategy choices regarding partici-
pation. Specifically, influencing factors comprise internal
enterprise factors and external environmental factors. 4.3.1 Analysis of the First Stage The interaction mechanism of influencing
factors on enterprises’ participation strategies is represented
b
h d
d li
i Fi
3
Table 1 Parameters and description
Player
Notation
Description
Enterprises (E)
퓍
The probability that the enterprise chooses “participation” in the first stage ( 0 ≤퓍≤1)
퓏
The probability that the enterprise chooses “long-term participation” in the second stage ( 0 ≤퓏≤1)
CE1
The cost of the enterprise when choosing “short-term participation” in emergency management
CE2
The cost of the enterprise when choosing “long-term participation” in emergency management ( CE2 > CE1)
CE
The fixed cost of the enterprise when choosing “non-participation” strategy
RE
The original benefit of the enterprise when choosing “non-participation” strategy
퓇
The reputation benefit of the enterprise when choosing “participation” strategy
θ퓇
The incremental benefit of reputation for the enterprise when choosing “long-term participation” strategy ( θ > 0)
ρ
The benefit of emergency management when enterprises choose “short-term participation” strategy
αρ
The incremental benefit of emergency management when enterprises choose “long-term participation” strategy
( α > 0)
Government (G)
퓎
The probability that the government chooses “long-term incentive” strategy ( 0 ≤퓎≤1)
S
The subsidies provided by the government for enterprises that choose “long-term participation” strategy; enter-
prises that choose “short-term participation” strategy cannot receive these subsidies
CG1
The collaboration cost input by the government when enterprises choose “short-term participation” strategy
CG2
The collaboration cost input by the government when enterprises choose “long-term participation” strategy
(CG2 > CG1)
풹
The emergency training cost paid for enterprises when the government chooses “long-term incentive” strategy
κρ
The incremental benefit of emergency management after the enterprises have been trained ( κ > 0)
RG
The benefit of emergency management when enterprises choose “not participation” strategy
CG
The cost of emergency management of the government when enterprises choose “not participation” strategy
Fig. 4.3.1 Analysis of the First Stage Whether enterprises should participate in emergency man-
agement is no longer a question. The real question is what
roles enterprises should play and how to improve emergency
management capabilities by establishing effective public-
private collaboration mechanisms. Therefore, we analyzed
the motivation of enterprises to participate and its influenc-
ing factors in the first stage of this evolutionary game model. In the process of emergency management, enterprises have
the option of priority, the participation of enterprises can be
regarded as event A, and the probability that event A occurs
is x. Then P(A) = x, P
(
A
)
= 1 −x. (3) The government will always adopt active emergency
management strategies toward social stability, security,
and sustainable development. (4) Enterprises are free to choose whether or not to partici-
pate in emergency management, respectively defined
as “participation” and “non-participation.” The game
will enter the second stage if and only if the enterprise
chooses the “participation” strategy in the first stage. i
(5) In the second stage, enterprises have two strategies:
“short-term participation” and “long-term participa-
tion.” Short-term participation refers to the tempo-
rary and random participation behavior of enterprises
in emergency management, which often occurs in Previous studies have shown that enterprise participation
in emergency management results from many influencing
and decisive factors (Lu and Zhu 2022; Luo et al. 2022; Wang et al. A Two-Stage Evolutionary Game Model for Collaborative Emergency Management 1034
Wang et al. A Two-Stage Evolutionary Game Model for Collaborative Emergency Management
Zhang and Kong 2022). First, according to the theory of
planned behavior (Luiza et al. 2020), some influencing fac-
tors enhance enterprises’ willingness to participate, thus
indirectly affecting their strategy choices regarding partici-
i
S
ifi
ll
i fl
i
f
i
i
l
of limited rationality, enterprises do not inevitably partici-
pate in emergency management even if their willingness to
do so improves. 4.3.2 Analysis of the Second Stage (4) Incentives from the government have a significant impact
on enterprise participation in emergency management
(Luo et al. 2022). They include a series of targeted poli-
cies, providing participation conveniences, and benefit such
as financial rewards and technical or policy support, which
are conductive to enterprises’ own economic development. Incentives from the government have a significant impact
on enterprise participation in emergency management
(Luo et al. 2022). They include a series of targeted poli-
cies, providing participation conveniences, and benefit such
as financial rewards and technical or policy support, which
are conductive to enterprises’ own economic development. Therefore, the second stage focuses mainly on sustainable
collaboration between the government and enterprises in
emergency management, considering the choice of govern-
ment incentives and the choice of enterprises’ participation
strategy, and exploring their behavior evolution rules and
stability strategies. The benefit of the government choosing the long-term
incentive strategy is EG1: The benefit of the government choosing the long-term
incentive strategy is EG1: (5)
EG1 =퓏[
(1 + α + κ)ρ −S −풹−CG2
]
+ (1 −퓏)
[
(1 + κ)ρ −풹−CG1
] (5) Therefore, the second stage focuses mainly on sustainable
collaboration between the government and enterprises in
emergency management, considering the choice of govern-
ment incentives and the choice of enterprises’ participation
strategy, and exploring their behavior evolution rules and
stability strategies. The benefit of the government choosing the short-term
incentive strategy is EG2: (6)
EG2 = 퓏[
(1 + α)ρ −CG2
] + (1 −퓏)
(ρ −CG1
) (6) Then the average expected revenue is recorded as EG: Then the average expected revenue is recorded as EG: Under the premise of bounded rationality, the evolution-
ary game of CEM between the government and enterprises
is a process of mutual learning and dynamic self-adaptation. Therefore, we used the replicated dynamic equation to simu-
late their decision-making process. The benefit of the enter-
prises choosing the long-term participation strategy is EE1: (7)
EG = 퓎EG1 + (1 −퓎)EG2 (7) The replicated dynamic equation of the government
choosing the long-term incentive strategy is: The replicated dynamic equation of the government
choosing the long-term incentive strategy is: Therefore, we used the replicated dynamic equation to simu-
late their decision-making process. 4.3.1 Analysis of the First Stage The
former includes emergency resources, experience, and capa-
bilities possessed by enterprises themselves, while the latter
are external, particularly participation policies, emergency
culture, and other participants. As an economic stakeholder Second, decisive factors include extra benefit ( 휔 ) and
cost (c) of participation for enterprises, which have a more
significant and direct impact on their decision on whether
to participate (Shi et al. 2023). Therefore, whether enter-
prises participate in emergency management depends International Journal of Disaster Risk Science 1035 Decisive
factors
Participation
willingness
Participation
behavior
Emergency resources
Emergency capabilities
Emergency culture
The second
stage
Influencing factors
Emergency experience
Other participants
Participation policies
Yes
No
No
Yes
End
Fig. 3 Evolution stability in stage 1. U(ω, 풸) = ω −풸 refers to the utility function of enterprises in the first stage, ω refers to the extra benefit of
enterprises’ participation in emergency management, 풸 refers to the extra cost of enterprises’ participation. Influencing factors Fig. 3 Evolution stability in stage 1. U(ω, 풸) = ω −풸 refers to the utility function of enterprises in the first stage, ω refers to the extra benefit of
enterprises’ participation in emergency management, 풸 refers to the extra cost of enterprises’ participation. (2)
EE2 = 퓎[
(1 + 휅)휌+ 퓇−CE1
] + (1 −퓎)
(휌+ 퓇−CE1
) ultimately on the utility function of participation, which
can be expressed as U(휔, c) = 휔−c . If and only if
U(𝜔, c) = 𝜔−c > 0, then c = 1 , and the enterprise will
choose participation strategy, with the evolutionary game then
entering the second stage. Otherwise, x = 0, and the enter-
prise chooses not to participate because the cost outweighs
the benefit; correspondingly, the government will choose the
non-incentive strategy (Fig. 3). (2) Then the average expected revenue is recorded as EE: Then the average expected revenue is recorded as EE: (3)
EE = 퓏EE1 + (1 −퓏)EE2 (3) The replicated dynamic equation of enterprises choos-
ing the long-term participation strategy is: (4)
풹퓏
풹퓉
=퓏퓉
(
EE1 −EE
)
= 퓏퓉
(
1 −퓏퓉
)
(
αρ + θ퓇+ CE1 −CE2 + S퓎퓉
) 4.3.3 Model Stability Test (11) The replicated dynamic equation needs to meet stability
requirements to ensure it will not significantly change due
to slight shifts in parameters. We therefore tested stability
before numerical analysis using the stability analysis method
suggested by Wei et al. (2012) and Sun et al. (2023): the sen-
sitivity index is calculated by changing 10% of key factors;
and if the sensitivity index is less than 1, the model is stable. The sensitivity index is calculated as follows: The stability of the equilibrium point in the differential
system can be calculated by analyzing the local stability of
the Jacobi matrix: (12)
J =
[ (1 −2퓏퓉
)(훼휌+ 휃퓇+ CE1 −CE2 + S퓎퓉
)
S퓏퓉
(1 −퓏퓉
)
−S퓎퓉
(1 −퓎퓉
)
(1 −2퓎퓉
)(휅휌−풹−S퓏퓉
)
] (12) Then the determinant of the Jacobi matrix can be calcu-
lated as follows: The sensitivity index is calculated as follows: (15)
SP = 1
n
n
∑
i=1
||||
dPt
Pt
⋅Ut
dUt
|||| (15) (13)
Dℯ𝓉(J) =(1 −2𝓏𝓉
)(1 −2𝓎𝓉
)(훼휌+ 휃𝓇+ CE1 −CE2 + S𝓎𝓉
)
(κρ −𝒹−S𝓏𝓉
) + 𝓏𝓉𝓎𝓉
(1 −𝓏𝓉
)(1 −𝓎𝓉
)S2 (13) where t denotes time; Sp refers to the sensitivity of system
state P to parameter U; Pt represents the system state at time
t; Ut is the value of the system parameter at time t; dPt and
dUt respectively denote the values of changes in system state
P and parameter U at time t; n is the number of sensitivity
test points, and n = 2. We tested the sensitivity of some key
model parameters (including 훼, 휃, CE1, CE2, κ, ρ, 풹, 퓇, andS )
by changing the value of each by +10% and −10% respec-
tively. As shown by the results in Fig. 4, all the sensitivity
indices are less than 1, which indicates that the model passed
the stability test. Next, the trace of the Jacobi matrix can be calculated
thus: (14)
T퓇(J) =(1 −2퓏퓉
)(훼휌+ 휃퓇+ CE1 −CE2 + S퓎퓉
)
+ (1 −2퓎퓉
)(휅휌−풹−S퓏퓉
) (14) According to the evolutionary game theory, if
Dℯ𝓉(J) > 0 and T퓇(J) < 0 are satisfied at equilibrium
points of the replicated dynamic equations, they constitute
ESS of the system. For each equilibrium point, the determi-
nant and trace of the system are shown in Table 2. 4.3.3 Model Stability Test According to the above hypothesis, as well as the deter-
minant and trace of equilibrium points in Table 2, the local
stability of this evolutionary game system can be judged. Specifically: 4.3.2 Analysis of the Second Stage The benefit of the enter-
prises choosing the long-term participation strategy is EE1: (8)
풹퓎
풹퓉
= 퓎퓉
(EG1 −EG
) = 퓎퓉
(1 −퓎퓉
)[(κρ −풹−S퓏퓉
) (8) (1)
EE1 =퓎[
(1 + 훼+ 휅)휌+ (1 + 휃)퓇+ S −CE2
]
+ (1 −퓎)
[
(1 + 훼)휌+ (1 + 휃)퓇−CE2
] (1) Therefore, the two-dimensional dynamic system of the
evolutionary game of CEM between the government and
enterprise is: The benefit of the enterprises choosing the short-term
participation strategy is EE2: 1036 Wang et al. A Two-Stage Evolutionary Game Model for Collaborative Emergency Management (2) If 𝛼𝜌+ 𝜃퓇+ CE1 −CE2 + S < 0and𝜅𝜌−풹> 0 , then (1,
0) is ESS of the system. (2) If 𝛼𝜌+ 𝜃퓇+ CE1 −CE2 + S < 0and𝜅𝜌−풹> 0 , then (1,
0) is ESS of the system. (9)
⎧
⎪
⎨
⎪⎩
풹퓏
풹퓉= 퓏퓉
EE1 −EE
= 퓏퓉
1 −퓏퓉
αρ + θ퓇+ CE1 −CE2 + S퓎퓉
풹퓎
풹퓉= 퓎퓉
EG1 −EG
= 퓎퓉
1 −퓎퓉
κρ −풹−S퓏퓉
(9) )
y
(3) If 𝛼𝜌+ 𝜃퓇+ CE1 −CE2 > 0 and 𝜅𝜌−풹−S < 0 , then
(0, 1) is ESS of the system. Let dy
/dt = 0, dz
/dt = 0 then on the plane
N = {(z, y); 0 ≤z, y ≤1} , this system has five equilibrium
points: (0, 0), (0, 1), (1, 0), (1, 1), and ( y∗
t,z∗
t ). Among them: y
(4) If 𝛼𝜌+ 𝜃퓇+ CE1 −CE2 + S > 0 and 𝜅𝜌−풹−S > 0 ,
then (1, 1) is ESS of the system. y
(4) If 𝛼𝜌+ 𝜃퓇+ CE1 −CE2 + S > 0 and 𝜅𝜌−풹−S > 0 ,
then (1, 1) is ESS of the system. y
(5) In any case, the equilibrium point ( 퓎∗
퓉
,퓏∗
퓉 ) cannot be an ESS of the system; and if
퓏∗
퓉퓎∗
퓉
(1 −퓏∗
퓉
)(1 −퓎∗
퓉
)S2 < 0 , then ( 퓎∗
퓉,퓏∗
퓉 ) is saddle
point of the system. (10)
퓎∗
퓉= CE2 −CE1 −훼휌−휃퓇
S (10)
퓎∗
퓉= CE2 −CE1 −훼휌−휃퓇
S
(11)
퓏∗
퓉= 휅휌−풹
S (10) (11)
퓏∗
퓉= 휅휌−풹
S 5.1 Numerical Analysis Under Different Situations To show the dynamic evolution process of enterprise and
government strategy selection more intuitively, we adopted
numerical simulation in this study, using MATLAB to
simulate the dynamic evolution trajectory from initial point
to equilibrium point in several situations. The simulation
results are shown in Fig. 5. g
}
Situation (c): When 𝛼𝜌+ 𝜃퓇> CE2 −CE1 , 𝜅𝜌< 풹+ S ,
and 훼= 0.4, 휃= 0.3, CE1 = 10, CE2 = 18, 휅= 0.7, 휌= 5,
풹= 2, 퓇= 10, S = 2, the evolution step of the system is
shown in Fig. 5c. In this situation, the sum of emergency
management benefit and reputation benefit exceeds the
incremental cost of participation, leading enterprises
to choose long-term participation strategy regardless
of which incentive strategy is pursued by the govern-
ment. For the government, however, the subsidies and
emergency management training cost paid to enterprises
pursuing long-term participation exceed the incremental
benefit for emergency management brought by trained
enterprises. Therefore, government will pursue the short-
term incentive strategy, and the evolution result of the
system finally converges to (1, 0). In this situation, the
ESS is {long-term participation, short-term incentive}. Situation (d): When 𝛼𝜌+ 𝜃퓇+ S > CE2 −CE1 , 𝜅𝜌> 풹+ S ,
and 훼= 0.5, 휃= 0.6, CE1 = 10, CE2 = 16, 휅= 0.8, 휌= 5,
풹= 1, 퓇= 10, S = 1, the evolution step of the system is
shown in Fig. 5d. This situation entails that the incremental
benefit of enterprises’ participation in emergency manage- g
Situation (c): When 𝛼𝜌+ 𝜃퓇> CE2 −CE1 , 𝜅𝜌< 풹+ S ,
and 훼= 0.4, 휃= 0.3, CE1 = 10, CE2 = 18, 휅= 0.7, 휌= 5,
풹= 2, 퓇= 10, S = 2, the evolution step of the system is
shown in Fig. 5c. In this situation, the sum of emergency
management benefit and reputation benefit exceeds the
incremental cost of participation, leading enterprises
to choose long-term participation strategy regardless
of which incentive strategy is pursued by the govern-
ment. For the government, however, the subsidies and
emergency management training cost paid to enterprises
pursuing long-term participation exceed the incremental
benefit for emergency management brought by trained
enterprises. Therefore, government will pursue the short-
term incentive strategy, and the evolution result of the
system finally converges to (1, 0). In this situation, the
ESS is {long-term participation, short-term incentive}. 5 Numerical Analysis This section analyzes the system’s evolution path under
different situations by adopting SD simulation methods, as
well as the effects of cost difference, government subsidies, (1) If 𝛼𝜌+ 𝜃퓇+ CE1 −CE2 < 0 and κρ −풹< 0 , then (0, 0)
is ESS of the system. Table 2 Dℯ𝓉(J) and T퓇(J) in equilibrium points
Equilibrium points
Dℯ𝓉(J)
T퓇(J)
(0, 0)
(훼휌+ 휃퓇+ CE1 −CE2
)
(κρ −풹)
(훼휌+ 휃퓇+ CE1 −CE2
) + (휅휌−풹)
(1, 0)
−(훼휌+ 휃퓇+ CE1 −CE2 + S)
(κρ −풹)
(훼휌+ 휃퓇+ CE1 −CE2 + S) −(휅휌−풹)
(0, 1)
−(훼휌+ 휃퓇+ CE1 −CE2
)
(κρ −풹−S)
−(훼휌+ 휃퓇+ CE1 −CE2
) + (휅휌−풹−S)
(1, 1)
(훼휌+ 휃퓇+ CE1 −CE2 + S)
(κρ −풹−S)
−(훼휌+ 휃퓇+ CE1 −CE2 + S) −(휅휌−풹−S)
(퓏∗
퓉, 퓎∗
퓉)
퓏∗
퓉퓎∗
퓉
(1 −퓏∗
퓉
)(1 −퓎∗
퓉
)S2
0 Table 2 Dℯ𝓉(J) and T퓇(J) in equilibrium points
Equilibrium points
Dℯ𝓉(J)
T퓇(J)
(0, 0)
(훼휌+ 휃퓇+ CE1 −CE2
)
(κρ −풹)
(훼휌+ 휃퓇+ CE1 −CE2
) + (휅휌−풹)
(1, 0)
−(훼휌+ 휃퓇+ CE1 −CE2 + S)
(κρ −풹)
(훼휌+ 휃퓇+ CE1 −CE2 + S) −(휅휌−풹)
(0, 1)
−(훼휌+ 휃퓇+ CE1 −CE2
)
(κρ −풹−S)
−(훼휌+ 휃퓇+ CE1 −CE2
) + (휅휌−풹−S)
(1, 1)
(훼휌+ 휃퓇+ CE1 −CE2 + S)
(κρ −풹−S)
−(훼휌+ 휃퓇+ CE1 −CE2 + S) −(휅휌−풹−S)
(퓏∗
퓉, 퓎∗
퓉)
퓏∗
퓉퓎∗
퓉
(1 −퓏∗
퓉
)(1 −퓎∗
퓉
)S2
0 Table 2 Dℯ𝓉(J) and T퓇(J) in equilibrium points 1037 International Journal of Disaster Risk Science shown in Fig. 5b. Under this situation, the sum of the
increase of emergency management benefit, reputation
benefit, and government subsidies obtained by choosing
long-term participation strategy is outweighed by the
incremental cost of participation. Consequently, enter-
prises will choose short-term participation strategy. For
the government, the incremental benefit of enterprises
supporting emergency management after training exceeds
the training cost paid by the government. Therefore, the
government will choose the long-term incentive strategy,
and the evolution result of the system finally converges to
(0, 1). In this situation, the ESS is {short-term participa-
tion, long-term incentive}. reputation benefit, and training cost on the evolutionary
game paths of long-term participation and short-term par-
ticipation, thereby further exploring the collaboration mech-
anism between enterprises and governments. Fig. 4 Parameter sensitivity
index chart 5.1 Numerical Analysis Under Different Situations Situation (d): When 𝛼𝜌+ 𝜃퓇+ S > CE2 −CE1 , 𝜅𝜌> 풹+ S ,
and 훼= 0.5, 휃= 0.6, CE1 = 10, CE2 = 16, 휅= 0.8, 휌= 5,
풹= 1, 퓇= 10, S = 1, the evolution step of the system is
shown in Fig. 5d. This situation entails that the incremental
benefit of enterprises’ participation in emergency manage- Situation (c): When 𝛼𝜌+ 𝜃퓇> CE2 −CE1 , 𝜅𝜌< 풹+ S ,
and 훼= 0.4, 휃= 0.3, CE1 = 10, CE2 = 18, 휅= 0.7, 휌= 5, Situation (a): When 𝛼𝜌+ 𝜃퓇< CE2 −CE1 , κρ < 풹 , and
훼= 0.2, 휃= 0.3, CE1 = 10, CE2 = 16, 휅= 0.4, 휌= 5,
풹= 4, 퓇= 10, S = 1, the evolution step of the system is
shown in Fig. 5a. In this situation, the incremental benefit
for the government to pay for training an enterprise in
emergency management is disproportionate to the train-
ing cost when pursuing a long-term incentive strategy. Consequently, the government is unwilling to pay train-
ing cost for enterprises and tends to pursue a short-term
incentive strategy. Meanwhile, the sum of incremental
emergency management benefit and reputation benefit for
enterprises from long-term participation is outweighed
by the extra cost paid. Therefore, as bounded rational
stakeholders, enterprises will choose the short-term par-
ticipation strategy to maximize self-interest. Finally, the
evolution result of the system converges to (0, 0), and the
ESS is {short-term participation, short-term incentive}. Situation (b): When 𝛼𝜌+ 𝜃퓇+ S < CE2 −CE1, 𝜅𝜌> 풹 , and
훼= 0.5, 휃= 0.5, CE1 = 10, CE2 = 17, 휅= 0.6, 휌= 5,
풹= 2, 퓇= 10, S = 2, the evolution step of the system is Situation (a): When 𝛼𝜌+ 𝜃퓇< CE2 −CE1 , κρ < 풹 , and
훼= 0.2, 휃= 0.3, CE1 = 10, CE2 = 16, 휅= 0.4, 휌= 5, Situation (a): When 𝛼𝜌+ 𝜃퓇< CE2 −CE1 , κρ < 풹 , and
훼= 0.2, 휃= 0.3, CE1 = 10, CE2 = 16, 휅= 0.4, 휌= 5, 풹= 4, 퓇= 10, S = 1, the evolution step of the system is
shown in Fig. 5a. In this situation, the incremental benefit
for the government to pay for training an enterprise in
emergency management is disproportionate to the train-
ing cost when pursuing a long-term incentive strategy. 5.2.1 Effect of Enterprise Participation Cost on Evolution
Results ment exceeds the training cost and subsidies paid by the
government. Consequently, whichever strategy enterprises
choose, the government will pursue long-term incentive
strategy. For enterprises, the sum of incremental emer-
gency management benefit, reputation benefit, and gov-
ernment subsidies exceeds the extra cost of pursuing long-
term participation. Therefore, enterprises will choose the
long-term participation strategy, and the evolution result of
the system finally converges to (1, 1), the ESS under this
situation is {long-term participation, long-term incentive}. Other parameters are: 훼= 0.6, 휃= 0.4, κ = 0.6, ρ = 5, 풹= 6,
퓇= 10, S = 15. T h e
i n i t i a l
p r o b a b i l i t y
i s
퓏0 = 0.5and퓎0 = 0.5 ; the incremental cost of enterprises
choosing long-term versus short-term participation is
Δ풸= CE2 −CE1 , varying between [0, 10]; and the evolution
result of the system is shown in Fig. 6a. The result of the
numerical analysis shows that if 0 ≤Δ풸≤6 , an enterprise’s
ESS converges to long-term participation, and the evolution
speed decreases as Δ풸 rises; if Δ풸≥8 , then the enterprises’
ESS converges to short-term participation, and the evolu-
tion speed increases as Δ풸 rises. Compared with short-term
participation, enterprises are willing to adopt long-term
participation strategy when the cost increment of long-term
participation is small. On the contrary, as the cost increment 5.1 Numerical Analysis Under Different Situations Consequently, the government is unwilling to pay train-
ing cost for enterprises and tends to pursue a short-term
incentive strategy. Meanwhile, the sum of incremental
emergency management benefit and reputation benefit for
enterprises from long-term participation is outweighed
by the extra cost paid. Therefore, as bounded rational
stakeholders, enterprises will choose the short-term par-
ticipation strategy to maximize self-interest. Finally, the
evolution result of the system converges to (0, 0), and the
ESS is {short-term participation, short-term incentive}. Situation (b): When 𝛼𝜌+ 𝜃퓇+ S < CE2 −CE1, 𝜅𝜌> 풹 , and
훼= 0.5, 휃= 0.5, CE1 = 10, CE2 = 17, 휅= 0.6, 휌= 5,
풹= 2, 퓇= 10, S = 2, the evolution step of the system is Fig. 4 Parameter sensitivity
index chart meter sensitivity Fig. 4 Parameter sensitivity
index chart Wang et al. A Two-Stage Evolutionary Game Model for Collaborative Emergency Management 1038 Fig. 5 Evolution steps of the system under different situations Fig. 5 Evolution steps of the system under different situations 5.2.1 Effect of Enterprise Participation Cost on Evolution
Results 5.2 Numerical Analysis of Parameter Variation With the initial probability unchanged, we analyzed the
effect of each parameter on the evolutionary game path. The
results of the numerical analysis are shown in Fig. 6. International Journal of Disaster Risk Science 1039 1039
ational Journal of Disaster Risk Science
ng-term participation increases gradually, enterprises
adopt short-term participation strategy. 5.2.2 Effect of Reputation Benefit on Evolution Results
Otherparametersare:훼= 0 6 휃= 0 4 κ = 0 6 ρ = 5 풹= 6
Effect of parameter variation on evolution results. Δ풸 refers to the incremental cost of enterprises choosing long-term versus short-term
pation strategy. Fig. 6 Effect of parameter variation on evolution results. Δ풸 refers to the incremental cost of enterprises choosing long-term versus short-
participation strategy. Fig. 6 Effect of parameter variation on evolution results. Δ풸 refers to the incremental cost of enterprises choosing long-term versus short-term
participation strategy. of long-term participation increases gradually, enterprises
will adopt short-term participation strategy. of long-term participation increases gradually, enterprises
will adopt short-term participation strategy. 5.2.2 Effect of Reputation Benefit on Evolution Results Other parameters are: 훼= 0.6, 휃= 0.4, κ = 0.6, ρ = 5, 풹= 6,
CE2 = 20, CE1 = 10, S = 15 . The initial probability is Other parameters are: 훼= 0.6, 휃= 0.4, κ = 0.6, ρ = 5, 풹= 6,
CE2 = 20, CE1 = 10, S = 15 . The initial probability is 1040 Wang et al. A Two-Stage Evolutionary Game Model for Collaborative Emergency Management 5.2.3 Effect of Government Subsidies on Evolution Results Other parameters are: 훼= 0.6, 휃= 0.4, κ = 0.6, ρ = 15, 풹= 6,
CE2 = 20, CE1 = 10, 퓇= 10 . The initial probability is
퓏0 = 0.5and퓎0 = 0.5 ; the value of government subsidies
S varies within the range [0, 10]; and the evolution result
of the system is shown in Fig. 6c. The result shows that if
0 ≤S ≤2 , the government’s ESS converges to long-term
participation, and the evolution speed decreases as S rises;
If S ≥4 , the government’s ESS converges to short-term par-
ticipation, and the evolution speed increases as S rises. For
enterprises, their ESS converges to long-term participation,
and the evolution speed increase as S rises (Fig. 6d). The
results indicate that as the government subsidies increase,
the government tends toward pursuing the short-term incen-
tive strategy. On the contrary, government subsidies have
positive effect on enterprises’ participation strategy, and the
more subsidies provided by the government, the less time it
takes for enterprises to converge to long-term participation. The simulation results reveal that in the second stage,
there are two situations in which the government will choose
long-term incentive strategy: the first is that although the
enterprises adopt short-term participation strategy, the incre-
mental benefit of enterprises supporting emergency man-
agement after training exceeds the training cost paid by the
government; the second is that the enterprises adopt long-
term participation strategy, and the incremental benefit of
enterprise participation in emergency management exceeds
the training cost and subsidies paid by the government. Correspondingly, there are also two situations in which the
enterprises will choose long-term participation strategy:
the first is that although the government adopt short-term
incentive strategy, the sum of emergency management ben-
efit and reputation benefit exceeds the incremental cost of
long-term participation; the second is that the government
adopt long-term incentive strategy, and the sum of incre-
mental emergency management benefit, reputation benefit,
and government subsidies exceeds the extra cost of pursuing
long-term participation. 6 Conclusion and Future Prospects 퓏0 = 0.5and퓎0 = 0.5 ; the value of the reputation benefit 퓇
obtained by enterprises for participating in emergency man-
agement varies within the range [0, 20], and the evolution
result of the system is shown in Fig. 6b. The result shows
that if 0 ≤퓇≤15 , the enterprise’s ESS converges to short-
term participation, and the evolution speed decreases as 퓇
rises; If 퓇≥20 , the enterprise’s ESS converges to long-term
participation, and the evolution speed increases as 퓇 rises. This indicates that increasing reputation benefit incentiv-
ize enterprises to pursue long-term participation; otherwise,
they will adopt the short-term participation strategy. In other
words, reputation benefit has a positive impact on enter-
prises’ strategy choice. With the complexity and impact of various emergencies
worldwide continuing to increase, enterprise participation
has become an indispensable part of effective response to
emergencies. However, the performance of enterprises par-
ticipating in emergency management needs to be further
improved. Considering the challenges of enterprise partici-
pation and government incentives, this study constructed a
two-stage evolutionary game model based on stakeholders’
bounded rationality to analyze the behavioral evolution char-
acteristics of both participants in the emergency manage-
ment system, and analyzed the system’s evolution path under
different situations using SD simulation, thereby exploring
the collaboration mechanism between the government and
enterprises. We found that although enterprise participation
in emergency management is affected by both influencing
and decisive factors, their final decision depends on the util-
ity function U(휔, 풸) : if and only if U(𝜔, 풸) = 𝜔−풸> 0, an
enterprise will choose the participation strategy and enter
the emergency management system, initiating the second
stage of the evolutionary game. 5.2.4 Effect of Training Cost on Evolution Results Other parameters are: 훼= 0.6, 휃= 0.4, κ = 0.6, ρ = 5, CE2 = 20,
CE1 = 10, 퓇= 10, S = 15. The initial probability is
퓏0 = 0.5 and 퓎0 = 0.5 ; training cost 풹 paid by the govern-
ment varies within the range [0.5, 3]; and the evolution result
of the system is shown in Fig. 6e. Some cases in this situ-
ation converge slowly or fail to converge to a stable state. Therefore, to show the long-term trend, evolution time 퓉 is
extended to [0, 50], and the evolution result of the system is
again shown in Fig. 6e. The results indicate that if 풹≥2 ,
the government’s ESS converges to short-term incentive
strategy; If 0.5 ≤풹≤1.5 , there is less stability in whether
the government chooses to pay the emergency management
training cost for enterprises. In addition, if other factors remain unchanged and the
cost increment of long-term participation is low, the enter-
prises’ ESS converges to long-term participation, whereas
as emergency management cost increases, enterprises tend
to opt for short-term participation. With the increase of
reputation benefit, enterprises will turn their ESS from
short-term participation into long-term participation. In other words, reputation benefit has a positive impact
on enterprises’ strategy choice. Similarly, government
subsidies such as tax reduction and preferential policies 1041 International Journal of Disaster Risk Science provide important references for government measures pro-
moting CEM performance through enterprise participation. However, this study also has some limitations that future
research should address. First, while this study considers
only enterprises and governments, CEM in practice also
includes social organizations, citizens, media, and other
stakeholders. Therefore, future research needs to compre-
hensively analyze collaboration relationship among multiple
agents in the emergency management process. Second, as
a complex system, the government-enterprise collabora-
tive relationship in emergency management is affected by
many factors that were given little attention in this study. Some influencing factors, such as relevant regulations and
polices need to be further discussed in future research. In
particular, the introduction of information and communica-
tion technology such as blockchain, 5G, and big data offers
great potential to improve CEM capability with multi-agent
participation. More studies are needed to further explore
how the application of these technologies affects the game
strategy choice of enterprises and governments for emer-
gency management. incentivize enterprises to pursue a long-term participation
strategy. 5.2.4 Effect of Training Cost on Evolution Results Overall, if and only if the ESS of the system con-
verges to {long-term participation, long-term incentive},
the benefit of CEM for both the government and enter-
prises outweigh the extra cost they paid. Therefore, the
most important thing is that the government should adopt
effective measures to promote the continuous participa-
tion of enterprises in emergency management. First, the
government can provide continuous financial subsidies
and promotion incentive for enterprises’ participation. Funding This work was supported by the Major Project of National
Social Science Foundation of China (Grant No. 21&ZD166), the
National Social Science Foundation of China (Grant No. 22VRC200),
and the China Scholarship Council (CSC, Grant No. 202206420064). 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/. 5.2.4 Effect of Training Cost on Evolution Results If other factors remain unchanged, the increase
in government subsidies will increase the evolution speed
of enterprises’ ESS converging to long-term participation,
but the ESS of the government simultaneously converges
to short-term incentive. Finally, when the cost of emer-
gency management training is low, there is less stability
in whether the government chooses to pay the training cost
for enterprises, but as the training cost rises, the ESS of
the government quickly converges to short-term incentive. y
gency management training is low, there is less stability
in whether the government chooses to pay the training cost
for enterprises, but as the training cost rises, the ESS of
the government quickly converges to short-term incentive. Overall, if and only if the ESS of the system con-
verges to {long-term participation, long-term incentive},
the benefit of CEM for both the government and enter-
prises outweigh the extra cost they paid. Therefore, the
most important thing is that the government should adopt
effective measures to promote the continuous participa-
tion of enterprises in emergency management. First, the
government can provide continuous financial subsidies
and promotion incentive for enterprises’ participation. Financial subsidies such as tax breaks can directly reduce
the cost of enterprise participation, promotion incentive
such as honor system can improve the reputation benefit of
enterprise participation, and both financial subsidies and
promotion incentive can promote continuous participation
behavior of enterprises in emergency management. Sec-
ond, the government also needs to optimize the commu-
nication mechanisms between itself and enterprises, and
provide diversified participation channels and information
support for enterprises. This can not only reduce the cost
of coordination for both the government and enterprises
in CEM, but also improve the benefit of CEM between the
government and enterprises, so as to establish long-term
collaborative relationships in emergency management. Third, the government should also provide systematic
emergency training for enterprises to improve their capa-
bilities to participate in emergency management, thereby
giving full play to the advantages and potential of enter-
prise participation. For enterprises, since corporate social
responsibility can positively and significantly influence
sustainable competitive advantages (Wang et al. 2023),
they should strengthen their emergency capabilities in
daily operation and management from the perspective of
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of corporate social responsibility on sustainable competitive
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Hautkrankheiten
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Archives of dermatological research
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Sonstige Dermatosen. l~Ialoney, William J. Uber die Beziehungen
der chemi-
schen
Zusammensetzung
der Muttermilch
zur Diarrhoe
u nd zum Ek zero d cr S~uglinge. The Practitioner. Febr. 1911. M a 1 on e y gelangt dureh seine Untersuchungen zu der Anschauung,
daft wohl Uberern~hrung des S~iuglings, allzu reicher Zuckergehal~ der
Milch oder fehlerhafte Zusammensetzung derselben infolge ~a]seher Er-
n~hrung dcr Mutter als Ursachc fiir das Zustandekommen der S~uglings-
ekzeme in Betracht komme; der Umstand jedocb, daft solche aueh bei
Feh|en dieser Momente beobachtct werden, spricht dafiir, daf noch andere,
mit der Ern~hrung nicht im Zusammenhange stehende Faktoren als Ur-
sachen anzusehen sind. Wilhelm B alb an (Wien). M a 1 on e y gelangt dureh seine Untersuchungen zu der Anschauung,
daft wohl Uberern~hrung des S~iuglings, allzu reicher Zuckergehal~ der
Milch oder fehlerhafte Zusammensetzung derselben infolge ~a]seher Er-
n~hrung dcr Mutter als Ursachc fiir das Zustandekommen der S~uglings-
ekzeme in Betracht komme; der Umstand jedocb, daft solche aueh bei
Feh|en dieser Momente beobachtct werden, spricht dafiir, daf noch andere,
mit der Ern~hrung nicht im Zusammenhange stehende Faktoren als Ur-
sachen anzusehen sind. Wilhelm B alb an (Wien). Poltowitsch, K. Ein Fall yon chronischem Ekzem. (Zur
Frage
des
Ursprunges,
des klinischen
Bildes und der
B h
dl
di
K
kh it ) R
k
W
t
h 1910 N
14
15 Poltowitsch, K. Ein Fall yon chronischem Ekzem. (Zur
Frage
des
Ursprunges,
des klinischen
Bildes und der
Behandlung dieser Krankheit.) Russky Wratsch. 1910. Nr. 14u. 15. Nichts Neues. P.A. Welikanow (Petersburg). Behandlung dieser Krankheit.) Russky Wratsch. 1910. Nr. 14u. 15. Nichts Neues. P.A. Welikanow (Petersburg). Roeaz. S~uglingsekzcm. Archives de Mcdecine des Enfants. T. XV. 1911. p. 81. Die Atiologie der Ekzeme ist bisher noch nlcht gekl~rt; man
nimmt eine Intoxikation als Ursache an, ohne fiber die Natur der Intoxi-
kation etwas Sicheres zu wissen. In Betracht kommen aliment~re
Seh~digungen, die besonders bci kiinstlich gen~hrten Kindcrn die Ursache
der Ekzeme sein k6nnen. Dcr Verf. meint, daft besonders grol~e Flfissig-
keitsmengen die Intoxikation begfinstigen. Auch h~lt Verf. es ffir mSg-
lich, dab durch einc unzweckm~fige Ern~hrung der Amine Toxine ent-
stehen k6nnen, die dureh die Milch den Kindern zugefiihrt werden und
zu Ekzemen ffihrcn k6nncn. Bei eincr Reihe yon Kindern kommt cs zu
Ekzemen, ohnc daf irgend einc Ern~hrungsst6rung vorliegt; in solchen
FMlen soll der Arthritismus der Eltern yon ~itiologischer Bedeutung sein. ]n manchen F~l|en yon Ekzem sollcn nach Ansicht des Yeff. Sonstige Dermatosen. St6rungen
der Dri~sen mit innerer Sekretion vorliegcn, was aus den thcrapeutischen 36* Bericht fiber die Leistungen auf dera Gebiete 564 Erfolgen, z. B. Behandlung tier Ekzeme mit Schildd~fisenpr~paraten zu
erschliel~en sei. Den Ubergang von den Ekzemen zu den Erythemen bildet eine
jfingst yon Moussous studierte Hautaffektion, der er den •amen 6ry-
th~me seborrheique gegeben hat. Das Wesen der Dermatose ist eine von
der Gefiil~gegend ausgehende, bisweilen universelle R6te der I-Iaut mit
Seborrhoe des Kopfes. C. L e i n e r. NIoro. Uber dieStellung
derErythrodermia
de squama-
tiva ~Leiner) im Kranktieitssystem,
Mfinchener mediz. Woehen-
schrift. 1911. Nr. 10. Die yon L ein er mit dem Namen ,Erythrodermia desquamativa"
bezeichnete eigenartige Dermatose tier S~uglinge hat More ia den letzten
3 Jahren in 11 F~llen beoloachtet und behandelt. Er h~ilt die Sonder-
stellung, die Leine r der Dermatose einr~umt, fiir zu weitgehend and
faint die Erkrankung als eine universelle Dermatitis ex intertrigine bei
jungen S~uglingen mit ausgesprochenem status seborrhoious auf exsuda-
flyer Grund]age auf. Oskar Mii IIe r (Reeklinghausen). •oro,
Uber Dermatitis
fibrinosa
faciei. Miinehener
mediz. Wochenschr. 1911. I~r. 6. More beschreibt eine Hauterkrankung, die bei Kindern an Kinn,
Oberlippe und am Nasene~ngang vorkommt und ein Analogen zu den
Stomatitis aphthosa bildet. Die Effloreszenzen zeigen die gleiehen Eigen-
tiimliehkeiten ~vie die Mundaphthen und bestehen in der Hauptsache aus
Fibrin und wenig Leukozyten. g
y
Verf. sehl~gt fi~r die noch nieht besehriebene Af[ektion den Namen
,Dermatitis fibrinosa" vor. Oskar M ii 1 l e r (Recklinghausen). Haslund, Poul. Studien fiber die Histologie
and die
Pathogenesis
der Psoriasis. Kopenhagen 1911. p. 150. Yerf. hat yon 25 Patienten mit klinlseh typischer Psoriasis din
versehiedenen Stadien der Effloreszenz untersuebt, um feststetlen zu
k6nnen, we der prim~ire Sitz der Krankheit ist and auch am das
mikroskopisehe Bild der versehiedenen Stadien genau zu zeiehnen. Zu diesem Zweeke hat er 6000 meistens in Serien gesehnittene
Pr~parate uutersueht und gezeigt, da$ die in dem ersten Stadium der
Krankheit nur im Mikroskope sichtbare u
der Haut eine
Oberhautentzfindung ist und zwar dadurch entsteht, dal~ sieh ein kleiner
ttaufe yon emigrierten Leukozyten gerade unter dem Stratum corneum
bfldet, cloch sieht man auch nicht selten einen wirklichen kleinen
,Abszel~ ~. Die gleiehzeitig gefundenen Yer~nderungen im Papilt~rkfrper
and im Korium sind deutlieh vorhanden, sic sind aber wahrseheinlich
yon sekundirer Bedeutung. Spiter kommt das allgemein bekannte Bild
yon typischer Psoriasis. y
yp
Yerf. schliefit sieh denn ziemlich nahe an die Ansehauungen, die
erst v. Sonstige Dermatosen. Munro und Sabouraud ausgesprochen haben, doch hat er hie
die yon diesen Forsehern gefundene primire Erosion gesehen und VerL
meint auch, daft das Bild sich nicht durch eine prim~re Erosion erkl~rea der Hautkrankheiten. 565 'l~Bt. Auch hat er --im Oegensatze zu den erw~hntea Autoren -- ge~
funden, dab eine ,Exoserose" ziemlieh h~ufig zu sehen ist. 'l~Bt. Auch hat er --im Oegensatze zu den erw~hntea Autoren -- ge~
funden, dab eine ,Exoserose" ziemlieh h~ufig zu sehen ist. g
Die Arbeit ist wegen der guten detaillierten Beschreibung des
mikroskopisehen Bildes besonders erw~hnenswert. g
Die Arbeit ist wegen der guten detaillierten Beschreibung des
mikroskopisehen Bildes besonders erw~hnenswert. Henrik Bang (Kopenhagen). Henrik Bang (Kopenhagen). Henrik Bang (Kopenhagen). g
p
g
Mayer~ A. Normales Schwangersehaftsserum
als Heil-
mittel gegen Schwangerschaftsdermatosen
im besonderen
und Schwangerschaftstoxikosen
iiberhaupt. Zentralbl. f. d. ges. Med. 1911. Nr. 9. Mayer~ A. Normales Schwangersehaftsserum
als Heil-
mittel gegen Schwangerschaftsdermatosen
im besonderen
und Schwangerschaftstoxikosen
iiberhaupt. Zentralbl. f. d. ges. Med. 1911. Nr. 9. ~Das Wesen der unter dem l~amen Sehwangerschaftstoxikosen zuo
sammengefaflten Krankheitsformen erblieken wir, u sehreibt Mayer,
,ganz allgemein gesagt darin, da~ gewisse durch den waehsenden Keim
in dem miitterliehen Organismus normaliter gebildete Giftstoffe diesem
in pathologischeu F~llen seh~idlieh werden kSnnen. Nach dieser Voro
stellung ist es flit den normalen Ab]auf der + Gravidit~t n5tig, da~ jene
Oift~toffe durch Gegengifte unseh~dlich gemaeht werden. Gesehieht das
nicht, dann entstehen blutfremde Stoffe im miitterliehen Blut, die zu
autotoxischeu Erkrankungen ffihren. Man kann also sagen~ dal} elne an
einer Gravldit~tstoxikose erkrankte Frau an einem D efizit yon ~egen-
giften leidet, die eine gesunde Schwangere besitzt." Durch diese Er-
~v~gung wurde M a y e r veranla~t, L i n s e r die Behandlungsmethode vor-
zuschlagen, die dieser erfolgreich inaugurierte. ~Das Wesen der unter dem l~amen Sehwangerschaftstoxikosen zuo
sammengefaflten Krankheitsformen erblieken wir, u sehreibt Mayer,
,ganz allgemein gesagt darin, da~ gewisse durch den waehsenden Keim
in dem miitterliehen Organismus normaliter gebildete Giftstoffe diesem
in pathologischeu F~llen seh~idlieh werden kSnnen. Nach dieser Voro
stellung ist es flit den normalen Ab]auf der + Gravidit~t n5tig, da~ jene
Oift~toffe durch Gegengifte unseh~dlich gemaeht werden. Gesehieht das
nicht, dann entstehen blutfremde Stoffe im miitterliehen Blut, die zu
autotoxischeu Erkrankungen ffihren. Sonstige Dermatosen. Man kann also sagen~ dal} elne an
einer Gravldit~tstoxikose erkrankte Frau an einem D efizit yon ~egen-
giften leidet, die eine gesunde Schwangere besitzt." Durch diese Er-
~v~gung wurde M a y e r veranla~t, L i n s e r die Behandlungsmethode vor-
zuschlagen, die dieser erfolgreich inaugurierte. g
Felix Miinchheimer (Wiesbaden). Mfiller, Rudolf. Lichen
ruber
aeuminatus
atypieus
eaplitis. Ikonographia dermatologica, Fuse. V. Tab. XL. Miil 1 e r besehreibt einen Fall yon Lichen rub. aeum. der Kopf-
haut, der sich yon den sonst beobachteten dadureh wesentlich unter-
scheidet, dab auch dab Kapillitium ,deutliche hyperkeratotisehe isolierte
KnStchen aufwies, wie sie ffir die fibrige Lokalisation des Lichen
charakteristiseh sind% w~hrend sonst stets ,das prim~ir infiltrierte Horn-
kuStchen vollst~indig fehlte und nur eine diffuse Pityriasis der Kopfhaut
beobaehtet wurde". Eiu 38j~ihr. Patient~ der a]s Kind an Kopfekzem gelitten hatte, er-
krankte vor 7 Monaten an einer Hautaffektion~ die sich fiber Kopf, Brust
und Vorderarme ausbreitete. Die Untersuchung des Kopfes ergab neben
einer zentralen Alopeeia pityroides das Vorhandensein verschieden gro~er
sehuppender Plaques, die sich am Rande in zahlreiche einzelne KnStchen
und Gruppen yon solchen aufl6sten. Die kleinsten KnStchen bestehen
aus einem Hornkege], der einem gelblichroten Infiltrate aufsitzt und
tragen z, T. eiu erhaltenes, z. T. ein abgebrochenes Haar; an anderen
KnStehen fehlen die Haare, an deren Stelle ein griibehenfSrmiger Defekt
siehtbar ist. Stellenweise sind die FoIlikelmfindungen erweitert oder
yon einem Schfippchen bedeckt. Vorderarme, Handrficken und Brust
tragen Kn6tchen, die z. T. die charakteristischen, an den Follikel-
mtindungen sitzenden Hornkegel zeigen. Unter energischer Arsenbehand-
lung heilte die Affektion ab. 566 Berieht fiber die Leistungen auf dem Gebiete Der histologische Befund zeigte eine starke Hyperkeratose der
Follikel und zirkumskripte kleinzellige Infiltration um den Follikelboden. Ferner f'~llt die dutch den Druck der hyperkeratotischen Schichten zu-
stande gekommene Yer~nderung der Epithelzellen auf im Sinne des AuG
treteas flacher Zellformen bet senkrechtem und yon Zylinderzellen bei
seitlichem Drueke. Wilhelm B a 1 b a n (Wien). Der histologische Befund zeigte eine starke Hyperkeratose der
Follikel und zirkumskripte kleinzellige Infiltration um den Follikelboden. Ferner f'~llt die dutch den Druck der hyperkeratotischen Schichten zu-
stande gekommene Yer~nderung der Epithelzellen auf im Sinne des AuG
treteas flacher Zellformen bet senkrechtem und yon Zylinderzellen bei
seitlichem Drueke. Wilhelm B a 1 b a n (Wien). Nobl. Zur Kenntnis
des Herpes
zoster
generalisatus
Wiener klinische Wochensehr. 1911. Nr. 1. Sonstige Dermatosen. An der Hand eines einschl~gigen Falles erfrtert N obl unsere
Kenntnisse fiber den Herpes zoster. Der engere pathogenetisehe Zu-
sammenhang der 5rtlichen systemisierten und der a]lgemeinen Bl~ischen-
eruption wird verst~ndlicher, wenn man die in allen Beobachtungen ver-
folgte Abh/ingigkeit der Ern~hrungsstfrung im Bereiche der disseminierten
Aussaat yon den intensiven Seh~digungen im Projektionsfeld eines
Spinalganglions in Berficksichtigung zieht. Man kann den Herpes zoster
als vasomotorisehes Ph~nomen betrachten, das ~ihnlich der neurotiscben
Hautgangriin auf sp~treflektorischem Wege zustandekommt. Viktor Bandler (Prag). Viktor Bandler (Prag). Kanera, Franz. Herpes
zoster
und l~ierenkolik. Dtsch. reed. Woch. Nr. 14. 1911. Kanera
h~lt das Zusammentreffen dieser beiden Erkrankungen
nieht fiir so selten, wie Bitt orf gelegentlieh der Verfffentliehung eines
~hnlichen Falles annimmt und beriehtet seinerseits fiber sine 56j~hrige
~atientin, die bet sonst gutem Kr~ftezustand wegen starker Sehmerzen
im r. Hypoehondrium seine Hilfe suehte. Verf. konsiatierte eine reehte
Wanderniere, der Harn war normal, der Stuhl aber oft verhalten. Eine
Woehe sparer entwickelte sich, you 2 roten Fleeken am Rfieken aus-
gehend, ein typiseher Herpes zoster, der unter entsprechender Behand-
lung bald heilte, wfihrend auch die Nierengeschwuls~ sieh in die Tiefe
verlor und nur noch als dumpfe Resistenz unter dem Rippenbogen zu
ffihlen war und die Schmerzen verschwanden. Kanera
h~lt das Zusammentreffen dieser beiden Erkrankungen
nieht fiir so selten, wie Bitt orf gelegentlieh der Verfffentliehung eines
~hnlichen Falles annimmt und beriehtet seinerseits fiber sine 56j~hrige
~atientin, die bet sonst gutem Kr~ftezustand wegen starker Sehmerzen
im r. Hypoehondrium seine Hilfe suehte. Verf. konsiatierte eine reehte
Wanderniere, der Harn war normal, der Stuhl aber oft verhalten. Eine
Woehe sparer entwickelte sich, you 2 roten Fleeken am Rfieken aus-
gehend, ein typiseher Herpes zoster, der unter entsprechender Behand-
lung bald heilte, wfihrend auch die Nierengeschwuls~ sieh in die Tiefe
verlor und nur noch als dumpfe Resistenz unter dem Rippenbogen zu
ffihlen war und die Schmerzen verschwanden. Max Joseph (Berlin). Max Joseph (Berlin). Max Joseph (Berlin). Bertarelli, E. und Parauhos, U. ~tiologiaehe
Unter-
suehungen
fiber den
Pemphigus
der
Tropengegenden. Zentralbl. f. Bakt. Bd. LVII. Heft 6. pag. 493. Uber den Tropenpemphigus liegen sehr wenige Angaben in der
Literatur vor. Verff. fassen die wiehtigsten kllnisehen Charaktere der
Krankheit kurz, wie folgt, zusammen:
,Der Tropenpemphigus ist eine
Hautkrankheit, charakterisiert dureh die Entstehung yon Blasen oder
Bl~isehen versehiedener Grfl]e, welehe ohne Reaktionserscheinungen von-
~eiten der umgebenden Haut auftreten und keine Gesehwfire und somit
keine Narben erzeugen. Sonstige Dermatosen. Die L~sion beginnt mit kleinen erythemat6sen,
stark juckenden Flecken, an deren Stelle naeh kurzer Zeit eharakteristisehe
Blasen
erscheinen. Diese
sind
anfangs
durehscheinend, gespannt,
gl~nzend; dann wird ihr serfser Inhalt trfibe, die W~inde erseMaffen und
die Blasen 5ffnen sieh spontan oder beim geringsten Druek. Diese Urn- Bertarelli, E. und Parauhos, U. ~tiologiaehe
Unter-
suehungen
fiber den
Pemphigus
der
Tropengegenden. Zentralbl. f. Bakt. Bd. LVII. Heft 6. pag. 493. Uber den Tropenpemphigus liegen sehr wenige Angaben in der
Literatur vor. Verff. fassen die wiehtigsten kllnisehen Charaktere der
Krankheit kurz, wie folgt, zusammen:
,Der Tropenpemphigus ist eine
Hautkrankheit, charakterisiert dureh die Entstehung yon Blasen oder
Bl~isehen versehiedener Grfl]e, welehe ohne Reaktionserscheinungen von-
~eiten der umgebenden Haut auftreten und keine Gesehwfire und somit
keine Narben erzeugen. Die L~sion beginnt mit kleinen erythemat6sen,
stark juckenden Flecken, an deren Stelle naeh kurzer Zeit eharakteristisehe
Blasen
erscheinen. Diese
sind
anfangs
durehscheinend, gespannt,
gl~nzend; dann wird ihr serfser Inhalt trfibe, die W~inde erseMaffen und
die Blasen 5ffnen sieh spontan oder beim geringsten Druek. Diese Urn- der Hautkrankheiten. 567 wandlung erfolgt durehschnittlich im Laufe yon 6--7 Tagen. Danach
bliittert die Epidermis auf Streeken yon einigen cm do und die IIaut er-
scbeint an den betreffenden Ste]len rosig und wie lacklert, wRhrend sich
feine IIautl~ppeben ablSsen. ~ Die Dermatose ~ritt schubwe~se auf und
verl~uft fieberlos; ihre ~_tiologie ist noch unklar. Sodann wird die
Krankheitsgeschiehte eines Falles mitgeteilt, aus dessen Material ver-
,sehiedene Untersuchungen ausgefiihrt wurden. Morphologische Unter-
suchungen der Blasenflfissigkeit, Krusten, Schuppen verliefen ergebnislos. Tierversuehe an der Haut einer Coebus-Art~ Meersehweinchen, Hund,
Kanlnchen waren negativ. Bei den Kaninchen kam es nach Inokulation
in die vordere Augenkammer und Infektion der Kornea nach 8 Tagen zu
IIyper~mie, Bindehautkatarrh, Keratitis und tIypopyon, in welehem
Staphylokokken naehgewiesen wurden, w~ihrend die bistologiscbe Unter-
suchung der IIornhautsehnitte keinen
besonderen Befund ergaben. Komplement-Ablenkungsversuche verliefen negativ. Alfred Kr au s (Prag). g
Feinberg, A. Ein Fall yon purpura variolosa. Wratschebn. Gazeta. 1910. Nr. 5. Beschreibung eines Falles bei einem 13j~ihrigen Knaben. Beschreibung eines Falles bei einem 13j~ihrigen Knaben. P
A W lik
(P t
b
) Beschreibung eines Falles bei einem 13j~ihrigen Knaben. P. A. Welikanow
(Petersburg). Wolkowitseh,
Nieolai. Zur
Statistik
und
dem
Vor-
kommen des Skleroms (Rhinoskleroms) in Rutland. Monats- Wolkowitseh,
Nieolai. Zur
Statistik
und
dem
Vor-
kommen des Skleroms (Rhinoskleroms) in Rutland. Monats-
sehrift f. Ohrenheilk. und Laryngo-Rhinologie. XLV. Jahrg. IIeft 1. Statistisehe Arbeit. Zum Referate nicht geeignet. Wilhelm B a i b a n (Wien). Sonstige Dermatosen. sehrift f. Ohrenheilk. und Laryngo-Rhinologie. XLV. Jahrg. IIeft 1. Statistisehe Arbeit. Zum Referate nicht geeignet. Wilhelm B a i b a n (Wien). Kahler. Zahnfleisehsklerom. (Demonstration in tier Wr. laryngol. Gesellsehaft.) Monatssehr. f. Obrenheilk. und Lar.-Rhinologie. XLV. Jabrg. 3. Heft. Krankendemonstration. Wilhelm B a 1 b a n (Wicn). K Kono, Michiyo. Beitrag zur Kasuistik
der Sklerodermie. Diss. GSttingen 1910. (Mediz. Klinik Hirsch.)
Fall yon Sklerodermie ohne weiteres Interesse. Weiler (Leipzig). Hartzell, B. Ein Fall yon Pellagra, der seinen Ursprung
in Pennsylvania
hatte. Philadelphia County Medical Society. 191l. 8. Februar. The Journal of the American Medical Association. 1911. 1. April. pag. 998. Bericht fiber einen Fall yon Pellagra mit ansehlielSender Diskussion
fiber Pellagra. Fritz J u li u s b e r g (Posen). Nice, Charles, Me Lester, James, Torrance, Gaston. Pell agr a
mit Salvarsan
behandelt. The Journal of the American Medical
Association. 1911. 25. M~irz. pag. 896, p g
Nice, Mc Lester
und Torrance
haben bei drei F~llen yon
Pellagra Salvarsan intraven6s angewendet und elne Besserung erzielt. Fritz Juliusberg
(Posen). Nice, Mc Lester
und Torrance
haben bei drei F~llen yon
Pellagra Salvarsan intraven6s angewendet und elne Besserung erzielt. Bericht fiber die Leistungen auf dem Gebiete 568 Cole, M. P. Transfusion
bet Pellagra. Ein Bericht fiber
20 Y~lle. The Journal of the American Medical Association. 1911. 25. Februar. pag. 584. Cole, M. P. Transfusion
bet Pellagra. Ein Bericht fiber g
20 Y~lle. The Journal of the American Medical Association. 1911. 25. Februar. pag. 584. g
20 Y~lle. The Journal of the American Medical Association. 1911. p g
Cole
kc, mmt zu folgenden Schliissen: Mit der Transfusion bet
20 F~llen yon Pel]a'gra baben wir hie direkt dutch die Operation schlechie
Resullate bet den Paiienien erlebt. Wir dfirfen zu der Transfusion greifea
bet schweren F~llen, die unter den erprobten Mitteln st~indige Ver-
schlechterung aufweisen. Es ist ganz gleich, ob man das Blur yon einem
Patienten nimmt, der Pellagra fiberstanden hat oder yon einem, der nie
Pellagra hatte. Heilungen gab es bet dem schweren Typus der Krank-
heir durch die Transfusion in 600/0, w~ihrend die sonstigen Behandlungs-
methoden nur einen Prozentsatz yon 10 bis 20 Heilungen fatten. Die
Anwenduug der Transfusion im Endstatium der Pellagra muB mit vollem
Bewu~tse~n der Schwierlgkeilen und der Gefahren der Methode vorge-
nommen werden. Nur sorgf~ltige Auswahl der F~ille und unvorein-
genommene Schliisse k5nnen das Verfahren vor eiuer Diskreditierung
bewabren, die es nicht verdient. Fritz Juliusberg
(Posen). Abbe beriehtet fiber zwei Fglle yon Ainhum. Die FMle spreehen
dafi~r, dal~ das Primgre der Erkrankung eine Trophoneurose ist.
F it
J
li
b
(P
) De Azfia, Juan.
Akuter 5demat5ser
and erythematfser
Jodismus
der kranken Zonen bei einem Fall
von erythe- Sonstige Dermatosen. Caccilli, A. Die Pellagra, wie wir sie in Italien sehen;
a 1 t e u n d n e u e T h e o r i e n. Bericht fiber F/ille aus New-York. Medical
Record. I911. ii. M~rz. pag. 421. Die Arbeit Caecinis
enth~ilt eine eingehende Darstellung der
Symptomatologie und der Ansichten fiber die Atiologie der Pellagra. Im
Anschlu~ werden s~cben in New-York beobaehtete F~lle besehrieben. Der Autcr kommt zu fo]genden Schlfissen:
1. Die Hauptursache der
Pellagra ist noch unbekannt. 9. Die Pellagra hat in den Vereinigten
Staaten in sporadischen F~llen schon lange Zeit bestanden, abet erst in
letzter Zeit hat sie sieh ausgebreitet and eincn epidemisehen Charakter
angenommen. 3. In den Vereinigten Staaten nimmt die Krankheit einen
akuieren Verlauf als in Europa und speziell in Italien, wo sie sehr h~ufig
einen ehronischen Charakter trfigt und das Erythem nie den schweren
Charakter aufweist, wie in den Vereinigiea Staaten. 4. In Amerika ist
die Kranl~heit relativ hfiufig unter den gut situierten Klassen, wahrend
sie in Europa auf die armen Kreise beschriinkt ist. Fritz J u 1 i u s b e r g (Posen). Wile, Udo. Uber
Granuloma
pyogenicum
(Pseudo-
B o t r y o m y k o s i s). U n n a s Dermatologlsehe Studien (Unna-Festschrif~
Bd. I), Bd. XX. pag. 333. Naoh ausfiihrlicher Darstellung zweier einschl~gigerF~lle kommt W i i e
zu folgendenSchliissen: 1. Dieganze Gruppevon sogenannterPseudo-Botryo-
mykosis, Granuloma simplex, Granuloma teleangiectodes und Granuloma
pyogenicum mag in eine Klasse zusammengefal~t und als Granulationsgewebe
betrachtet werden, we]e]~es aus vor]~ufig unbestimmten Grfinden eine
ungewehnliche Form angenommen hat. 2. Als iitiologischer Faktor tritt
bier nicbt ein Fungus, sondern wahrscheinlich der Staphylococcus aureus
auf, mSglicherweise auf ungfinstigem Nfihrboden oder in abgeschw~ichter
Form. S. Die histologischen Ver/inderungen sind wesentlich die~elben der Hautkrankheiten. 569 ~in allen Formen ; der einzige Unterschied, der konstatiert werden kann,
ist ein quantitativer Unterschied in Vasku]aritiit und Entzfindung. 4. In
keinem Falle 1M~t sieh ein Zusammenhang zwischen Tumor und Schwei•-
drfisen nachweisen. 5. 0bgleich hie b6sartig, hat doeh die Geschwulst
eine Neigung zu rezidivieren, wenn bei der Enffernung der Stumpf
nicht kaaterisiert worden ist. 6. Granaloma pyogenicum ist nuch des
Autors Ansicht anter allen ffir dieses Krankheitsbild vorgeschlagenen
Named der passendste. Fritz Juliusberg
(Posen). ~in allen Formen ; der einzige Unterschied, der konstatiert werden kann,
ist ein quantitativer Unterschied in Vasku]aritiit und Entzfindung. 4. Sonstige Dermatosen. In
keinem Falle 1M~t sieh ein Zusammenhang zwischen Tumor und Schwei•-
drfisen nachweisen. 5. 0bgleich hie b6sartig, hat doeh die Geschwulst
eine Neigung zu rezidivieren, wenn bei der Enffernung der Stumpf
nicht kaaterisiert worden ist. 6. Granaloma pyogenicum ist nuch des
Autors Ansicht anter allen ffir dieses Krankheitsbild vorgeschlagenen
Named der passendste. Fritz Juliusberg
(Posen). Gjorgjevi6. Beitrag
zur Kenntnis
der Streptothrix-
erkrankungen
des Menschen. Wiener klinische Wochenschrift. 1911. Nr. 6. Der Autor berichtet in extenso fiber 4 beobachtete Fiille. Die
einze]nen Organe liel~en entsprechend dem makroskopischen Knftchen-
befand mikroskopiseh das Bild yon Eiterherden erkennen; erwiihnenswert
erscheint der Befund yon Riesenzellen. Wie in gef~rbten Priiparaten so
auch im Mngenden Tropfen crkennt man echte u
der
Stature tier Fiiden hat die gleicbe Dicke wie die abgehenden Zweige,
welche an de~ Enden die Andeutung einer leiehten ko|bigen Anschwel]ung
zcigen kfnnen. Die Pilze erweisen sich als siiarefest, aber nieht als
alkoholfest. Was die kultare]len Eigensehaften anbe]angt, so unter-
seheidet sich die Pilzart yon den meisten his jetzt bekannten dureh das
tkkultativ an~irobe Waehstum; die meiste ubereinstimmung zeigt sich
mit den yon Eppinger,
Aoyama and Miyamoto,
A. Horst , Mac
Collum und A. Schabad beschriebenen Stihnmen, Yiktor Bundler (Prag). Yiktor Bundler (Prag). Yiktor Bundler (Prag). Sutton, Richard. Cheilitis
glandularis
apostematosa. (Eine klinische und histopathologische Studie.) Unnas Dermatologische
Studien (Unna-Festsehrift Bd. I)~ Bd. XX. pug. 612. Sutton
kommt in seinen Untersuchangen fiber die zuerst yon
V o I km a n n beschriebene Cheilitis glandularls apostematosa zu folgenden
Sehlfissen: Die Cheilitis glandularis apostematosa ist eino adenomatfse
Yeriinderung der Sehleimdriisen der Lippe, die histologlseh char~kterisiert
ist durch eine enorme Dilatation nnd Hyperfrophie der Driisengiinge,
wclche gewfhnlich begleitet ist yon einer starken Zunahme des Drfisen-
gewebes. Es ist sehr wahrscheinlich, da~ die u
ursprfinglich
eine kongenitale ist, der dann degenerative Ver~nderungen und Zunahme
des elastischen Gewebes in derumgebenden Haut folgen. Diebefriedigendst0
Art der Behandlung ist die Exzision unter loka]er oder allgemeiner
Aniisthesie. Fritz J u I i u s b e r g (Posen). g
Abbe, Truman. Zwel F~lle yon Ainhum. Medical Record. 1911. 18. Miirz. long. 478. Abbe, Truman. Zwel F~lle yon Ainhum. Medical Record. 1911. 18. Miirz. long. 478. Abbe beriehtet fiber zwei Fglle yon Ainhum. Die FMle spreehen
dafi~r, dal~ das Primgre der Erkrankung eine Trophoneurose ist. De Azfia, Juan. Akuter 5demat5ser
and erythematfser
Jodismus
der kranken Zonen bei einem Fall
von erythe- Berieht fiber die Lelstungen auf dem Gebiete 570 mat68er
Anetodermie
Jadassohn. Sammelreferat, in dem yon Hauterschelnungen Pigmenta~ionen~
Pruritus~ Tumoren~ erythematSse, exsudative Effloreszenzen und Gesehw[ire
erw~hnt werden.
F. L e w a n d o w s k y (Hamburg). Sonstige Dermatosen. Unnas
Dermatologisehe
Studien (Unna-Festschrift Bd. I)~ Bd. XX. pag. 216. (
)
p g
D e Az5a berichtet fiber eine 28i~hrige Patientin, die seit drei
Jahren an einer Hautaffektion leidet, die folgendermal]en verlfiuft: Zu-
n{iehst erscheinen runde und ovale Herde yon dunkelroter Farbe, einer
vermehrten Konsistenz, wie oberfi~chliche Papeln mit glatter Oberfl{iche. 8ie sind absolut schmerzlos. Nach Wochen oder Monaten verlieren sie
ihren papulSsen Charakter, um dann ira Niveau der Haut zu sitzen. Dann wird ihre Farbe bl~sser, die Epidermis verdfinnt sich und man
fiihlt eine Resistenzverminderung. Dieser Zustand wird immer aus-
gesproehener und als Endstadium finder sich eine scharf begrenzte
Depression~ innerhalb der der Finger in eln tiefes Loeh eindringt. Diese
Effloreszenzen haben dieselbe Ausdehnung wie der urspr[iugliche Herd. Der Autor stellte die Diagnose: Atrophie maeuleuse en plaques consg-
cutives ~ un processus chronique ind6termin6 de type inflammatoire
6rythemateux. Der Fall entspricht der Anet~dermia erythematosa
Jadassohn. Der Autor gab Jod 0"5. Einige Stunden darauf trat eine
Sehwellung der kranken Partien auf und eine starke RStung derselben. Drei Tage naehher war wieder der Status quo antea eingetreten. Eine
erneute Joddarreichung hatte dieselben Fo]gen. Bernhardt, 1%. Lupus erythematosus
linearis. Gazeta
lek, 1911. Nr. 5. Siehe dieses Archiv Bd
CVIIL pag
55 Bernhardt, 1%. Lupus erythematosus
linearis. Gazeta
lek, 1911. Nr. 5. Siehe dieses Archiv Bd
CVIIL pag
55 lek, 1911. Nr. 5. Siehe dieses Archiv Bd. CVIIL pag. 55. Brinitzer. Fall yon hoehgradiger
Akne. Altonaer iirztl. Verein. Sffzung v. 22. Februar 1911. 94j~hriger Mann seit 10 Jahren an Akne vulgarls pustulosa mit
perifollikul~ren Abszessen leidend. Ausgedehnte Narbenbildungen; an
manchen Stellen des Riiekens kaum noch gesunde Hautstellen im er-
krankten Gebiet. F. L e w a n d o w s k y (Hamburg). Fabian,
E. Die
Lymphogranulomatosis
(Paltauf~
Sternberg). Zentralbl. f. allg. PathoL u. pathol. Anat. Bd. XXII. pag. 145. 1911. Sammelreferat, in dem yon Hauterschelnungen Pigmenta~ionen~
Pruritus~ Tumoren~ erythematSse, exsudative Effloreszenzen und Gesehw[ire
erw~hnt werden. F. L e w a n d o w s k y (Hamburg).
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Shifting Desulfurization Equilibria in Ionic Liquid–Oil Mixtures
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Email: jalil.hussein@epu.edu.krd; Email: jalil.hussein@epu.edu.krd; Shifting desulfurisation equilibria in ionic liquid-oil mixtures 1Department of Chemistry, University of Leicester, Leicester LE1 7RH, UK. E-mail: apa1@le.ac.uk;
2Petroleum Technology Department, Erbil Technology Institute, Erbil Polytechnic University,
Erbil, Iraq. 1Department of Chemistry, University of Leicester, Leicester LE1 7RH, UK. E-mail: apa1@le.ac.uk;
2Petroleum Technology Department, Erbil Technology Institute, Erbil Polytechnic University,
Erbil, Iraq. Abstract Ionic liquids (ILs) and deep eutectic solvents have been used for the extraction of molecules,
particularly natural products. An often studied system is that for thiophene removal from oil. In the
current study, ILs have been used for the removal of thiophene (Th), benzothiophene (BT) and
dibenzothiophene (DBT) by liquid-liquid extraction. The equilibrium can be shifted by polymerising
the thiophenic compounds both electrochemically and chemically. A 1:1 mixture of 1-butyl-3-
methylimidazolium chloride (Bmim)Cl and FeCl3 was used to extract Th and BT from alkane layers
using electropolymerisation to shift the position of the equilibrium. While the process could be carried
out, the kinetics of polymer formation were too slow to make this a viable process. The final part of
the study used a 1:2 (Bmim)Cl: FeCl3 mixture to chemically remove sulfur containing compounds
from alkane layer. It was shown that the rate of the chemical polymerisation reaction of Th is around
1500 times faster than electrochemical polymerisation. Issues associated with the regeneration of the
liquid are discussed. Key words: Thiophene, liquid-liquid extraction, electrochemical polymerisation, desulfurization,
imidazolium ionic liquids. 1 Experimental All materials and reagents employed in this work: ferric chloride, 1-Butyl-3-methylimidazolium
chloride, n-decane, thiophene and dibenzothiophene (all Sigma-Aldrich ≥99 %), benzothiophene
(Alfa Aeser > 98%), were all used as received.(Bmim)FeCl4 eutectic mixture has been made
according to the literature procedure.7 In the extraction experiments, different solutions of Th was prepared in n-decane as a model oil and
then stirred and extracted with that of each of the mixture of ILs addition at different conditions of
ILs. The same conditions and experiments were repeated with BT and DBT. The S-content with respect to Th, BT, and DBT in decane was measured by GC-FID with a fused
silica capillary column (PE Elite-5, 29.45 m long, 0.25 mm in diameter) connected to gas
chromatograph (Perkin Elmer Autosystem XL) using the Totalchrom software. The operational
temperature of the FID was 320 ºC, and that of the injector was 310 ºC. For the first three minutes,
the temperature of column was set at 50 ºC, increased to 300 ºC at a rate of 15 ºC min-1, kept at 300
C for 2 min. Helium was the carrier gas at a flow rate of 1ml min-1. The quantities of S-compounds
in a model fuel were determined from peak areas corresponding to these sulfur species on the gas
chromatograph. According to the GC setup, the retention time chromatograms for Th, BT and DBT
were around 3.14, 9.88 and 14.70 min respectively. The amount extracted was determined using a
calibration plots. Good linear correlations were achieved for analytical technique, with an R2 value
of more than 0.99 for GC-FID. The error bars for most of the data are within the size of the plot
symbols showing that replicate results are accurate. All electrochemical experiments were carried out using a three electrode set up (Pt working electrode
(WE), a Ag wire pseudo-reference electrode (RE) and Pt mesh counter electrode (CE). CVs were
carried out at a polished 0.5 mm diameter (1.963 x 10-3 cm2) Pt disc working electrode immersed in
a (Bmim)Cl FeCl3 mixture as a function of sweep rate. Before each experiment, the WE was cleaned and washed with deionised water, polished with
alumina paste and after sonicating in deionised water for 5 minutes; it was dried with N2 gas. The
WE acts as a substrate for electro-deposition of polythiophene. Introduction Ionic liquids and deep eutectic solvents (DESs) have been proposed as solvents for extraction
although one obvious drawback is that the solutes are difficult to remove from the ionic phases as
they have low vapour pressures.1 Liquid-liquid extraction has been extensively studied for the
removal of metals from aqueous solutions using hydrophobic ILs and the removal of sulfur containing
compounds from oils using hydrophilic ILs.2 While numerous solutes have been extracted, relatively
little is known about the specificity and thermodynamics of extraction. A recent study on the extraction of thiophenic compounds from alkane phases into DESs showed that
extraction was limited by enthalpic change and driven by entropic change. In most cases the enthalpy
of extraction was endothermic limited by the energy required to create a hole in which to put the
solute. The highest partition coefficients were found with the liquids with the lowest surface tensions
and therefore the lowest cohesive energy densities.3 The extraction of thiophene and its polyaromatic analogues has been studied by several groups in
different DESs and while efficient extraction has been observed little is known about the process. Lewis acidic tetrachloroferrate based ILs, particularly those containing imidazolium cations, are very
efficient at chemically removing sulfur containing compounds from alkane based liquids. The
advantage of using an iron-based catalyst is naturally that it is sustainable and hence has a low cost. Dharaskar and coworkers used a 1:1(BmimCl): FeCl3 misture and found that 59.2% of Th was
extracted.4 The authors proposed a specific interaction between thiophene and the FeIII-based anion. Li et al.5 and Yang et al.,6 studied the adsorption of thiophene using a Ag+ or Cu+-Y zeolite. They
found that the metals interacted with the thiophene π orbitals with a donation into the vacant σ orbitals
of the metal and a back donation of electron charge to the π* orbital of thiophene from the d orbitals
of metals. The authors believed that the same mechanism occur with the Lewis acidic FeCl3 based-
triethyl ammonium chloride. In the current study, the extraction and polymerisation of thiophene (Th), benzothiophene (BT) and
dibenzothiophene (DBT) was studied from alkanes. Initially, these compounds are extracted and then
electropolymerised from decane in an equimolar (Bmim)Cl- FeCl3 mixture. In the second part, the
extraction and chemical polymerisation was carried out in 1:2 (Bmim)Cl: FeCl3 to compare and
determine the rate of their reactions. 2 Experimental Since the polymeric film was
deposited by an oxidative process, an inert electrode (Pt) was requested so as not to oxidise
concurrently with the aromatic thiophene monomer.8 3 3 Results and Discussion Extractive electropolymerisation of Th by 1:1 (Bmim)Cl: FeCl3 Extractive electropolymerisation of Th by 1:1 (Bmim)Cl: FeCl3 The extraction of Th, BT and DBT has previously been shown to be possible into a range of DESs. Th
showed the best extraction efficiency followed by BT and then DBT and this was found to be related
to the size of the solute (the latter requiring a larger hole).3 The equilibrium can be driven by making
a solid product from the thiophenic compounds by polymerisation of thiophene to form polythiophene
(PTh). Previous work has converted the thiophene to a solid thiophene sulfoxide catalysed by DESs in the
presence of H2O2 as an oxidising agent.9 The issue with this approach is that H2O is introduced as an
aqueous solution as a by-product, contaminating the oil. In the current study it is proposed that Th could be converted into a solid by polymerising it to
polythiophene (PTh). This is an interesting material which has been used in numerous electronic
devices as a semiconductor and electrochromic material.8, 10 Attempts to drive the equilibrium by
electropolymerising the thiophenic molecules in non-metallic DES were unsuccessful. Electropolymerisation of several monomers has successfully been achieved in a variety of ionic
liquids although polymer formation can be sensitive to the medium. This topic has been reviewed in
more depth by Pringle.11 Csihony et al.,12 showed that thiophene can be successfully partitioned into an ionic liquid composed
of a 1:1 (Bmim)Cl: FeCl3 mixture from a decane layer. We propose that the efficiency of this
partitioning occurs because (C4mim)FeCl4 has a relatively low surface tension (46.4 mN m-1)
meaning that the enthalpy of hole formation is decreased by adding ferric chloride to the liquid. In this study, a DES composed of 1:2 molar mixture of choline chloride: ethylene glycol (1:2 ChCl:
EG) has been used to extract and electro-polymerise thiophene by cyclic voltammetry (CV). 1:2
ChCl: EG was used with and without FeCl3, using Pt working and counter electrodes and a Ag wire
reference electrode. The cyclic voltammetric response of thiophene in 1:2 ChCl: EG is shown in
Figure 1a. 4 Figure 1: CVs for polymerisation of (0.15 mol/L) thiophene in (a) in 1:2 ChCl: EG,
(b) 1:2 ChCl: EG with (0.077 mol/L) of FeCl3,, (c) 0.5 mol/L Th in 1:1
FeCl3/(Bmim)Cl and (d) as (c) but with 0.4 mol/L (0.8mmol) Th in decane. All 40
cycles at 25 ºC, scan rate: 50 mV s-1. -0.4
0.0
0.4
0.8
1.2
1.6
2. Extractive electropolymerisation of Th by 1:1 (Bmim)Cl: FeCl3 5 5 The 1:1 (Bmim)Cl: FeCl3 mixture should be sufficiently aprotic to enable electropolymerisation of
Th. Figure 1c shows a cyclic voltammogram of 0.5 mol/L Th in a 1:1 (Bmim)Cl: FeCl3 mixture and
the characteristic shape is indicative of the polymerisation of Th on the electrode surface. Figure 1d
shows the same experiment run using Th in decane (0.4 mol/L). A similar voltammogram is obtained
to the one where Th was in the ionic liquid phase. This shows that the Th is effectively extracted from
the decane layer. The addition of decane should dilute the total concentration of thiophene in the
system, so the charge observed should decrease. The observation that the charges with and without
decane are similar shows that the partition coefficient must be > 1. The kinetics of polymerisation
can be estimated from the charge data. A charge equivalent to 2.25 electrons per molecule will lead
to the addition of one molecule of thiophene and 0.25 doping anions (Cl-).14 Consequently, the
electropolymerisation charge, QTh, 14-15 can be written as: 𝑄𝑇𝑛𝑛𝑛 The 1:1 (Bmim)Cl: FeCl3 mixture should be sufficiently aprotic to enable electropolymerisation of
Th. Figure 1c shows a cyclic voltammogram of 0.5 mol/L Th in a 1:1 (Bmim)Cl: FeCl3 mixture and
the characteristic shape is indicative of the polymerisation of Th on the electrode surface. Figure 1d
shows the same experiment run using Th in decane (0.4 mol/L). A similar voltammogram is obtained
to the one where Th was in the ionic liquid phase. This shows that the Th is effectively extracted from
the decane layer. The addition of decane should dilute the total concentration of thiophene in the
system, so the charge observed should decrease. The observation that the charges with and without
decane are similar shows that the partition coefficient must be > 1. The kinetics of polymerisation
can be estimated from the charge data. A charge equivalent to 2.25 electrons per molecule will lead
to the addition of one molecule of thiophene and 0.25 doping anions (Cl-).14 Consequently, the
electropolymerisation charge, QTh, 14-15 can be written as: 𝑄𝑛𝑛𝑛 (1) 𝑄𝑄𝑇𝑇ℎ= 𝑛𝑛𝑛𝑛𝑛𝑛 where, n is the number of electron reduced or oxidised, N is the number of moles of thiophene formed
and F is the Faraday constant. where, n is the number of electron reduced or oxidised, N is the number of moles of thiophene formed
and F is the Faraday constant. Extractive electropolymerisation of Th by 1:1 (Bmim)Cl: FeCl3 -9
-6
-3
0
3
6
9
12
15
Current (mA) x10
-1
Potential (V)
c)
20 cycles
0.0
0.2
0.4
0.6
0.8
1.0
1.2
-1
0
1
2
3
4
5
Current (mA) x10
-2
Potential (V)
B
0.2
0.4
0.6
0.8
1.0
1.2
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
Current (mA) x10
-3
Potential (V)
A
-0.4
0.0
0.4
0.8
1.2
1.6
2
-9
-6
-3
0
3
6
9
12
15
Current (mA)X10
-1
Potential (V)
20 cycles
d) 0.0
0.2
0.4
0.6
0.8
1.0
1.2
-1
0
1
2
3
4
5
Current (mA) x10
-2
Potential (V)
B
0.2
0.4
0.6
0.8
1.0
1.2
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
Current (mA) x10
-3
Potential (V)
A B Current (mA)X10
-1 Potential (V) Figure 1: CVs for polymerisation of (0.15 mol/L) thiophene in (a) in 1:2 ChCl: EG,
(b) 1:2 ChCl: EG with (0.077 mol/L) of FeCl3,, (c) 0.5 mol/L Th in 1:1
FeCl3/(Bmim)Cl and (d) as (c) but with 0.4 mol/L (0.8mmol) Th in decane. All 40
cycles at 25 ºC, scan rate: 50 mV s-1. FeCl3/(Bmim)Cl and (d) as (c) but with 0.4 mol/L (0.8mmol) Th in decane. All 40
cycles at 25 ºC, scan rate: 50 mV s-1. Figure 1a shows the clear oxidation and reduction of the thiophene with a half-wave potential of
approximately 0.6 V. Scans to 1.2 V do not show the further oxidation to form the polymer growing
on the electrode surface. The main reason for this is probably because 1:2 ChCl: EG is a protic solvent
containing a hydroxyl group and it could also possibly contains small amounts of water. A large
number of studies have shown that the presence of traces of water in the medium precludes the
electropolymerisation of thiophene through reaction of the water molecule with thiophene radical
cations which make a passivating layer on the working electrode.13 The same experiment was repeated using ferric chloride as an electrocatalyst. Ferric chloride is known
to be a good chemical catalyst for the polymerisation of thiophene. A redox process is again observed
in Figure 1b, but this time the redox half wave potential is at a lower over-potential (0.4 V) suggesting
that it is the FeII/III redox couple. Again no signal for the polymerisation of Th was observed which
again is probably due to the protic nature of 1:2 ChCl: EG. Extractive electropolymerisation of Th by 1:1 (Bmim)Cl: FeCl3 So as to compare the amount of Th converted to polymer in Figure 1c and d, the reaction rate has
been calculated.16 The electropolymerisation rates of Th in (Bmim)FeCl4 were calculated from
Figure 1c and d and are shown in Table 1. The rate of the reaction of consumed Th should be equal
to the reaction rate of produced PTh. However, owing to the biphasic nature of the system, it was
difficult to estimate the amount of Th in the IL phase after 20 cycles from reaction of polymerisation,
therefore, reaction rates were calculated in terms of the products. The data from Table 1 show that the rate of PTh formation in monophasic system is only marginally
higher than that from the biphasic system. Given that an equal volume of decane to (Bmim)FeCl4 was
used the charge should decrease by half, but the observation that it decreases by less than 10% shows
that the partition coefficient must be significantly greater than 1. Table 1: Reaction rate of electropolymerisation of thiophene. Scan rate 50mVs-,
20 scans, potential window:-0.4 – 1.9V. Q (C)
N of PTh (mmol) from
R (mmol/min)
Fig. Qox
Qred
Ox. Red. Ox. Red. c
0.0183
0.0188
7.6x10-4
8.70x10-5
2.48x10-5
2.84x10-6
d
0.0162
0.0186
6.7x10-4
8.55x10-5
2.2x10-5
2.79x10-6 Table 1: Reaction rate of electropolymerisation of thiophene. Scan rate 50mVs-,
20 scans, potential window:-0.4 – 1.9V. On the other hand, as can be seen in Figure 1(c) and 1(d), as the film thickness of the polymer
increases, the voltammetric wave for the reduction of the polymer shifts to more negative potentials
and for the oxidation peak shifts to more positive values with consecutive scans which is characteristic 6 of polymer growth. This is possibly due to the polymer conductivity which increases the resistivity
of the film on the electrode surface due to slows counter-ion mobility and electron transfer kinetics.17 The Fe-based ionic liquids cannot chemically polymerise thiophene until the molar ratio of FeCl3 and
(Bmim)Cl is above 1:1. Above this molar ration the liquid becomes more Lewis acidic and
coincidentally less viscous. The concentration of free Cl- is significantly decreased as FeCl4-
dominates. This also prevents the chloride ion reacting with the radical cation causing polymer
termination. Optimisation of electrocatalytic polymerisation Optimisation of electrocatalytic polymerisation Optimisation of electrocatalytic polymerisation 7 It is interesting to note that while the current increased in 1:1 volume ratio, there was no significant
difference in the amount of PTh deposited when compared with that obtained from 1:7 volume ratio
which suggests that polymerisation is rate limiting rather than the solubility of Th in the DES. The reaction rates shown in Table 2 are relatively slow which is probably because
electropolymerisation is a heterogeneous process.18 In most homogeneous solution polymerisation
reactions, the kinetics of addition reactions are first order in monomer.19 Assuming the charge is
proportional to concentration and the sweep rate can be related to the time of polymerisation then a
pseudo first order kinetic plot could be approximated. 𝑅𝑑𝑑𝑑𝑑𝑘𝐴 𝑅𝑅= −
𝑑𝑑𝑑𝑑
𝑑𝑑𝑑𝑑= 𝑘𝑘[𝐴𝐴] (2) 𝐴 𝑅𝑅= −
𝑑𝑑𝑑𝑑
𝑑𝑑𝑑𝑑= 𝑘𝑘[𝐴𝐴] (2) where, k is the rate constant of the polymer reaction and [𝐴𝐴] is the initial concentration of monomer. It can be seen from Figure 3 that a plot of ln [Th] vs t gives an approximately straight line correlation
suggesting that the process is pseudo first order with a rate constant of 1.4 x 10-5 min-1. where, k is the rate constant of the polymer reaction and [𝐴𝐴] is the initial concentration of monomer. It can be seen from Figure 3 that a plot of ln [Th] vs t gives an approximately straight line correlation
suggesting that the process is pseudo first order with a rate constant of 1.4 x 10-5 min-1. Figure 3: Plot of a) deposition charge (from reduction peak) against scan rate, b)
ln(unpolymerised Th) vs time for the electrochemical polymerisation of thiophene
into 6.87 mmoles of 1:1 (Bmim)Cl: FeCl3, with 0.25 mmoles Th in 2ml decane, at 30
ºC, 15 cycles. Optimisation of electrocatalytic polymerisation Equi-volume amounts of IL and oil are clearly impractical and pre-concentration of Th into the IL
are clearly required. This was investigated using different rations of decane to IL. Figure 2 shows the
cyclic voltammograms of two different IL: decane ratios. The number of moles of Th converted to
polymer in Figure 2, was calculated from the charge under the voltammogram using equation 1 and
the data are summarised in Table 2. Figure 2: CVs of a) 1:7 v/v of DES: decane: 0.3 ml includes1.43 mmoles of FeCl3
into 1-1 Fe-DES, 2ml of Th -containing decane (0.21mol/L), b) 1:1 v/v of DES:
decane, Th in 2ml of decane is 0.21 mol/L, 25 cycles, mV s-1, 25 ºC. -0.4
0.0
0.4
0.8
1.2
1.6
2. -1.0
-0.5
0.0
0.5
1.0
1.5
2.0
Current (mA)
Potential (V)
25 cycles
b)
-0.4
0.0
0.4
0.8
1.2
1.6
2. -1.0
-0.5
0.0
0.5
1.0
1.5
2.0
Current (mA)
Potential (V)
25 cycles
a) -0.4
0.0
0.4
0.8
1.2
1.6
2. -1.0
-0.5
0.0
0.5
1.0
1.5
2.0
Current (mA)
Potential (V)
25 cycles
a) -0.4
0.0
0.4
0.8
1.2
1.6
2. -1.0
-0.5
0.0
0.5
1.0
1.5
2.0
Current (mA)
Potential (V)
25 cycles
b) Current (mA) Figure 2: CVs of a) 1:7 v/v of DES: decane: 0.3 ml includes1.43 mmoles of FeCl3
into 1-1 Fe-DES, 2ml of Th -containing decane (0.21mol/L), b) 1:1 v/v of DES:
decane, Th in 2ml of decane is 0.21 mol/L, 25 cycles, mV s-1, 25 ºC. Table 2: Stoichiometry effect on the rate of the electropolymerisation reaction of Th
after extraction from decane. Scan rate 50 mV s-1, 25 scans, potential window:-0.4 –
1.9V. IL : Decane
(v/v)
Q (C)
N of Th oxidised (mmol)
R (mmol/min)
Qox
Qred
Ox. Red. Ox. Red. 1:7
0.020
0.022
8.09x10-4
1.01x10-4
2.11x10-5 2.62x10-6
1:1
0.023
0.025
9.36x10-4
1.15x10-4
2.44x10-5
3.0x10-6 Table 2: Stoichiometry effect on the rate of the electropolymerisation reaction of Th
after extraction from decane. Scan rate 50 mV s-1, 25 scans, potential window:-0.4 –
1.9V. Table 2: Stoichiometry effect on the rate of the electropolymerisation reaction of Th
after extraction from decane. Scan rate 50 mV s-1, 25 scans, potential window:-0.4 –
1.9V. Extractive chemical polymerisation of Th and its analogues in 1:2 (Bmim)Cl: FeCl3 For several decades, the lowest sulfur content achieved by HDS of fuels was around 500 mg/kg
(around 6 mmoles/L). 20 To demonstrate the efficacy of the DESs to chemically remove aromatic
sulfur compounds mixtures of Th, BT and DBT were prepared in decane. Previously, the process
could be estimated to be a dynamic equilibrium between the two phases, however with the Lewis
acidic liquids, the Th in the IL phase is constantly being oxidised to form PTh and driving the
equilibrium towards to IL phase. By analysing the effect of some factors on the pre-equilibrium, the
rate of extraction polymer formation can be determined. Optimisation of electrocatalytic polymerisation 20
40
60
80
100
0.01
0.02
0.03
0.04
Charge (C)
Scan rate (mV.s
-1)
a)
10
20
30
40
50
60
4.8274
4.8275
4.8276
4.8277
4.8278
4.8279
4.8280
4.8281
4.8282
Ln(unpolymerised)
Time (min)
b)
K= 1.4X10
-5 min
-1 10
20
30
40
50
60
4.8274
4.8275
4.8276
4.8277
4.8278
4.8279
4.8280
4.8281
4.8282
Ln(unpolymerised)
Time (min)
b)
K= 1.4X10
-5 min
-1 20
40
60
80
100
0.01
0.02
0.03
0.04
Charge (C)
Scan rate (mV.s
-1)
a) Figure 3: Plot of a) deposition charge (from reduction peak) against scan rate, b)
ln(unpolymerised Th) vs time for the electrochemical polymerisation of thiophene
into 6.87 mmoles of 1:1 (Bmim)Cl: FeCl3, with 0.25 mmoles Th in 2ml decane, at 30
ºC, 15 cycles. To determine the amount of Th that could be turned over in a given time, the rate of oxidation from
Table 2 (1:7) was used with the initial monomer concentration (0.21M) to calculate the rate constant. This was found to be 1.0 x 10-7 moles min-1. Taking a 1 L sample of decane containing 1wt % of Th
and 10 x10 cm WE, the time to remove 75 % of this Th from 1wt % in 1 L fuel using
electropolymerisation would be 4.5 hrs. This is clearly far too slow to be practically viable. 8
Removal of BT from decane into IL mixture followed by electropolymerisation was also attempted
and no polymer could be formed with this monomer which was thought to be due to the high reactivity 8 of radical BT cations, which can undergo fast reaction with the chloride anions or with the
imidazolium cation. Electropolymerisation of DBT, was also found to be impossible in this medium. Effect of temperature and stirring Table 3 shows the ability of 1:2 (Bmim)Cl: FeCl3 to extract Th, BT and DBT. It can be seen that the
amount of each component extracted after 5 min decreases when the temperature is raised. This shows
that the extraction must be an exothermic process as the position of the equilibrium is shifted toward
the reagents when the temperature is increased as per Le Chateliers’s principle. It is not possible to
calculate the thermodynamics of phase transfer as the system is not at equilibrium, but it is possible
to contrast the data with those for extraction in different DESs where the enthalpy of extraction was
endothermic (10 – 70 kJ mol-1). 3 Spontaneous polymerisation processes are always strongly
exothermic and it could be this driving the extraction of the monomer. Table 3: Extractive desulfurisation of 35 mmol L-1 of each one of Th, BT and DBT
into 1:2 (Bmim)Cl: FeCl3 mixture, as a function to time, 500 rpm, fuel/IL mass
ratio: 2:1. Table 3: Extractive desulfurisation of 35 mmol L-1 of each one of Th, BT and DBT
into 1:2 (Bmim)Cl: FeCl3 mixture, as a function to time, 500 rpm, fuel/IL mass
ratio: 2:1. Table 3: Extractive desulfurisation of 35 mmol L-1 of each one of Th, BT and DBT
into 1:2 (Bmim)Cl: FeCl3 mixture, as a function to time, 500 rpm, fuel/IL mass
ratio: 2:1. Temperature (oC)
25
40
Time (min)
5
10
5
10
20
Extracting
(%)
Th
85.7 ± 5
˃99.9
76.3 ± 2
95 ± 4
˃99.9
BT
67.5 ± 3
˃99.9
75.3 ± 3
92.8 ± 4
˃99.9
DBT
58 ± 6
˃99.9
65.8 ± 3.8
90.6 ± 2
˃99.9 Transfer of BT and DBT are quite similar once experimental errors are taken into account but either
way neither are significantly affected by temperature. This could suggest that the equilibrium is
governed by the rate at which the monomer is converted to the polymer (oligomer). After 10 min,
the extraction at 25 oC is all but complete whereas those at 40 ºC have still not quite gone to
completion. This further suggests that the transfer is slightly exothermic. Contrasting these data with 9 those obtained for the DESs where after 1 h, a significant proportion of Th was still in the alkane
phase. It can be seen that converting Th to PTh helps in extracting the Th from the decane phase. Effect of temperature and stirring The previous study of thiophene extraction in DESs showed that the surface tension gives a measure
of the cohesive energy density in ionic liquids and this in turn controls the viscosity and conductivity
of the liquid. The lower the surface tension the larger the void volume and the lower the viscosity. 3
Measuring the surface tension of two different molar ratios of the 2:1 and 1:1 FeCl3: (Bmim)Cl it was
found that they were both 46.4 mN m-1. Previously it was found that the best solvent for extracting
of Th, BT and DBT was a mixture of pentylene glycol and choline chloride (4:1) which had a surface
tension of 46 mN m-1. The ferric chloride decreases the surface tension of BmimCl (71 mN m-1) by
delocalising the charge and it also has a stronger interaction with Th than occurs in a non-metallic
DES and these two factors are sufficient to change the enthalpy of transfer from endothermic to
exothermic which makes the partition coefficient larger. those obtained for the DESs where after 1 h, a significant proportion of Th was still in the alkane
phase. It can be seen that converting Th to PTh helps in extracting the Th from the decane phase. In the previous studies, the reactivity of organosulfur rings in HDS processes has been reported by
some researchers and it was investigated that the reactivity follows the order: Th˃ BT ˃ DBT.21 This
could be due to two factors, increasing the size of the solute molecule increase the size of the hole
required to accommodate the solute, the interaction between the iron species and the heterocyclic
sulfur atom is probably precluded due to steric factors. This also explains why BT and DBT are slower
to partition into the ionic liquid since polymerisation of these monomers will be slower due to steric
hindrance and deactivation. The influence of the stirring speed on the sulfur compound extractions is statistically significant as
seen in Figure 4. Doubling the rotation speed of the stirrer in the ionic liquid phase ensured that all
the S-containing species were extracted from the decane at 25 ºC within 5 min which took about
double the time with a slower stirring rate. Figure 4: Effect of stirring speed on the extraction of sulfur compounds, IL: fuel
mole proportion 1:2. Extraction time was 5 min. Effect of temperature and stirring 500
1000
0
20
40
60
80
100
S-removal (%)
Mixing speed (rpm)
Th
BT
DBT Figure 4: Effect of stirring speed on the extraction of sulfur compounds, IL: fuel
mole proportion 1:2. Extraction time was 5 min. 10 Effect of phase ratio Effect of phase ratio To investigate the effect of the mass ratio of the organic and DES phases on the extraction efficiency,
different mass ratios were employed and the results are shown in Figure 5. All were mechanically
agitated (500 rpm) with a magnetic stirrer for 5 min and then left to settle for 5 min to ensure complete
thermodynamic equilibrium at 25 ºC. Figure 5: Desulfurisation of a mixture of sulfur compounds from decane containing
35 mmol L-1 of Th, BT and DBT into (Bmim)FeCl4. 1:1
1:2
1:3
1:4
0
20
40
60
80
100
S-removal (%100)
Mass ratio of IL:fuel
Th
BT
DBT Figure 5: Desulfurisation of a mixture of sulfur compounds from decane containing
35 mmol L-1 of Th, BT and DBT into (Bmim)FeCl4. It can clearly be seen that the amount of (Bmim)FeCl4 was an important factor in EDS. Since the iron
chloride is still in vast excess to thiophene it is probably a mass transport effect which is still
controlling the conversion rate. It can clearly be seen that the amount of (Bmim)FeCl4 was an important factor in EDS. Since the iron
chloride is still in vast excess to thiophene it is probably a mass transport effect which is still
controlling the conversion rate. Effect of time A mixtures of Th, BT and DBT were extracted with different oil to IL ratios, all in one mixture tried
to elucidate the rate limiting factor. Figure 6a shows the efficiency of extracting 35 mmol L-1 of Th,
BT and DBT from the decane phase into 2:1 FeCl3: (Bmim)Cl ionic liquids in an unstirred system. This was done at an IL: oil mass ratio of 1:1. 11 Figure 6: Extractive desulfurisation of Th and its derivatives as a mixture from
decane into 2:1 FeCl3: (Bmim)Cl at 25 ºC, with equimass ratio of IL to fuel: a)
extracting S-compound (%), b) plot of ln (monomer) vs time. 6min 12min 1/2h
1h
2h
4h
8h
1day
0
20
40
60
80
100
Th
BT
DBT
Extracting amount (%)
Time
a)
0
100
200
300
400
500
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Th (K= 2.13X10
-2 min
-1)
BT (K= 5.7X10
-3 min
-1)
DBT (K= 1.3X10
-3 min
-1)
Ln(unreacted)
Time (min)
b) 6min 12min 1/2h
1h
2h
4h
8h
1day
0
20
40
60
80
100
Th
BT
DBT
Extracting amount (%)
Time
a) y
0
100
200
300
400
500
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Th (K= 2.13X10
-2 min
-1)
BT (K= 5.7X10
-3 min
-1)
DBT (K= 1.3X10
-3 min
-1)
Ln(unreacted)
Time (min)
b) Figure 6: Extractive desulfurisation of Th and its derivatives as a mixture from
decane into 2:1 FeCl3: (Bmim)Cl at 25 ºC, with equimass ratio of IL to fuel: a)
extracting S-compound (%), b) plot of ln (monomer) vs time. After 6 min, 38 % of Th had been extracted and it required about 4 hours to extract all the Th (Figure
6a). Comparing this with the stirred system in Figure 5 it can be seen that mass transport is a very
important factor governing extraction and conversion to the polymer. With respect to BT, after 1 h,
only 35 % had been extracted and it only reached 100 % after 8 hrs. Removal of DBT was the slowest
process with only 34 % removed after 4 hrs. This fits in with the earlier observation that the
polymerisation of Th is the fastest followed by BT and DBT. Strong interaction between the Lewis
acid and S-species made the extraction easier even when the reaction takes place in the absence of
stirring. Effect of time The overall removal of the sulfur containing species is given by; Th(decane) ↔ Th(DES)
Th(DES) →PTh(s) Th(decane) ↔ Th(DES) Th(DES) →PTh(s) The reaction could either be limited by the transfer of species into the DES or by the activation of the
monomer. If it is the latter of these then the reaction could be thought of as a pseudo-first order
reaction since the catalyst is present in excess. The rate equation for this type of reaction should
therefore approximate to: 𝑇𝑘𝑘 [𝑇𝑇ℎ]
[𝑇𝑇ℎ]0 = −𝑘𝑘𝑘𝑘 (5) [𝑇𝑇ℎ]
[𝑇𝑇ℎ]0 = −𝑘𝑘𝑘𝑘 (5) A plot of ln [S-compound] should give a straight line plot. The data in Figure 6a were tested in this
way and the results are shown in Figure 6b. It can be seen that the extraction of all of the sulfur 12 containing compounds follow this pseudo first order plot. However, it should be stressed that this is
by no means proof that this is the definite mechanism for the extraction process. The data do not,
however fit well to a second order plot or to a half order plot as may be appropriate if diffusion was
the limiting factor. It can therefore only be suggested that a pseudo first order polymerisation may be
an appropriate model that fits the observed data relatively well. Finally, it is important to note the comparative rate constants for the electrochemical and chemical
rates of polymer production. Comparing the data for Th from Figure 6b with those presented in
Figure 3b, it can be seen that the rate for the chemical production of PTh is approximately 1500 times
faster than the electrochemical process. This would be expected given that the chemical process is a
3-D rather than a 2-D reaction as would be the case for the electrocatalytic process. It should also be
noted that the concentration of FeCl3 is significantly higher for the chemical process. The main difficulty of using ILs to extract thiophenic compounds from oils is that the polymerisation
is a stoichiometric reaction with iron and to make it a catalytic process the Fe(II) produced would
have to be reoxidised. If all the Fe(III) is reduced then the IL becomes more viscous and eventually
solidifies. It is difficult to extract the polymer from the ionic liquid due to the high viscosity. Conclusion This study has shown that the main factor controlling the partitioning of species between an oil and
an ionic liquid is the enthalpy of solvation of the solute and the enthalpy of hole formation in the
DES. These are controlled by the relative solvent-solvent and solvent-solute interactions. Using a
mixture of (Bmim)Cl and FeCl3 causes more thiophene to partition into the ionic phase than when
non-metallic DESs are used. This is thought to be due to the low surface tension but also the specific
interaction between FeCl3 and thiophene. It is shown for the first time that electropolymerisation of
thiophene can be carried out in the in the 1:1 (Bmim)Cl: FeCl3 phase by cyclic voltammetry. This can
drive the equilibrium to pull the Th from the alkane phase as it is polymerised in the IL phase. This study has shown that the main factor controlling the partitioning of species between an oil and
an ionic liquid is the enthalpy of solvation of the solute and the enthalpy of hole formation in the
DES. These are controlled by the relative solvent-solvent and solvent-solute interactions. Using a
mixture of (Bmim)Cl and FeCl3 causes more thiophene to partition into the ionic phase than when
non-metallic DESs are used. This is thought to be due to the low surface tension but also the specific
interaction between FeCl3 and thiophene. It is shown for the first time that electropolymerisation of
thiophene can be carried out in the in the 1:1 (Bmim)Cl: FeCl3 phase by cyclic voltammetry. This can
drive the equilibrium to pull the Th from the alkane phase as it is polymerised in the IL phase. While electrochemical polymerisation of Th is possible it is not practically viable due to the slow
reaction kinetics. A 1:2 (Bmim)Cl: FeCl3 mixture was successfully used to polymerise Th, BT and
DBT. The extraction Th into 1:2 (Bmim)Cl: FeCl3 is exothermic and occurs 1500 times faster than
electropolymerisation in 1:1 (Bmim)Cl: FeCl3. While electrochemical polymerisation of Th is possible it is not practically viable due to the slow
reaction kinetics. A 1:2 (Bmim)Cl: FeCl3 mixture was successfully used to polymerise Th, BT and
DBT. The extraction Th into 1:2 (Bmim)Cl: FeCl3 is exothermic and occurs 1500 times faster than
electropolymerisation in 1:1 (Bmim)Cl: FeCl3. Regeneration of used IL mixture was difficult because polythiophene does not separate from the ionic
liquid (due to similar densities and high viscosity). Conclusion The polymer causes the ionic liquid to gel which
decreases mass transport and stops the polymerisation reaction. To enable ionic liquids to be used for
ultra-desulfurisation of fuel the phase behaviour of the catalyst needs to be addressed and a facile
method of reoxidation needs to be found. Effect of time Regeneration of FeCl3 were attempted using solvent extraction and an electrochemical set up using a
liquid-liquid junction to enable electron transfer.22 Both of these approaches were ultimately
unsuccessful and the ionic liquid solidified with extended use. To improve the viability of ionic
liquids for preparing ultra-low sulfur fuels it is important to focus on the reoxidation of the ionic
liquid. The main issue behind this is maintaining liquid phase behaviour for the ionic liquid and
establishing a catalytic method of reoxidation. Nie and coworkers investigated the EDS process using ILs on commercial diesel samples.23 The
authors suggested that S-removal from commercial fuel is much harder than desulfurisation of model
fuel without giving the reasons.24 Similarly, Eßer et al, 25 reported the Kp for S-removal of commercial
diesel is less than Kp of a model oil. Similarly, Xu et al. and Yu et al. also found that in addition the
relatively high moisture content in commercial diesel fuel necessitates additives which have a
negative impact on the removal of sulfur compounds.26 For practical applications the other components in fuels need to be taken into account when preparing
a sulfur extraction process. Commercial diesel was used but it was spiked with Th, BT, and DBT each
at a concentration of 35 mmol L-1. The concentrations of the thiophenic compounds were determined
by GC at the same operational reaction conditions as those described above. During the process, the
sulfur levels in the upper phase of each one of Th, BT and DBT could be extracted to (27.8, 18.8 and
16.3 mmol L-1) respectively through the optimisation of reaction conditions; which means 79.3, 53.8 13 and 46.5 % of Th, BT and DBT respectively were removed just after 5 min. This compared with 85.7,
67.5 and 58% respectively for the corresponding extraction from decane. As expected, the extraction
data for removing Th, BT and DBT from commercial diesels are lower than from decane. This may
be attributed to the coexistence of many aromatics, oxygen and nitrogen additives which compete
with the partition of S-compounds in the (C4mim)FeCl4 and saturate the ionic liquid phase.27 References g
,
( ),
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Imputation of Ancient Whole Genome Sus scrofa DNA Introduces Biases Toward Main Population Components in the Reference Panel
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Frontiers in genetics
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Imputation of Ancient Whole Genome Sus scrofa DNA Introduces Biases Toward Main
Population Components in the Reference Panel
Erven, Jolijn; Çakirlar, Canan; Raemaekers, Daan; Madsen, Ole; Bradley, Daniel Imputation of Ancient Whole Genome Sus scrofa DNA Introduces Biases Toward Main
Population Components in the Reference Panel
Erven, Jolijn; Çakirlar, Canan; Raemaekers, Daan; Madsen, Ole; Bradley, Daniel Published in:
Frontiers in Genetics DOI:
10.3389/fgene.2022.872486 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below. Document Version
Publisher's PDF, also known as Version of record Publication date:
2022 Link to publication in University of Groningen/UMCG research database Citation for published version (APA):
Erven, J., Çakirlar, C., Raemaekers, D., Madsen, O., & Bradley, D. (2022). Imputation of Ancient Whole
Genome Sus scrofa DNA Introduces Biases Toward Main Population Components in the Reference Panel. Frontiers in Genetics, 13, Article 872486. https://doi.org/10.3389/fgene.2022.872486 Copyright
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number of authors shown on this cover page is limited to 10 maximum. ORIGINAL RESEARCH
published: 12 July 2022
doi: 10.3389/fgene.2022.872486 Imputation of Ancient Whole Genome
Sus scrofa DNA Introduces Biases
Toward Main Population Components
in the Reference Panel
J. A. M. Erven 1*, C. Çakirlar 1, D. G. Bradley 2, D. C. M. Raemaekers 1 and O. Madsen 3
1Groningen Institute of Archaeology, University of Groningen, Groningen, Netherlands, 2Smurfit Institute of Genetics, Trinity
College Dublin, Dublin, Ireland, 3Animal Breeding and Genomics, Wageningen University and Research, Wageningen,
Netherlands Sequencing ancient DNA to high coverage is often limited by sample quality and cost. Imputing missing genotypes can potentially increase information content and quality of
ancient data, but requires different computational approaches than modern DNA
imputation. Ancient imputation beyond humans has not been investigated. In this
study we report results of a systematic evaluation of imputation of three whole
genome
ancient
Sus
scrofa
samples
from
the
Early
and
Late
Neolithic
(~7,100–4,500 BP), to test the utility of imputation. We show how issues like genetic
architecture and, reference panel divergence, composition and size affect imputation
accuracy. We evaluate a variety of imputation methods, including Beagle5, GLIMPSE, and
Impute5 with varying filters, pipelines, and variant calling methods. We achieved genotype
concordance in most cases reaching above 90%; with the highest being 98% with
~2,000,000 variants recovered using GLIMPSE. Despite this high concordance the
sources of diversity present in the genotypes called in the original high coverage
genomes were not equally imputed leading to biases in downstream analyses; a trend
toward genotypes most common in the reference panel is observed. This demonstrates
that the current reference panel does not possess the full diversity needed for accurate
imputation of ancient Sus, due to missing variations from Near Eastern and Mesolithic wild
boar. Imputation of ancient Sus scrofa holds potential but should be approached with
caution due to these biases, and suggests that there is no universal approach for
imputation of non-human ancient species. Edited by:
Xiangdong Ding,
China Agricultural University, China
Reviewed by:
Luca Ermini,
Luxembourg Institute of Health,
Luxembourg
Huashui Ai,
Jiangxi Agricultural University, China
*Correspondence:
J. A. M. Erven
jolijn_erven@hotmail.com Edited by:
Xiangdong Ding,
China Agricultural University, China Reviewed by:
Luca Ermini,
Luxembourg Institute of Health,
Luxembourg
Huashui Ai,
Jiangxi Agricultural University, China *Correspondence:
J. A. M. Erven
jolijn_erven@hotmail.com Specialty section:
This article was submitted to
Livestock Genomics,
a section of the journal
Frontiers in Genetics Keywords: imputation, ancient DNA (aDNA), Sus scrofa, animal husbandry, Neolithic Received: 09 February 2022
Accepted: 20 May 2022
Published: 12 July 2022 Keywords: imputation, ancient DNA (aDNA), Sus scrofa, animal husbandry, Neolithic 2.1 Data Description and Preparation
2.1.1 Ancient Samples A number of factors are known to influence imputation
ranging from reference panel characteristics to demographic
history; assessing the potential and limitations of imputation
of species beyond model species like humans is valuable to aid our
understanding of not only imputation performance but also
recent evolutionary events. This paper assesses the power of imputation to increase the
quality and information potential of low coverage aDNA samples,
using Sus scrofa as a case study. This species is an intensively
studied livestock species in terms of aDNA, particularly in the
context of expansion of animal husbandry into Europe and
significantly enhancing our understanding of how farming
started in Europe (Larson et al., 2007; Ottoni et al., 2013;
Frantz et al., 2019). Investigations have indicated that ancient
Near Eastern domestic pigs lost their Near Eastern genomic
signatures after their introduction to Europe (Larson et al.,
2007; Frantz et al., 2019). Obtaining HQ samples to pinpoint
the pace and nature of this turnover in different regions and
shorter timescales in relation to larger societal and economic
developments is necessary, but it remains a challenge due to poor
preservation and contamination. To address this challenge, a
systematic evaluation of different imputation methods was
performed on whole genome ancient Sus scrofa DNA using
data from a recent study consisting of ancient whole genomes
of pigs sequenced to an appropriate depth for imputation (Frantz
et al., 2019). Imputation achieved high genotype concordance but
this is paired with biases toward a fraction of the reference panel. These biases might be related to the size and diversity of the
reference panel, the reference genome, or the genetic architecture
of pigs, and they impose limitations on the interpretive power of
imputed data in terms of the proposed genomic turnover of this
species in particular and in general the evolution of animal
husbandry in Neolithic Europe. Contamination from prokaryotes and humans was assessed by
calculating percent identity score and coverage per read with
BLAST + Blastn Megablast v2.10.1 on prokaryotes, human and
Sus scrofa databases (Camacho et al., 2008). Reads were
considered contaminants if the percent identity (E-value) and
coverage of the contaminants (prokaryotes and humans) was
higher than the percent identity and coverage of Sus scrofa. Contaminated reads were removed from the BAM file with a
custom-made python script. 2.1 Data Description and Preparation
2.1.1 Ancient Samples 2009; Prüfer et al., 2010; Ginolhac et al., 2011; Sánchez-Quinto
et al., 2012; Parks and Lambert, 2015; Kistler et al., 2017). One
way to counter these problems is imputation, which is a powerful
way to improve the quality of data and can potentially maximize
the power of analysis that require dense genotypes such as runs of
homozygosity (ROH), in depth admixture and trait association
analyses (Gamba et al., 2014; Martiniano et al., 2017). Imputation
is widely employed in studies of modern data (Van den Berg et al.,
2019; Ye, et al., 2019), targeting allele frequencies from a set of
reference individuals to infer allele frequencies at unknown or
missing sites (Browning and Browning, 2007; Ausmees, 2019). Seven archaeological samples with high-coverage data and four
archaeological samples with moderate coverage from Frantz et al. (2019) were used (Table 1; Supplementary Table S1). Raw
FASTQ reads were downloaded from the ENA (accession
numbers see Supplementary Table S1). Raw reads were
trimmed using cutadapt v2.10 (Martin, 2011) for quality
(<20),
length
(<20)
and
adapters
used
in
the
library
preparation (Meyer and Kircher, 2010). FastQC v0.11.9 quality
reports were made for the raw and trimmed data (Andrews,
2010). The trimmed reads were aligned applying the Burrows-
Wheeler algorithm (BWA) aln v0.7.17 (Li and Durbin, 2009) to
the Sus scrofa 11.1 reference genome (Warr et al., 2020), with
default parameters apart from disabling the seed option (−l 1024),
increasing the maximum number of gap opens (−o 2) and
changing the maximum edit distance (−n 0.01). Duplicates
were removed with Picard MarkDuplicates v2.18.17 (http://
broadinstitute.github.io/picard) and BAM files from different
sequencing lanes were merged using SAMtools merge v0.1.19
(Li
et
al.,
2009). Duplicates
were
removed
with
FilterUniqueSamCons.py for the merged BAM files (Kircher,
2012). Indels
were
realigned
with
GATK
3.8
RealignerTargetCreator
and
IndelRealigner
with
default
parameters (Van der Auwera et al., 2013). Depth of coverage
and
quality
were
computed
using
Qualimap
v2.2.1
(Okonechnikov et al., 2015). Molecular damage was assessed
using MapDamage2.0 using default parameters (Jónsson et al.,
2013). In aDNA studies, imputation has been applied on human
genomes and achieved high levels of concordance between
imputed genotypes and their high-quality (HQ) counterparts
(>99%) (Gamba et al., 2014; Martiniano et al., 2017; Ausmees
et al., 2021). Imputation of aDNA beyond humans is lacking;
livestock aDNA is critical to understand pivotal moments in
recent evolution such as domestication and pose an excellent case
study. Citation: Recent advances in sequencing techniques led to a dramatic increase in the amount of retrievable
ancient DNA (aDNA) from archaeological remains (Kircher, 2012), providing new insights into
recent evolutionary history (Slatkin and Recimo, 2016; MacHugh et al., 2017; Brunson and Reich,
2019; McHugo et al., 2019). Poor preservation and contamination of exogenous DNA restricts
sequence quality, reliability, and coverage of aDNA from archaeological bones (Pääbo et al., 2004;
Prüfer et al., 2010). Furthermore, the damaged nature of aDNA poses computational challenges and
introduces biases to the analysis of aDNA (Höss et al., 1996; Brotherton et al., 2007; Briggs et al., Erven JM, Çakirlar C, Bradley DG,
Raemaekers DCM and Madsen O
(2022) Imputation of Ancient Whole
Genome Sus scrofa DNA Introduces
Biases Toward Main Population
Components in the Reference Panel. Front. Genet. 13:872486. doi: 10.3389/fgene.2022.872486 July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 1 Imputation of Ancient Sus scrofa Erven et al. 2.1 Data Description and Preparation
2.1.1 Ancient Samples Imputation was assessed by comparing imputed genotypes to
their corresponding HQ genotypes, similar to previous studies
(Gamba et al., 2014; Martiniano et al., 2017; Ausmees et al., 2021). Three of the seven samples with high-coverage data (KD033,
KD037, and VEM185) were downsampled with Picard v2.18.17
(http://broadinstitute.github.io/picard), to create low coverage
samples for imputation ranging from 0.5 to 2× with steps of 0. 5×. Three methods were used to assess the accuracy of
imputation: Method 1, imputation with variant sites; Method
2, imputation with all confident sites; and Method 3, added to
achieve
higher
genotype
concordance
which
called
only
genotypes present in the reference panel. HQ genotypes were
created from the high-coverage samples to create a golden
standard. Genotype likelihoods were called with the Genome
Analysis Toolkit (GATK) UnifiedGenotyper v3.8.0 (Van der
Auwera et al., 2013) using either each alignment data of the
ancient samples individually or by joined SNP calling. Genotype
likelihoods were called with a minimum quality of 25, with output
mode EMIT_VARIANTS_ONLY for Method 1, EMIT_ALL_ 2 MATERIALS AND METHODS Evaluating imputation of Sus scrofa aDNA by comparing three
tools, two pipelines, and three variant calling methods. July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 2 Imputation of Ancient Sus scrofa Erven et al. TABLE 1 | Sample information. ID, origin, period and ancestry taken from Frantz et al. (2019). ID
Origin
Period
Ancestry
Genome coverage
KD033
Germany-Herxheim
Neolithic
~46% European, ~54% Near Eastern
6.9
KD037
Germany-Herxheim
Neolithic
~91% European, ~9 Near Eastern
21.6
VEM185
England-Durrington Walls
Neolithic/Bronze Transition
~90% European, ~10 Near Eastern
21.7
TABLE 2 | Reference panels with their respective number of individuals/population. Reference panel
Number of individuals
Main references
Dutch wild boar-European wild boar (EUW)
12
Italian wild boar-European wild boar (EUW)
6
French wild boar-European wild boar (EUW)
1
Pig breeds-European Domestic (EUD)
25
Greek wild boar (BLW)
4
Near Eastern + Turkish wild boar- Near Eastern wild boar (NEW)
3
Total
51
Main + ancient references
Main reference
51
Near Eastern-Ancients (ANC)
5
European-Ancients (ANC)
3
Total
59 TABLE 1 | Sample information. ID, origin, period and ancestry taken from Frantz et al. (2019). ID
Origin
Period
Ancestry
Genome coverage
KD033
Germany-Herxheim
Neolithic
~46% European, ~54% Near Eastern
6.9
KD037
Germany-Herxheim
Neolithic
~91% European, ~9 Near Eastern
21.6
VEM185
England-Durrington Walls
Neolithic/Bronze Transition
~90% European, ~10 Near Eastern
21.7 TABLE 1 | Sample information. ID, origin, period and ancestry taken from Frantz et al. (2019). TABLE 2 | Reference panels with their respective number of individuals/population. Reference panel
Number of individuals
Main references
Dutch wild boar-European wild boar (EUW)
12
Italian wild boar-European wild boar (EUW)
6
French wild boar-European wild boar (EUW)
1
Pig breeds-European Domestic (EUD)
25
Greek wild boar (BLW)
4
Near Eastern + Turkish wild boar- Near Eastern wild boar (NEW)
3
Total
51
Main + ancient references
Main reference
51
Near Eastern-Ancients (ANC)
5
European-Ancients (ANC)
3
Total
59 TABLE 2 | Reference panels with their respective number of individuals/population. Number of individuals effect of the reference composition on the imputation, multiple
reference panels were considered. The main reference panel
consists of modern pig breeds, European wild boar, and Near
Eastern wild boar (51 individuals, 12,737,362 variants—Table 2;
Supplementary Table S2). 2 MATERIALS AND METHODS In order to avoid
introducing a possible bias from nucleotide misincorporations
due to post-mortem damage, the generated VCF (Variant Call
Format) files were filtered to exclude all sites where the most likely
genotype could have been inferred from a deaminated allele with
a custom-made python script. For C→T deaminations, C↔T
SNPs were excluded from further analyses if the most likely
genotype contained a T allele, and for G→A deaminations, G↔A
SNPs were excluded from further analyses if the most likely
genotype contained an A allele. Genotypes were not filtered in
Method 3 when using GLIMPSE, because this software only
imputes genotypes present in the target VCF, they were
instead kept as no calls (./.). 2.1.2 Reference Panel The reference material used for imputation consisted of the wild
boar and pig breeds collection of Wageningen University and two
Iberian samples from Ramírez et al. (2015) (Supplementary
Table S1). Pig breeds that have no known introgression with
Asian breeds were selected to avoid potential bias. Genotype
likelihoods were called with the Genome Analysis Toolkit (GATK)
UnifiedGenotyper v3.8.0 (Van der Auwera et al., 2013), with a
minimum
quality
of
15,
calling
SNPs,
with
the
mode
EMIT_VARIANTS_ONLY
for
Method
1
and
EMIT_ALL_CONFIDENT_SITES for Method 2. The reference
panel was filtered to only include autosomal biallelic SNPs, a
minimum quality of 30, and a minimum depth of 4, a call rate of
0.8, and removal of repetitive elements. In order to evaluate the 2 MATERIALS AND METHODS To deduce the effect of ancient
samples on imputation, eight ancient individuals were added
to the main reference panel, consisting of two Near Eastern
samples,
three
ancient
Near
Eastern,
and
three
ancient
European
samples
(59
individuals,
10,823,257
variants—Table 2, Supplementary Table S2), called Main +
ancient reference. Moreover, the main reference panel was
divided into several subsets to pinpoint the effect of reference
bias on imputation (See Supplementary Material-Subsets of
reference panel). Additionally, to deduce the effect of Asian
haplotypes on imputation, Asian wild boars, Asian domestic
pigs and South-East Asian Sus were added to the reference
panel (See Supplementary Material-Including Asian samples). Different filters and combinations of filters were used on the
reference panel to optimize the imputation workflow and deduce
the effects of these filters on imputation. These filters consisted of
removing 1) transversions, 2) transitions, 3) filtering for minor
allele frequency (MAF) bins {<0.05, 0.05–0.1, 0.1–0.3, >0.05, >0.3,
No MAF}, and their various combinations. Results of all
combinations can be found in Supplementary Table S2. The
reference panels were phased with Beagle5 (Browning et al.,
2018), using default parameters apart from changing the
effective population size (Ne) to 20,000 (Groenen et al., 2012). CONFIDENT_SITES for Method 2 and output mode EMIT_
ALL_SITES
and
genotyping
mode
GENOTYPE_GIVEN_
ALLELES for Method 3, with -alleles genotypes from the
reference panel. Variants were filtered to keep only autosomal,
biallelic SNPs, and a minimum quality of 30. In order to avoid
introducing a possible bias from nucleotide misincorporations
due to post-mortem damage, the generated VCF (Variant Call
Format) files were filtered to exclude all sites where the most likely
genotype could have been inferred from a deaminated allele with
a custom-made python script. For C→T deaminations, C↔T
SNPs were excluded from further analyses if the most likely
genotype contained a T allele, and for G→A deaminations, G↔A
SNPs were excluded from further analyses if the most likely
genotype contained an A allele. Genotypes were not filtered in
Method 3 when using GLIMPSE, because this software only
imputes genotypes present in the target VCF, they were
instead kept as no calls (./.). CONFIDENT_SITES for Method 2 and output mode EMIT_
ALL_SITES
and
genotyping
mode
GENOTYPE_GIVEN_
ALLELES for Method 3, with -alleles genotypes from the
reference panel. Variants were filtered to keep only autosomal,
biallelic SNPs, and a minimum quality of 30. 2.2 Genetic Map A genetic map was created using the recombination frequencies
that Johnsson et al. (2020) estimated based on nine genotyped
pedigrees on the Sus scrofa 11.1 reference genome. These July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 3 Erven et al. Imputation of Ancient Sus scrofa calculated by dividing the amount of imputed genotypes to
the total amount of HQ genotypes. recombination frequencies were converted to cM using the
Haldane
formula
(Haldane’s
Mapping
Function,
2008). Genetic maps were made for each chromosome in the plink
format with bins of 1 MB (Supplementary Table S3). 2.6 Reference Affinity Correct and incorrect imputed genotypes were compared to their
HQ counterpart to assess whether imputed genotypes show a
systematic bias toward the reference genome. Reference bias was
measured as the presence/absence of different ancestral/origin
groups between the correct and incorrect imputed genotypes and
their HQ counterpart. 2.3 Imputation p
For Methods 1–3 imputation was performed using Impute5 and
Beagle5, using default parameters, with a phased reference panel
(Supplementary Table S2), with a Ne of 20,000 and, --div-select
and –out-gp-field parameters for Impute5 (Rubinacci et al., 2020)
and window = 40, overlap = 4 and gp = true parameters for
Beagle5 (Browning et al., 2018). Imputation was performed for
chromosome 1–18, individually and using sliding windows (See
Supplementary
Material—Chromosomal
imputation). The
effect of including multiple ancient samples on imputation was
evaluated by imputing joint ancient samples and was compared to
individual
imputation
(Supplementary
Material—Joined
Imputation). The
focus
was
on
individual
imputation. Imputation was performed using two different imputation
pipelines: 1) the original one-step pipeline used in Ausmees
et al. (2021) and 2) the two-step pipeline used for low
coverage samples in Hui et al. (2020). The two-step pipeline
adds another filtering step prior to imputation that accounts for
genotype probability. Beagle 4.1 was used to calculate genotype
probabilities for the target downsampled VCF using default
parameters, with the same phased reference panel that was
used for imputation (Supplementary Table S2), with a Ne of
20,000 and gprobs = true parameters. Variants with a genotype
probability (GP) < 0.99 were removed from the target
downsampled VCF, leaving only confident genotype calls. The
imputed genotypes were filtered for an imputation score of 1
(highest imputation accuracy). For Method 3, GLIMPSE v1.1.1
(Rubinacci, et al., 2021) was also used with similar settings as
applied in ancient human imputation and the pipeline proposed
by Rubinacci, et al. (2021), with default parameters, and a phased
reference panel (Supplementary Table S2). Variants with an
imputation
score
of
<1
were
removed
from
the
target
downsampled VCF, leaving only confident imputed genotype
calls. GLIMPSE v1.1.1 was only tested with Method 3 because of
the incompatibility with the other two methods/pipelines. 2.5 Downstream Analysis Downstream
analyses
were
performed
to
investigate
the
difference
and/or
similarity
between
imputed
and
HQ
genotypes. Data were pruned with PLINK 1.9 (Purcell et al.,
2007) with the parameters—geno 0.10. A principal component
analysis (PCA) was performed on diploid genotypes consisting of
the reference panel, the HQ samples and the imputed samples
using PLINK 1.9 pca on autosomes only. Eigenvalues and vectors
were plotted with the use of Mathplotlib (Hunter, 2007) and
Seaborn (https://zenodo.org/record/883859#.XSdFFugza01). An
admixture analysis was performed using the same dataset as the
PCA analysis, however separately for downsampled, imputed and
HQ genotypes. ADMIXTURE v1.3.0 (David et al., 2009) was used
with standard parameters and K ranging from 2 till 5. Furthermore,
bootstrapping
was
performed
using
the
parameter
-B. Identical
By
Descent
(IBD)
analysis
was
conducted on the same dataset as the admixture analysis. IBDseq v2.0 with standard parameters was used to calculate
IBD segments between samples (Browning and Browning,
2013). A regions of homozygosity analysis was performed
using the same dataset as the admixture analysis using plink
–homozyg with the parameters –homozyg-kb 10, --homozyg-gap
10, --homozyg-snp 100, --homozyg-window-het 2, --homozyg-
window-snp 100 --homozyg-window-missing 1. DetectRUNS
(https://cran.r-project.org/package=detectRUNS) was used to
visualize and calculate ROH statistics. 2.4 Genotype Concordance Genotype concordance was calculated for three imputation tools,
two pipelines and three variant calling methods to test the best
method to approach imputation in Sus scrofa. Downstream
analyses were performed to assess the accuracy and power of
imputation. 2.4 Genotype Concordance
Imputation accuracy was assessed by genotype concordance
defined as the fraction of genotypes that were imputed
correctly. This was measured by dividing the incorrectly
imputed SNPs with all imputed SNPs and was measured
separately for each sample. The correctly and incorrectly
imputed SNPs were derived from comparing the imputed
SNPs to their HQ counterpart similar to the approach of
Picard GenotypeConcordance. The HQ genotypes used are
pre-deamination
filtered,
to
keep
confident
transitions
(transitions that also occurred in the reference panel), and
not transitions arising from deamination. The incorrectly
imputed
SNPs
were
classified
into
incorrect
positions
(positions not occurring in HQ) and incorrect genotypes
(genotypes
different
from
HQ
genotypes). Information
content, that is, the amount of gained genotypes, was Frontiers in Genetics | www.frontiersin.org 3.1 Genotype Concordance 3.1.1 Tools: Beagle5 Versus Impute5 g
p
Genotype concordance was higher for Beagle5 compared to
Impute5 for KD037 and VEM185 but lower for KD033
(Figure 1). For both tools, KD037 and VEM185 performed
better than KD033, this being more pronounced for Beagle5. The amount of correctly imputed variants differed greatly
between the tools, with Beagle5 being systematically lower
(Figures 1IC,D). Impute5 imputed 25%–34% of the total
amount of HQ genotypes, while Beagle5 imputed around 5%. July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 4 Erven et al. Imputation of Ancient Sus scrofa FIGURE 1 | Genotype concordance and fraction of high-quality (HQ) genotypes (portrayed in decimals, total HQ genotypes: VEM185: 4,701,683, KD037:
4,531,126, KD033: 3,887,848) using different imputation tools and reference panels. Imputed from 1× downsampled coverage genomes of VEM185, KD037, and
KD033. I) Genotype concordance for Impute 5 (A), Beagle5 (B), Fractions of HQ genotypes covered by imputation in Impute5 (C), and Beagle5 (D). II) Genotype
concordance and Fraction of HQ genotypes of various MAF bins of Main + 2-step + Anc + all reference panel. Genotype concordance for Impute 5 (A), Beagle5, (B)
Fractions of HQ genotypes covered by imputation in Impute5 (C), Beagle5 (D) and, Genotype concordance of only heterozygotic variants of various MAF bins imputed
with Impute5 (E) III) Filters on target downsampled vcf Genotype concordance for Impute 5 (A) Beagle5 (B) Fractions of HQ genotypes covered by imputation in
p FIGURE 1 | Genotype concordance and fraction of high-quality (HQ) genotypes (portrayed in decimals, total HQ genotypes: VEM185: 4,701,683, KD037:
4,531,126, KD033: 3,887,848) using different imputation tools and reference panels. Imputed from 1× downsampled coverage genomes of VEM185, KD037, and
KD033. I) Genotype concordance for Impute 5 (A), Beagle5 (B), Fractions of HQ genotypes covered by imputation in Impute5 (C), and Beagle5 (D). II) Genotype
concordance and Fraction of HQ genotypes of various MAF bins of Main + 2-step + Anc + all reference panel. Genotype concordance for Impute 5 (A), Beagle5, (B)
Fractions of HQ genotypes covered by imputation in Impute5 (C), Beagle5 (D) and, Genotype concordance of only heterozygotic variants of various MAF bins imputed
with Impute5 (E). III) Filters on target downsampled vcf. Genotype concordance for Impute 5 (A), Beagle5 (B), Fractions of HQ genotypes covered by imputation in
Impute5, (C) Beagle5 (D). 3.1 Genotype Concordance IV) Genotype concordance (A) and fraction of HQ genotypes (B) from GLIMPSE, Impute5 and Beagle5 for Method 3. * Genotype concordance
of Method 3 was done on all sites and cannot be directly compared to the other methods. Main, original one-step pipeline on main reference panel; 2-step, two-step
pipeline with an extra filtering step; Anc, Ancient samples included in the reference panel; TV, transversions. FIGURE 1 | Genotype concordance and fraction of high-quality (HQ) genotypes (portrayed in decimals, total HQ genotypes: VEM185: 4,701,683, KD037:
4,531,126, KD033: 3,887,848) using different imputation tools and reference panels. Imputed from 1× downsampled coverage genomes of VEM185, KD037, and
KD033. I) Genotype concordance for Impute 5 (A), Beagle5 (B), Fractions of HQ genotypes covered by imputation in Impute5 (C), and Beagle5 (D). II) Genotype
concordance and Fraction of HQ genotypes of various MAF bins of Main + 2-step + Anc + all reference panel. Genotype concordance for Impute 5 (A), Beagle5, (B)
Fractions of HQ genotypes covered by imputation in Impute5 (C), Beagle5 (D) and, Genotype concordance of only heterozygotic variants of various MAF bins imputed
with Impute5 (E). III) Filters on target downsampled vcf. Genotype concordance for Impute 5 (A), Beagle5 (B), Fractions of HQ genotypes covered by imputation in
Impute5, (C) Beagle5 (D). IV) Genotype concordance (A) and fraction of HQ genotypes (B) from GLIMPSE, Impute5 and Beagle5 for Method 3. * Genotype concordance
of Method 3 was done on all sites and cannot be directly compared to the other methods. Main, original one-step pipeline on main reference panel; 2-step, two-step
pipeline with an extra filtering step; Anc, Ancient samples included in the reference panel; TV, transversions. Beagle5 achieved the highest genotype concordances in KD037
and VEM185, but produced less correctly imputed variants. Impute5, on the other hand, achieved the highest genotype
concordance in KD033 and produced more correctly imputed
variants. Furthermore,
genotype
concordance
between
chromosomes was more uniform in Beagle5 compared to
Impute5 (Supplementary Table S4). These results are based
on the default one-step pipeline. The main one-step pipeline
was extended to the two-step pipeline to test various settings for both tools that could influence imputation accuracy changing one
element at a time. 3.1.3 Reference Panel: With and Without Ancient
Samples Genotype concordance was ~5% higher in the two-step pipeline
when using the reference panel including ancient samples for
Impute5, whereas the inclusion of ancient samples only provided
a 0.5% different genotype concordance for Beagle5 (Figures
1IA,B;
Supplementary
Table
S4). Similarly,
genotype
concordance between chromosomes showed more uniformity
with
ancient
samples
than
without
ancient
samples
for
Impute5, but not for Beagle5 (Supplementary Table S4). The
amount of correctly imputed variants with respect to the
inclusion of ancient samples had no effect for Impute5 but
decreased for Beagle5 (Figures 1IC,D). this analysis were IMP1 and IMP2. Filtering for transitions pre-
imputation, thus only keeping transversions, had the most
favorable
effect
on
genotype
concordance. The
highest
genotype concordance for Beagle5 was filtering for transitions
pre-
and
post-imputation,
whereas
the
highest
genotype
concordance for Impute5 was filtering for transitions pre-
imputation
(Figures
1IIIA,B). The
amount
of
correctly
imputed variants of the pre- and post-imputation filters
decreased drastically compared to using no filter on the target
VCF for both tools (Figures 1IIIC,D). The imputed variants were
further filtered for well-known positions, namely the 50 k porcine
SNP-Chip (Yang et al., 2017), the transversions only, and main
SNP-sets from the study of Frantz et al. (2019). These filters did
not improve genotype concordance and decreased the amount of
correctly
imputed
variants
(Supplementary
Figure
S3
— Methods Known genomic positions). 3.1.5 Reference Panel: Pre-Imputation Filters Versus
Standard Reference panels filtered for transversions and transitions showed
similar genotype concordance, with transversions only having the
lowest genotype concordance (Supplementary Figures 2A,B). The amount of correctly imputed variants decreased drastically
for only transitions and only transversions with roughly 50%, in
both tools (Supplementary Figures 2C,D). The uniformity of
concordance between chromosomes was equal for all filters
(Supplementary Table S4). Reference panels filtered for MAF showed variation in genotype
concordance, where MAF bins <0.05 and 0.05–0.10 had the highest
and MAF >0.3 the lowest genotype concordance for Impute5
(Figure 1IIA). This contrasted with Beagle5, where MAF <0.05
had the lowest genotype concordance and MAF >0.3 the highest
(Figure 2B). Filtering for MAF (Beside the common variant >0.05
filter) drastically decreased the amount of correctly imputed
variants in both tools (Figures 1IIC,D). Genotype concordance
of heterozygotes did not show the same trend as all variant
genotype concordance for Impute5 (Figure 1IIE), ~30% of the
total imputed genotypes were heterozygotes (Supplementary
Table S4). Genotype concordance of MAF bin <0.05 decreased
drastically, while the other MAF bins decreased more modestly. 3.1.2 Pipeline: One-Step Versus Two-Step Genotype concordance in the two-step pipeline was higher compared
to the one-step pipeline for both tools (Figures 1IA,B). The amount of
correctlyimputedvariants increased for Beagle5 whereastheamountof
correctly imputed variants for Impute5 stagnated (Figures 1IC,D). Genotype concordance between chromosomes was more uniform in July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 5 Imputation of Ancient Sus scrofa Erven et al. | Principal component analysis comparing high-quality, imputed genotypes and downsampled data together with samples from the reference panel. MP4 (B), and IMP5 (C). onent analysis comparing high-quality, imputed genotypes and downsampled data together with samples from the reference panel. P5 (C). FIGURE 2 | Principal component analysis comparing high-quality, imputed genotypes and downsampled data together with samples from the reference panel. IMP1 (A), IMP4 (B), and IMP5 (C). July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 6 Imputation of Ancient Sus scrofa Erven et al. TABLE 3 | Reference panel abbreviations. ID
Reference panel
IMP1
Main + 2-step + Ancients + All confident sites, Impute5
IMP2
Main + 2-step + Ancients + All confident sites, Beagle5
IMP3
Main + 2-step + Ancients + All confident sites + MAF<0.05, Impute5
IMP4
Main + 2-step + Ancients + All confident sites + MAF>0.3, Beagle5
IMP5
Main + 2-step + Ancients + All, GLIMPSE the
two-step
pipeline
compared
to
the
one-step
pipeline
(Supplementary Table S4) (variation of filter combinations used
for the comparisons can be found in Supplementary Table S2). 3.1.4 Reference Panel: Variant Sites Versus All
Confident Sites Category (All) Using the all confident sites category, method 2, slightly increased
genotype concordance for both tools (Supplementary Figures
S1A,B). The amount of correctly imputed variants was larger in
the all confident sites category compared to the variant sites
category, method 1, for both tools (Supplementary Figures
S1C,D). The uniformity of genotype concordance between
chromosomes was more equal in the all confident sites
category compared to the variant sites category for Beagle5,
except for KD037 (Supplementary Table S4). 3.1.7 Target VCF: All Sites (Sites Present in Reference
Panel) A method that has been shown to achieve high genotype
concordance in ancient human imputation (Martiniano et al.,
2017; Hui, et al., 2020; Method 3) was also applied. This method
was tested on three imputation tools, Beagle5, Impute5, and
GLIMPSE. GLIMPSE
achieved
the
highest
genotype
concordance, reaching 98% in KD037 and VEM185 and 96%
in KD033 (Figure 1IVA). Genotype concordance for Beagle5
stayed constant for VEM185 and KD037 but increased for KD033
compared to the two-step pipeline. Genotype concordance for
Impute5 decreased for all samples compared to the two-step
pipeline. The amount of correctly imputed variants increased for
all samples and all tools, reaching roughly 50% (Figure 1IVB). Furthermore, the genotype concordance between chromosomes
was more constant for this method compared to the two-step
pipeline (Supplementary Table S4). 3.2 Downstream Analysis Downstream analyses were performed on the imputed genotypes
IMP1, 2, 3, 4, and 5 (Full descriptions found in Table 3). PCA were
performed to pinpoint and compare the genetic affinities of
imputed, HQ and downsampled samples. Variation captured by
the first two principal components of IMP1 shows that the imputed
genotypes of KD033, KD037, and VEM185 cluster closer to their
HQ counterparts in the first principal component (PC1, 9.5%
variation) but tend to have a bias toward European wild boar in the
second principal component (PC2, 4.1% variation) (Figure 2A). This bias is greater for KD033 compared to KD037 and VEM185. PCA of IMP2 shows that the downsampled genotypes and the HQ 3.1.6 Target VCF: Filters Versus No Filters
The target VCF was filtered pre- and post-imputation to deduce
the effect on genotype concordance. The reference panels used for 3.1.6 Target VCF: Filters Versus No Filters
The target VCF was filtered pre- and post-imputation to deduce
the effect on genotype concordance. The reference panels used for 3.1.6 Target VCF: Filters Versus No Filters 3.1.6 Target VCF: Filters Versus No Filters The target VCF was filtered pre- and post-imputation to deduce
the effect on genotype concordance. The reference panels used for The target VCF was filtered pre- and post-imputation to deduce
the effect on genotype concordance. The reference panels used for July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 7 Imputation of Ancient Sus scrofa Erven et al. FIGURE 3 | Admixture analysis with K = 3, comparing high-quality and imputed data from IMP1-5 together with samples from the reference panel. NE, Near East;
EU, European. The colors represent the three ancestral groups, where blue is European domestic pig, red is European wild boar, and yellow is Near Eastern. FIGURE 3 | Admixture analysis with K = 3, comparing high-quality and imputed data from IMP1-5 together with samples from the reference panel. NE, Near East;
EU, European. The colors represent the three ancestral groups, where blue is European domestic pig, red is European wild boar, and yellow is Near Eastern. genotypes cluster closer to each other than to the imputed
genotypes, the latter showing a bias toward the domestic pigs in
the first principal component (PC1, 9% variation) (Supplementary
Figure S4). PCA of IMP3 shows that rare alleles have a bias toward
the domestic cluster in the first principal component (PC1, 9%
variation) and a domestic and European wild boar bias in the
second
principal
component
(PC2,
3.8%
variation)
(Supplementary Figure S5). The PCA for IMP4 has the same
trend as IMP2, where the downsampled genotypes and the HQ
genotypes cluster closer to each other than to the imputed
genotypes. However, the imputed genotypes of KD037 and
VEM185 show a decreased bias toward the domestic pigs on
the
second
principal
component
(PC2,
4.2%
variation)
(Figure 2B). The PCA for IMP5 shows a bias toward the
European wild boar cluster for KD033 and VEM185 in both
principal
components,
where
the
imputed
genotypes
of
VEM185 cluster closer with the HQ genotypes than the
downsampled
genotypes
(Figure
2C). This
trend
is
not
observed in KD033. The imputed genotypes of KD037 cluster
closely toward the HQ counterpart, showing a slight bias on PC2
toward the European wild boar cluster. However, there seems to be
a slight bias introduced in the HQ and downsampled genotypes,
which is more evident for KD037 and VEM185. Frontiers in Genetics | www.frontiersin.org 3.1.6 Target VCF: Filters Versus No Filters They are clustering
more toward the European domestic cluster than in their previous
PCA (IMP1–4). Beagle5 showed a similar trend as GLIMPSE but
with an elevated bias toward the downsampled genotypes
(Supplementary Figure 6SA). Impute5 showed an increased
amount
of
bias
toward
the
European
wild
boar
cluster
(Supplementary Figure 6SB). and two ancestral groups in KD033. The ancestral groups of
KD037 and VEM185 are similar, with VEM185 having a slightly
larger “European domestic pig” component, whereas KD033
consists of a larger Near Eastern and smaller European
component. Admixture analysis of IMP1 shows an increase in
the
European
component,
and
a
decrease
of
the
other
components of all imputed samples, highlighting a bias toward
European samples. Admixture analysis of IMP2 shows a decrease
of the European component in KD037 and VEM185 and an
increase in KD033. The most noticeable difference between IMP2
and HQ is the increased component of “European domestic pigs”
in all three imputed samples, again highlighting a potential bias
toward “European domestic pigs.” KD033 showed most bias
losing
almost
half
of
its
Near
Eastern
component
and
increasement of both European wild boar and domestic pig’s
components. Admixture analysis of IMP3 shows only one
ancestral component, namely, the Near Eastern component
(Figure 3). This potential bias toward the Near Eastern
component could have arisen from a low amount of variants
present in the imputed genotypes. Admixture analysis of IMP4 is
similar to IMP2, with a slight decrease in Near Eastern ancestral
component and an increase in European ancestral component,
showing a bias toward the “European domestic pigs” component
in all samples and a bias toward the European wild boar
component in KD033. Admixture analysis for Method 3
shows a deviation between the two HQ, where the HQ in
Method 3 shows a decrease in Near Eastern components and
an increase in European wild and domestic components. Therefore, IMP5 was compared to the HQ of Method 3. Admixture
analysis
of
IMP5
shows
an
increase
in
the
European wild boar component in all samples and a decrease
of “European domestic pig” and Near Eastern component in Admixture analysis of three ancestral groups (K = 3) shows the
genetic ancestry of reference panel and HQ samples and indicates
a presence of all three ancestral groups in KD037 and VEM185, July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 8 Imputation of Ancient Sus scrofa Erven et al. 3.1.6 Target VCF: Filters Versus No Filters TABLE 4 | ROH statistics of the IMP1 reference panel for all autosomes shown per class of 0–0.5 mb, 0.5–1 mb, and >1 mb. 0–0.5
0.5–1
>1
Count
Sum kb
Froh
Count
Sum kb
Froh
Count
Sum kb
Froh
KD033_HQ
4,496
232,157
0.1025
1
516
0.0002
NA
NA
NA
KD033_Imp1
3,287
343,670
0.1517
17
13,152
0.0058
2
2,377
0.0010
KD037_HQ
4,434
193,928
0.0856
NA
NA
NA
NA
NA
NA
KD037_Imp1
2,743
285,325
0.1259
12
8,626
0.0038
1
1404
0.0006
VEM185_HQ
3,071
124,173
0.0548
NA
NA
NA
NA
NA
NA
VEM185_Imp1
2,585
266,071
0.1174
14
9,909
0.0044
1
1600
0.0007
Each class has three statistics, ROH count, total sum of kb and Froh. TABLE 4 | ROH statistics of the IMP1 reference panel for all autosomes shown per class of 0–0.5 mb, 0.5–1 mb, and >1 mb. FIGURE 4 | Genotype concordance and fraction of high-quality (HQ) genotypes (total HQ genotypes: VEM185:4,701,683, KD037: 4,531,126, KD033, 3,887,848)
of imputed samples for different levels of coverage for Impute5 and Beagle5. Imputation was performed using IMP1 and IMP2. Bars are standard errors. Genotype
concordance (A), Fraction of HQ genotypes (B). FIGURE 4 | Genotype concordance and fraction of high-quality (HQ) genotypes (total HQ genotypes: VEM185:4,701,683, KD037: 4,531,126, KD033, 3,887,848)
of imputed samples for different levels of coverage for Impute5 and Beagle5. Imputation was performed using IMP1 and IMP2. Bars are standard errors. Genotype FIGURE 4 | Genotype concordance and fraction of high-quality (HQ) genotypes (total HQ genotypes: VEM185:4,701,683, KD037: 4,531,126, KD033, 3,887,848)
of imputed samples for different levels of coverage for Impute5 and Beagle5. Imputation was performed using IMP1 and IMP2. Bars are standard errors. Genotype
concordance (A), Fraction of HQ genotypes (B). KD037 and VEM185, whereas KD033 only has a decrease in the
Near Eastern component. IBD segments. IMP3 and 4 did not have enough depth to perform
a proper IBD analysis. ROH analysis shows a similar but less
drastic trend. The amount of ROHs was smaller in the imputed
samples but they consisted of longer stretches (Table 4). The
imputed samples had considerably larger ROHs, some larger than
1 MB, while the HQ samples had smaller fragmented ROHs. The
elongated ROH stretches in the imputed samples attributed to a
higher Froh compared to the HQ samples (Table 4). 4 DISCUSSION The analyses revealed that for imputation of Sus scrofa aDNA data:
1) genotype concordance is relatively high, similar to modern
imputation, with a minor increase in information content (fraction
of gained genotypes in relation to HQ) in imputed genotypes and
2) imputation performance showed inaccuracies in downstream
analyses. These results have a variety of implications for our
understanding of the potency of imputation of non-human
ancient DNA in terms of its performance and limitations. 3.4 Effects of Reference Bias The HQ genotypes were considered a baseline of the true
genotypes that overlap with the groups in the reference panel
(Supplementary Figure S7). For Impute5, the correctly imputed
genotypes showed a bias toward the genotype that is most
common in the reference panel and occurs across all groups
(Ancients, EUW, EUD, and NEW), while the incorrectly imputed
genotypes showed a bias toward the EUD; EUW and EUW
groups
(Supplementary
Figures
S8,9). For
Beagle5,
the
correctly imputed genotypes showed a bias toward ANC;
EUD; EUW, EUD; EUW, EUD; EUW; NEW, and, ANC;
EUD, which is similar to the bias shown in the incorrectly
imputed genotypes (Supplementary Figures S10,11). The
incorrectly
imputed
genotypes
were
randomly
divided
throughout the chromosomes (Supplementary Figure S12). When only looking at genotype concordance the imputation
performance of imputation of Sus scrofa aDNA could be deemed
sufficient. However, there are potential shortcomings. High
genotype concordance obtained in the imputed genotypes does
not result in an equal representation of genotypes from the original
high coverage genome and consists of only a subset, covering
roughly 5%–50%. Moreover, imputed genotypes showed greater
affinity with populations that are overrepresented in the reference
panel as seen in downstream analyses (e.g., PCA, Admixture). One
example of the unequal representation of genotypes from the
original HQ genome is apparent from genotype concordances
on different MAF bins. Genotype concordance in rare alleles (MAF
< 0.05) reached 97% but resulted in a bias toward main
components in the reference panel in downstream analyses. This is potentially due to the representation of only ~5% of the
original HQ genotypes in the imputed genotypes It is therefore
essential to look beyond genotype concordance and focus on
multiple aspects like fraction of HQ genotypes obtained by
imputation and potential biases in downstream analysis. 3.3 Effects of Coverage on Imputation Coverage levels vary in genotype concordance, reaching 0.94 for
KD037 and VEM185, and 0.75 for KD033 using Beagle5, where
2× reached the highest genotype concordance (Figure 4A). This
trend is opposite for Impute5, which reached a genotype
concordance of 0.92 for KD037, 0.91 for VEM185, and 0.88
for KD033, with the lowest coverage 0.5×. Imputed genotypes
increased with increasing coverage (Figure 4B) All tools achieved high genotype concordance but differed in
amount of information gained. Moreover, Beagle5 showed less
variation
in
imputation
of
repeated
downsampled
VCFs
compared to Impute5, showing that Beagle5 might be less
affected
by
the
randomness
of
downsampling. Genotype
concordance increased with the two-step imputation pipeline. This was specifically designed for genomes with low coverage
(Hui et al., 2020). Our results indicate that non-model species
and species without an extensive reference panel could also
benefit from this approach. Furthermore, genotype concordance
increased when ancient samples were added to the reference panel,
adding to the number and diversity of individuals. Finally, genotype
concordance and number of correctly imputed genotypes increased
when using all confident sites but this increased computational time
and memory significantly. GLIMPSE achieved the highest genotype
concordance with Method 3, that consisted of reference panel called
genotypes in the three target downsampled samples. However, this
method did not improve the genotype concordance for Impute5 and
Beagle5, but did improve amount of genotypes recovered in all tools. 3.1.6 Target VCF: Filters Versus No Filters However,
the ROHs in the imputed samples overlap with the HQ samples Identity-By-Descent (IBD) analysis shows that the imputed
genotypes of IMP1 share large IBD segments with each other,
covering whole chromosomes. These IBD segments are not
present in their HQ or downsampled counterparts. IMP2
showed more variation with the imputed genotypes resulting
in more fragmented IBD segments when compared to IMP1. However, most of the IBD segments do not overlap with the HQ July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 9 Imputation of Ancient Sus scrofa Erven et al. downsampled to a similar coverage, excluding this possibility
(Supplementary Methods- Downsampling KD037 and VEM185). Moreover, KD033 showed most deviation when downsampled
multiple times, showing that the ancestry components of this
sample seem to be a factor in the level of accuracy in imputation. but consists of longer stretches (Supplementary Appendix
ROH). The ROH analysis was only performed for IMP1,
because Beagle5 had a low amount of variants. 3.3 Effects of Coverage on Imputation
Coverage levels vary in genotype concordance, reaching 0.94 for
KD037 and VEM185, and 0.75 for KD033 using Beagle5, where
2× reached the highest genotype concordance (Figure 4A). This
trend is opposite for Impute5, which reached a genotype
concordance of 0.92 for KD037, 0.91 for VEM185, and 0.88
for KD033, with the lowest coverage 0.5×. Imputed genotypes
increased with increasing coverage (Figure 4B) 4.1 Imputation Performance The relatively high genotype concordance of 0.95 for Beagle5, 0.925
for Impute5, and 0.98 for GLIMPSE at 1× coverage on ancients is
along the lines of genotype concordance in imputation of modern
breeds (see Song et al., 2019; Ye et al., 2019; Wang et al., 2021). The
higher genotype concordance in KD037 and VEM185 compared to
KD033 might be explained by their difference in ancestry. Frantz et al. (2019) have shown that KD033 possessed ~54% Near Eastern
ancestry, while KD037 and VEM185 possessed only ~10% Near
Eastern ancestry (Frantz et al., 2019). The larger component of Near
Eastern ancestry in KD033 may have caused the lower performance
due to the reference panel being skewed toward European
individuals. Another explanation could be the difference in
coverage between the samples, KD037 and VEM185 both have
coverages >20×, whereas KD033 has a coverage of ~7x. However,
KD037 and VEM185 had the same genotype concordance when Downstream analyses can identify how imputed genotypes act
in comparison to their HQ counterpart. The PCA of IMP1
resulted in accurate clustering of imputed and HQ genotypes
with only a slight bias toward the European wild boar component. This same analysis for Beagle5 resulted in a stronger bias toward
the European domestic pig component. This illustrated that a
high genotype concordance does not necessary lead to accurate
downstream analyses. The imputed genotypes are correct, but
introduce bias in subsequent downstream analyses because they
are from specific regions of the genome and are not informative
enough to detect genetic variation among samples. This trend is
also apparent in the admixture analysis, where imputed
genotypes
have
biases
toward
European
wild
boar
and
domestic pig components. IMP3 is an exception that might be July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 10 Imputation of Ancient Sus scrofa Erven et al. European wild boar, Near Eastern wild boar and domestic pigs to
the reference panel. attributed to the high amount of missing genotypes pulling it
toward the Near Eastern component that featured missingness,
due to low coverage and ancient individuals. IMP5 achieved the
highest genotype concordance and resulted in the most accurate
clustering for KD037 in the PCA, suggesting that imputation of
ancient Sus is feasible. 5 CONCLUSION The use of imputation of ancient low-coverage Sus scrofa data
resulted in relatively high genotype concordance and a moderate
increase in information content. However, the imputed genotypes
represented only a fraction, roughly 5%–50%, of all genotypes
called in the HQ ancient genomes and featured biases toward the
main population components in the reference panel. Our analysis
indicated
that
these
can
lead
to
misidentifications
or
overrepresentation of ancestry components and selective traits
in imputed genotypes. This is especially significant considering
the weight archaeological debates place on ancestral relationships
and admixture patterns of domesticated animals to understand the
mechanisms of emergence and dispersal of early animal husbandry
throughout the Neolithic across Europe and the Near East. This
further highlights the measures needed to interpret the results and
biases introduced by imputation and difficulty of imputation of
admixed individuals. A more diverse reference panel is one of the
most important priorities in ancient Sus imputation and
particularly, introducing diversity present in ancient Sus could
elevate accuracy and limit bias. 4.2 Factors Limiting the Power of Imputation
of Sus scrofa aDNA One of the limitations is size of the reference panel (59 individuals),
but more specifically diversity in the reference panel. Studies on both
humans and pigs showed that a larger and more diverse reference
panel increase imputation accuracy (Jostins et al., 2011; Pistis et al.,
2015; Van Den Berg et al., 2019; Ausmees et al., 2021; Wang et al.,
2021). Ancient human imputation studies had a minimum of ~250
individuals to perform successful imputation (Ausmees, 2019). Adding individuals to the reference panel, that do not add genetic
diversity to target samples does not increase genotype concordance,
as seen from the results when adding Asian samples to the reference
panel. The current reference panel lacks diversity, as the main groups
in the reference panel consisted of European wild boar, European
domestic pigs and Near Eastern wild boar, with (Dutch) European
wild boar and European domestic pig dominating. A study on ancient
human imputation observed a lower genotype concordance and
similarity in their PCA for hunter-gatherer genomes of which
ancestry is more or less absent in the reference panel (Ausmees
et al., 2021). This was also found in imputation of pig breeds where
rarer pig breeds had a lower genotype concordance and dosage score
than breeds that were common in the reference panel (Wang et al.,
2021). In this study genotype concordance improved when adding
five ancient samples with Near Eastern ancestry to the reference
panel. Improving and mitigating current biases of the reference panel
should aid imputation. This could be achieved by including
Mesolithic wild boar, Iberian, British, Scandinavian and East 4.1 Imputation Performance However, VEM185 had a similar genotype
concordance as KD037 but showed the most bias in downstream
analyses for this specific method, implying that high genotype
concordance does not preclude bias across samples. Another potential limitation that is associated with the
reference panel is the available reference genome. The Sus
scrofa 11.1 reference genome, is from a Duroc individual, with
known Asian introgression. Moreover, the nature of the reference
genome could potentially increase the rate of false genotyping
leading to errors in haplotypes and LD structure, which could
result in decreasing imputation accuracy. p
p
The IBD analysis shows that imputed genotypes of different
samples from Impute5, share large IBD segments, sometimes
even stretching chromosome wide. This could be a result of: 1)
samples which lost their individual variation and became more
similar due to imputation and/or 2) imputed genotypes that did
not have enough depth for IBD analysis. The second explanation
is unlikely, as imputed genotypes for Beagle5, did not show these
large IBD segments. The ROH analysis shows that there are
longer homozygous stretches throughout the genome in imputed
genotypes compared to their HQ counterparts. Causes for this
could be that the imputed genotypes were predominantly
homozygous
with
little
representation
of
heterozygotes,
contributing to long ROHs and that the imputed genotypes
have less markers than the HQ counterparts, resulting in an
unequal density of markers. Thus, interpretation of ROHs in
imputed ancient Sus should be taken with caution as it can be a
result of the increase in homozygosity for Impute5. Overall, these
downstream analyses highlight that there are biases and
limitations toward imputation of Sus scrofa aDNA. One final potential limitation is the genetic architecture of pigs. Accuracy of imputation is dependent on LD, recombination,
genetic
distance,
and
MAF
(Stephens
and
Scheet,
2005;
Browning et al., 2018; Ye et al., 2019). These factors are different
in pigs compared to humans and even other livestock species, where
average heterozygosity is lower and, LD and genetic distance are
significantly greater (Zhang and Plastow, 2011). The recombination
rate used in this study was based on nine breeding lines, all having
introgression with Asian domestic pigs. This potentially introduced
inaccuracies but is mitigated as the recombination was divided into
bins of 1 MB, which might not be at a size resolution to introduce
inaccuracies between wild and domestic pigs. DATA AVAILABILITY STATEMENT Publicly available datasets were analyzed in this study. These data
can be found here: https://www.ebi.ac.uk/ena/browser/view/
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bioinformatics/btp324 Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., et al. (2009). BLAST+: Architecture and Applications. BMC Bioinforma. 10 (421). doi:10.1186/1471-2105-10-421 Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., et al. (2009). The Sequence Alignment/Map Format and SAMtools. Bioinformatics 25 (16),
2078–2079. doi:10.1093/bioinformatics/btp352 Frantz, L. A. F., Haile, J., Lin, A. T., Scheu, A., Geörg, C., Benecke, N., et al. (2019). Ancient Pigs Reveal a Near-Complete Genomic Turnover Following Their
Introduction to Europe. Proc. Natl. Acad. Sci. U. S. A. 116 (35), 17231–17238. doi:10.1073/pnas.1901169116 MacHugh, D. E., Larson, G., and Orlando, L. (2017). AUTHOR CONTRIBUTIONS JE, DB, and OM conceptualized and designed the study. JE
collected and assembled the data. JE, DB, and OM analyzed July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 11 Erven et al. Imputation of Ancient Sus scrofa ACKNOWLEDGMENTS the data. JE wrote the manuscript and JE, CÇ, OM, DB, and DR
contributed to reviewing and editing the manuscript. DR and CÇ
contributed to project oversight. The authors would like to thank the Smurfit Institute of Genetics
and the EDAN project for their helpful advice during discussions. The authors would also like to thank the reviewers for their
comments and efforts towards improving our manuscript. The Supplementary Material for this article can be found online at:
https://www.frontiersin.org/articles/10.3389/fgene.2022.872486/
full#supplementary-material AncestralWeave). SUPPLEMENTARY MATERIAL This study is supported by the Dutch Research Council Open
Competition (Grant No. 406.18.HW.026) and the European
Research Council award to DB under the European Union’s
Horizon 2020 research and innovation programme (885729-
AncestralWeave). This study is supported by the Dutch Research Council Open
Competition (Grant No. 406.18.HW.026) and the European
Research Council award to DB under the European Union’s
Horizon 2020 research and innovation programme (885729-
AncestralWeave). The Supplementary Material for this article can be found online at:
https://www.frontiersin.org/articles/10.3389/fgene.2022.872486/
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(R47), R47. doi:10.1186/gb-2010-11-5-r47 Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest. Purcell, S., Neale, B., Todd-Brown, K., Thomas, L., Ferreira, M. A. R., Bender, D.,
et al. (2007). PLINK: A Tool Set for Whole-Genome Association and
Population-Based Linkage Analyses. Am. J. Hum. Genet. 81 (3), 559–575. doi:10.1086/519795 Publisher’s Note: All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations, or those of
the publisher, the editors and the reviewers. Any product that may be evaluated in
this article, or claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher. Ramírez, O., Burgos-Paz, W., Casas, E., Ballester, M., Bianco, E., Olalde, I., et al. (2015). Frontiers in Genetics | www.frontiersin.org July 2022 | Volume 13 | Article 872486 REFERENCES Genome Data from a Sixteenth Century Pig Illuminate Modern Breed
Relationships. Hered. (Edinb) 114, 175–184. doi:10.1038/hdy.2014.81 Rubinacci, S., Delaneau, O., and Marchini, J. (2020). Genotype Imputation Using
the Positional Burrows Wheeler Transform. PLoS Genet. 16, e1009049. doi:10. 1371/journal.pgen.1009049 Copyright © 2022 Erven, Çakirlar, Bradley, Raemaekers and Madsen. 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. Rubinacci, S., Ribeiro, D. M., Hofmeister, R. J., and Delaneau, O. (2021). Efficient
Phasing and Imputation of Low-Coverage Sequencing Data Using Large
Reference Panels. Nat. Genet. 53, 120–126. doi:10.1038/s41588-020-00756-0 Sánchez-Quinto, F., Schroeder, H., Ramirez, O., Ávila-Arcos, M. C., Pybus, M., Olalde, I.,
et al. (2012). Genomic Affinities of Two 7,000-Year-Old Iberian Hunter-Gatherers. Curr. Biol. 22 (16), 1494–1499. doi:10.1016/j.cub.2012.06.005 July 2022 | Volume 13 | Article 872486 Frontiers in Genetics | www.frontiersin.org 13
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Asthma Mortality Among Persons Aged 15–64 Years, by Industry and Occupation — United States, 1999–2016
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Asthma Mortality Among Persons Aged 15–64 Years, by Industry and
Occupation — United States, 1999–2016 Opal Patel, MPH1,2; Girija Syamlal, MBBS2; John Wood, MS2; Katelynn E. Dodd, MPH2; Jacek M. Mazurek, MD, PhD2 In 2015, an estimated 18.4 million U.S. adults had current
asthma, and 3,396 adult asthma deaths were reported (1). An
estimated 11%–21% of asthma deaths might be attributable to
occupational exposures (2). To describe asthma mortality among
persons aged 15–64 years,* CDC analyzed multiple cause-of-death
data† for 1999–2016 and industry and occupation information
collected from 26 states§ for the years 1999, 2003, 2004, and 2007–
2012. Proportionate mortality ratios (PMRs)¶ for asthma among
persons aged 15–64 years were calculated. During 1999–2016, a
total of 14,296 (42.9%) asthma deaths occurred among males and
19,011 (57.1%) occurred among females. Based on an estimate
that 11%–21% of asthma deaths might be related to occupational
exposures, during this 18-year period, 1,573–3,002 asthma deaths
in males and 2,091–3,992 deaths in females might have resulted
from occupational exposures. Some of these deaths might have
been averted by instituting measures to prevent potential workplace
exposures. The annual age-adjusted asthma death rate** per 1 mil-
lion persons aged 15–64 years declined from 13.59 in 1999 to 9.34
in 2016 (p<0.001) among females, and from 9.14 (1999) to 7.78
(2016) (p<0.05) among males. The highest significantly elevated
asthma PMRs for males were for those in the food, beverage, and
tobacco products manufacturing industry (1.82) and for females
were for those in the social assistance industry (1.35) and those in
community and social services occupations (1.46). Elevated asthma
mortality among workers in certain industries and occupations
underscores the importance of optimal asthma management and
identification and prevention of potential workplace exposures. persons aged 15–64 years. Asthma deaths were identified from
death certificates using International Classification of Diseases,
10th Revision underlying cause-of-death codes J45 (asthma) and
J46 (status asthmaticus). Death rates per 1 million persons aged
15–64 years by sex, race, ethnicity, and year were age-adjusted
using the 2000 U.S. Census standard population. Time trends
were assessed using a first-order autoregressive linear regression
model to account for the serial correlation. Industry and occupa-
tion information available from 26 states for the years 1999, 2003,
2004, and 2007–2012†† was coded§§ using the U.S. Census 2000
Industry and Occupation Classification System. PMRs, adjusted
by 5-year age groups and race, were generated by industry and
occupation for males and females. In addition, 95% confidence
intervals (CIs) were calculated assuming Poisson distribution of
the data. Morbidity and Mortality Weekly Report Morbidity and Mortality Weekly Report †† Information on industry and occupation was available only for decedents from
26 states for the years 1999, 2003, 2004, and 2007–2012. https://www.cdc.
gov/niosh/topics/noms/default.html.
§§ https://webappa.cdc.gov/ords/norms-glossary.html#ind-occ.
¶¶ For 98 residents of these 26 states, deaths occurred in states that did not
provide the industry and occupation information to the National Institute
for Occupational Safety and Health. Retired, unemployed, and non-paid (229
males and 687 females) and unknown or not reported (90 males and 78
females) industries, and retired, students, volunteers, homemakers and
unemployed (233 males and 688 females) and unknown or not reported (78
males and 68 females) occupations were excluded from PMR analyses. * https://www.dol.gov/general/topic/youthlabor/agerequirements.
† Decedents who had the International Classification of Diseases, 10th Revision
codes J45 (asthma) or J46 (status asthmaticus) assigned as the underlying
cause of death (the disease or injury that initiated the chain of events that led
directly and inevitably to death). https://wonder.cdc.gov/.
§ Colorado, Florida, Georgia, Hawaii, Idaho, Indiana, Kansas, Kentucky,
Louisiana, Michigan, Nebraska, Nevada, New Hampshire, New Jersey, New
Mexico, North Carolina, North Dakota, Ohio, Rhode Island, South Carolina,
Texas, Utah, Vermont, Washington, West Virginia, and Wisconsin. States
represent the state where the death took place.
¶ PMR was defined as the observed number of deaths from asthma in a specified
industry/occupation, divided by the expected number of deaths from asthma.
The expected number of deaths was the total number of deaths in industry
or occupation of interest multiplied by a proportion defined as the number
of asthma deaths in all industries and/or occupations, divided by the total
number of deaths in all industries/occupations. The asthma PMRs for each
sex were internally adjusted by 5-year age groups and race.
** Age-adjusted death rates were calculated by applying age-specific death rates
to the 2000 U.S. Census standard population age distribution. https://wonder.
cdc.gov/wonder/help/mcd.html#Age-AdjustedRates. †† Information on industry and occupation was available only for decedents from
26 states for the years 1999, 2003, 2004, and 2007–2012. https://www.cdc.
gov/niosh/topics/noms/default.html.
§§ h
b
d
d
l
h
l
d US Department of Health and Human Services/Centers for Disease Control and Prevention y
y
p
g
§ Colorado, Florida, Georgia, Hawaii, Idaho, Indiana, Kansas, Kentucky,
Louisiana, Michigan, Nebraska, Nevada, New Hampshire, New Jersey, New
Mexico, North Carolina, North Dakota, Ohio, Rhode Island, South Carolina,
Texas, Utah, Vermont, Washington, West Virginia, and Wisconsin. States
represent the state where the death took place. * https://www.dol.gov/general/topic/youthlabor/agerequirements.
† Decedents who had the International Classification of Diseases, 10th Revision
codes J45 (asthma) or J46 (status asthmaticus) assigned as the underlying
cause of death (the disease or injury that initiated the chain of events that led
directly and inevitably to death). https://wonder.cdc.gov/. g
§§ https://webappa.cdc.gov/ords/norms-glossary.html#ind-occ.
¶¶
d
f h
d
h
d ,
,
,
,
d
d,
u
,
Texas, Utah, Vermont, Washington, West Virginia, and Wisconsin. States
represent the state where the death took place.
¶ PMR was defined as the observed number of deaths from asthma in a specified
industry/occupation, divided by the expected number of deaths from asthma.
The expected number of deaths was the total number of deaths in industry
or occupation of interest multiplied by a proportion defined as the number
of asthma deaths in all industries and/or occupations, divided by the total
number of deaths in all industries/occupations. The asthma PMRs for each
sex were internally adjusted by 5-year age groups and race.
** Age-adjusted death rates were calculated by applying age-specific death rates
to the 2000 U.S. Census standard population age distribution. https://wonder.
cdc.gov/wonder/help/mcd.html#Age-AdjustedRates. ¶¶ For 98 residents of these 26 states, deaths occurred in states that did not
provide the industry and occupation information to the National Institute
for Occupational Safety and Health. Retired, unemployed, and non-paid (229
males and 687 females) and unknown or not reported (90 males and 78
females) industries, and retired, students, volunteers, homemakers and
unemployed (233 males and 688 females) and unknown or not reported (78
males and 68 females) occupations were excluded from PMR analyses. Asthma Mortality Among Persons Aged 15–64 Years, by Industry and
Occupation — United States, 1999–2016 Retired, unemployed, and nonpaid workers and those
with information that was unknown or not reported for industry
or occupation were excluded from PMR analyses. During 1999–2016, a total of 33,307 U.S. decedents aged
15–64 years had asthma or status asthmaticus assigned as the
underlying cause of death (Table 1) for an overall death rate of 8.89
per 1 million persons. The highest asthma death rates were among
adults aged 55–64 years (16.32 per 1 million persons), females
(9.95 per 1 million persons), persons who were not Hispanic or
Latino (9.39 per 1 million), and blacks or African Americans
(25.60 per 1 million persons). The age-adjusted asthma death
rate per 1 million persons aged 15–64 years decreased 24.6%
from 11.41 in 1999 to 8.60 in 2016 (p<0.01). The age-adjusted
asthma death rates among females aged 15–64 years decreased
from 13.59 per 1 million in 1999 to 9.34 in 2016 (p<0.001), and
among males decreased from 9.14 (1999) to 7.78 (2016) (p<0.05). By state, annualized age-adjusted asthma death rates ranged from
4.59 per 1 million in Maine to 14.72 in the District of Columbia
for males and from 6.70 per 1 million in North Dakota to 15.30
in Mississippi for females (Figure). National Vital Statistics System’s multiple cause-of-death data
for 1999–2016 were analyzed to examine asthma mortality among Industry and occupation data were available for 3,393¶¶
(97.2%) of 3,491 asthma deaths, (1,398 of 1,435 [97.4%] MMWR / January 19, 2018 / Vol. 67 / No. 2 US Department of Health and Human Services/Centers for Disease Control and Prevention 60 Morbidity and Mortality Weekly Report TABLE 1. Number of asthma deaths* and age-adjusted asthma death rates† among persons aged 15–64 years, by sex and selected characteristics —
United States, 1999–2016§
Characteristic
Males
Females
Overall
No. of deaths
(% of asthma deaths) Death rate
No. of deaths
(% of asthma deaths)
Death rate
No. US Department of Health and Human Services/Centers for Disease Control and Prevention Asthma Mortality Among Persons Aged 15–64 Years, by Industry and
Occupation — United States, 1999–2016 of deaths
(% of asthma deaths) Death rate
Overall (% of all asthma deaths)
14,296 (42.9)
7.78
19,011 (57.1)
9.95
33,307 (100.0)
8.89
Age group (yrs)¶
15–24
1,731 (12.1)
4.42
1,035 (5.4)
2.78
2,766 (8.3)
3.62
25–34
2,272 (15.9)
6.12
1,818 (9.6)
4.97
4,090 (12.3)
5.55
35–44
2,874 (20.1)
7.55
3,692 (19.4)
9.60
6,566 (19.7)
8.58
45–54
3,853 (27.0)
10.28
6,284 (33.1)
16.22
10,137 (30.4)
13.30
55–64
3,566 (24.9)
12.39
6,182 (32.5)
19.98
9,748 (29.3)
16.32
Race**
American Indian or Alaska Native
138 (1.0)
6.28
198 (1.0)
9.15
336 (1.0)
7.75
Asian or Pacific Islander
525 (3.7)
5.67
439 (2.3)
4.23
964 (2.9)
4.92
Black or African American
5,695 (39.8)
25.21
6,463 (34.0)
25.76
12,158 (36.5)
25.60
White
7,938 (55.5)
5.28
11,911 (62.7)
7.74
19,849 (59.6)
6.52
Ethnicity††
Hispanic or Latino
1,348 (9.4)
5.49
1,474 (7.8)
6.37
2,822 (8.5)
5.96
Not Hispanic or Latino
12,862 (90.0)
8.21
17,468 (91.9)
10.48
30,330 (91.1%)
9.39
Unknown
86 (0.6)
N/A
69 (0.4)
N/A
155 (0.5)
N/A
Year
1999
824
9.14
1,257
13.59
2,081
11.41
2000
878
9.60
1,150
12.24
2,028
10.95
2001
792
8.47
1,192
12.41
1,984
10.49
2002
872
9.14
1,148
11.71
2,020
10.49
2003
828
8.54
1,162
11.62
1,990
10.12
2004
770
7.82
1,044
10.21
1,814
9.06
2005
720
7.21
1,102
10.59
1,822
8.96
2006
721
7.12
1,039
9.81
1,760
8.52
2007
745
7.22
908
8.51
1,653
7.89
2008
667
6.47
931
8.54
1,598
7.52
2009
699
6.69
996
9.08
1,695
7.92
2010
747
7.04
982
8.86
1,729
7.97
2011
732
6.82
953
8.45
1,685
7.67
2012
850
7.91
988
8.71
1,838
8.31
2013
852
8.01
999
8.77
1,851
8.43
2014
875
8.19
1,089
9.63
1,964
8.94
2015
885
8.14
997
8.65
1,882
8.43
2016
839
7.78
1,074
9.34
1,913
8.60
p-value§§
0.72
<0.05
0.004
<0.001
0.11
<0.001
Abbreviation: N/A = not available. * Decedents who had International Classification of Diseases, 10th Revision codes J45 (asthma) or J46 (status asthmaticus) assigned as the underlying cause of death (i.e., the
disease or injury that initiated the chain of morbid events leading directly to death, or the circumstances of the accident or violence that produced the fatal injury). † Age-adjusted asthma death rates per 1 million persons calculated using the 2000 U.S. Census standard population. § National Vital Statistics System. https://wonder.cdc.gov/. ¶ Age-specific asthma death rates per 1 million persons. Discussion The annual number of asthma deaths among persons aged
15–64 years has declined significantly from 1999 through
2016, most likely reflecting improvements in asthma manage-
ment and effectiveness of prevention efforts (3,4). For example,
replacing powdered latex gloves with powder-free natural rub-
ber latex or nonlatex gloves reduced latex allergen exposure and
substantially reduced work-related asthma*** among health
care workers (4). Differences in asthma mortality by age, sex,
and race/ethnicity have been previously reported (5). Based on
an estimate that 11%–21% of asthma deaths might be attribut-
able to occupational exposures (2), an estimated 3,664–6,994
asthma deaths during 1999–2016 (1,573–3,002 among males
and 2,091–3,992 among females) might have been job-related,
and therefore potentially preventable. population aged 15 64 years, by sex and state
United States,
1999–2016¶
>10.06
8.89–10.06
8.07–8.88
6.99–8.06
5.90–6.98
<5.90
Males
Females Males
Females Males p
y p
Female workers in the health care industry and male work-
ers in the construction industry accounted for the highest
industry-related numbers of asthma deaths. The PMRs were
significantly elevated among males in the food, beverage, and
tobacco products manufacturing, other retail trade, and miscel-
laneous manufacturing industries; and among females in the
social assistance industry and in the community and social ser-
vices occupations. A higher proportion of females with current
asthma and a high frequency of exposures associated with work-
related respiratory diseases have been observed in the health
care and social assistance industries (6,7). National survey data
indicate that approximately 9.1% (1.3 million) of 13.9 million
female workers in the health care and social assistance indus-
tries, and 4.2% (394,000) of 9.4 million male workers in the
construction industry, have current asthma.††† Approximately
13.4% of health care and social assistance workers, 51.1% of
construction workers, 31.8% of food manufacturing workers,
36.1% of beverage and tobacco product manufacturing work-
ers, 40.0% of miscellaneous manufacturing workers, 21.5% of
retail trade workers, and 3.7% of community and social services
workers are frequently exposed to vapors, gas, dust, or fumes
in the workplace (6). Workplace exposures to asthma-causing
agents,§§§ such as cleaners, disinfectants, antibiotics, natural
rubber latex among health care workers, and welding fumes
and isocyanates (e.g., paints) among construction workers¶¶¶
have been associated with work-related asthma (8,9). Higher Females >10.06
8.89–10.06
8.07–8.88
6.99–8.06
5.90–6.98
<5.90
Females * Age-adjusted death rates were calculated by applying age-specific death rates
to the 2000 U.S. Census standard population age distribution. https://wonder. cdc.gov/wonder/help/mcd.html#Age-Adjusted Rates. *** Work-related asthma includes occupational asthma (i.e., new-onset asthma
caused by factors related to work) and work-exacerbated asthma (i.e.,
preexisting or concurrent asthma worsened by factors related to work).
https://www.cdc.gov/niosh/topics/asthma/default.html.
††† https://wwwn.cdc.gov/eworld/Grouping/Asthma/97.
§§§ Association of Occupational and Environmental Clinics list of occupational
asthmagens. http://www.aoecdata.org/ExpCodeLookup.aspx.
¶¶¶ Occupational Safety and Health Administration. Health Hazards in
Construction. https://www.osha.gov/dte/grant_materials/fy09/sh-19495-09/
health_hazards_workbook.pdf. Asthma Mortality Among Persons Aged 15–64 Years, by Industry and
Occupation — United States, 1999–2016 of asthma deaths* and age-adjusted asthma death rates† among persons aged 15–64 years, by sex and selec
9 2016§ ge-adjusted asthma death rates† among persons aged 15–64 years, by sex and selected characteristics — Abbreviation: N/A not available. * Decedents who had International Classification of Diseases, 10th Revision codes J45 (asthma) or J46 (status asthmaticus) assigned as the underlying cause of death (i.e., the
disease or injury that initiated the chain of morbid events leading directly to death, or the circumstances of the accident or violence that produced the fatal injury). † Age-adjusted asthma death rates per 1 million persons calculated using the 2000 U.S. Census standard population. § N ti
l Vit l St ti ti
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t
htt
//
d
d
/ males and 1,995 of 2,056 [97.0%] females) among persons
aged 15–64 years that occurred in residents of 26 states during
1999, 2003, 2004, and 2007–2012 (Table 2). By industry, the
highest number of asthma deaths occurred among males in the
construction industry (184; 13.2% of asthma deaths in males) and among females in the health care industry (279; 14.0% of
asthma deaths in females). By occupation, the highest number
of asthma deaths occurred among male construction trades
workers (149; 10.7%) and among female office and adminis-
trative support workers (186; 9.3%). By industry, PMRs were MMWR / January 19, 2018 / Vol. 67 / No. 2 rtment of Health and Human Services/Centers for Disease Control and Prevention 61 61 Morbidity and Mortality Weekly Report FIGURE. Annualized age-adjusted asthma death rate* per 1 million
population aged 15–64 years,† by sex and state§ — United States,
1999–2016¶ FIGURE. Annualized age-adjusted asthma death rate* per 1 million
population aged 15–64 years,† by sex and state§ — United States,
1999–2016¶ US Department of Health and Human Services/Centers for Disease Control and Prevention MMWR / January 19, 2018 / Vol. 67 / No. 2 US Department of Health and Human Services/Centers for Disease Control and Prevention **** https://www.cdc.gov/nchs/data/misc/hb_occup.pdf. Discussion Industries and occupations with ≥25 asthma*
2007–2012
Characteristic
Industry
Male (n = 1,079)
Food, beverage, and tobacco products manufacturing
Other retail trade
Miscellaneous manufacturing
Arts, entertainment and recreation
Public administration
Health care
Repair and maintenance
Professional, scientific, technical and management services
Transportation and warehousing
Accommodation and food services
Educational services
Construction
Transportation equipment
Administrative and support, and waste management services
All other industries
Female (n = 1,230)
Social assistance
Arts, entertainment and recreation
Food and beverage stores
Private households
Health care
Other retail trade
Public administration
Accommodation and food services
Administrative and support, and waste management services
Transportation and warehousing
Finance and Insurance
Personal and laundry services
Educational services
Miscellaneous manufacturing
Professional, scientific, technical and management services
All other industries Discussion significantly elevated among males working in food, beverage,
and tobacco products manufacturing (1.82; CI = 1.22–2.61),
other retail trade (1.65; CI = 1.29–2.10), and miscellaneous
manufacturing (1.45; CI = 1.13–1.86); and among females
working in social assistance (e.g., individual and family ser-
vices and child day care services) (1.35; CI = 1.00–1.79). By
occupation, the PMR was significantly elevated among female
community and social services workers (1.46; CI = 1.02–2.01). MMWR / January 19, 2018 / Vol. 67 / No. 2 US Department of Health and Human Services/Centers for Disease Control and Prevention 62 Morbidity and Mortality Weekly Report TABLE 2. Industries and occupations with ≥25 asthma* deaths among persons aged 15–64 years, by sex — 26 states,† 1999, 2003, 2004, and
2007–2012
Characteristic
No. of deaths
PMR§,¶ (95% CI) TABLE 2. Industries and occupations with ≥25 asthma* deaths among persons aged 15–64 years, by sex — 26 states,† 1999, 2003, 2004, and
2007–2012
Characteristic
No. of deaths
PMR§,¶ (95% CI)
Industry
Male (n = 1,079)
Food, beverage, and tobacco products manufacturing
29
1.82 (1.22–2.61)**
Other retail trade
69
1.65 (1.29–2.10)**
Miscellaneous manufacturing
66
1.45 (1.13–1.86)**
Arts, entertainment and recreation
29
1.30 (0.88–1.87)
Public administration
52
1.09 (0.83–1.45)
Health care
40
1.04 (0.74–1.42)
Repair and maintenance
46
1.01 (0.73–1.34)
Professional, scientific, technical and management services
34
1.00 (0.69–1.39)
Transportation and warehousing
89
0.98 (0.79–1.21)
Accommodation and food services
66
0.96 (0.75–1.23)
Educational services
29
0.95 (0.64–1.37)
Construction
184
0.92 (0.79–1.07)
Transportation equipment
28
0.78 (0.52–1.12)
Administrative and support, and waste management services
36
0.66 (0.46–0.91)
All other industries
282
—
Female (n = 1,230)
Social assistance
49
1.35 (1.00–1.79)**
Arts, entertainment and recreation
26
1.29 (0.84–1.89)
Food and beverage stores
27
1.19 (0.78–1.73)
Private households
31
1.16 (0.79–1.64)
Health care
279
1.12 (1.00–1.27)
Other retail trade
96
1.10 (0.89–1.34)
Public administration
69
1.06 (0.83–1.35)
Accommodation and food services
116
1.01 (0.84–1.21)
Administrative and support, and waste management services
42
0.97 (0.70–1.31)
Transportation and warehousing
37
0.90 (0.63–1.24)
Finance and Insurance
48
0.90 (0.66–1.19)
Personal and laundry services
29
0.86 (0.58–1.24)
Educational services
94
0.85 (0.69–1.04)
Miscellaneous manufacturing
29
0.75 (0.50–1.07)
Professional, scientific, technical and management services
35
0.66 (0.46–0.92)
All other industries
223
—
See table footnotes on page 64 TABLE 2. Industries and occupations with ≥25 asthma* deaths among persons aged 15–64 years, by sex — 26 states,† 1999, 2003, 2004, and
2007–2012 TABLE 2. See table footnotes on page 64. * Decedents who had the International Classification of Diseases, 10th Revision codes J45 (asthma) or J46 (status asthmaticus) assigned as the underlying cause of
death (i.e., the disease or injury that initiated the chain of morbid events leading directly to death, or the circumstances of the accident or violence that produced
the fatal injury). j
y)
† Colorado, Florida, Georgia, Hawaii, Idaho, Indiana, Kansas, Kentucky, Louisiana, Michigan, Nebraska, Nevada, New Hampshire, New Jersey, New Mexico, North
Carolina, North Dakota, Ohio, Rhode Island, South Carolina, Texas, Utah, Vermont, Washington, West Virginia, and Wisconsin. States represent the state where the
death took place. y
† Colorado, Florida, Georgia, Hawaii, Idaho, Indiana, Kansas, Kentucky, Louisiana, Michigan, Nebraska, Nevada, New Hampshire, New Jersey, New Mexico, North
Carolina, North Dakota, Ohio, Rhode Island, South Carolina, Texas, Utah, Vermont, Washington, West Virginia, and Wisconsin. States represent the state where the
death took place. § PMR is defined as the observed number of deaths from asthma in a specified industry/occupation, divided by the expected number of deaths from asthma. The
expected number of deaths is the total number of deaths in industry or occupation of interest multiplied by a proportion defined as the number of asthma deaths
in all industries or occupations, divided by the total number of deaths in all industries/occupations. The asthma PMRs were internally adjusted by 5-year age groups
and race. CIs were calculated assuming Poisson distribution of the data. and race. CIs were calculated assuming Poisson distribution of the data. ¶ Retired, unemployed, and unpaid (229 males and 687 females) and unknown or not reported (90 males and 78 females) workers in industries, and retired, students,
volunteers, homemakers and unemployed (233 males and 688 females) and unknown or not reported (78 males and 68 females) occupations were excluded from
PMR analyses. and race. CIs were calculated assuming Poisson distribution of the data. See table footnotes on page 64. The findings in this report are subject to at least five limita-
tions. First, asthma and status asthmaticus diagnoses could
not be validated. It is possible that some decedents were
misdiagnosed. However, given the potential impact of asthma
diagnosis and status asthmaticus on patients’ lives, it seems
likely that asthma would be accurately recorded on death
certificates. Second, no information was available to assess
whether workplace exposures triggered asthma attacks that
led directly to death. Some attacks might have been triggered
by exposures outside of the work environment. Third, to the
extent that asthma attacks were triggered by workplace expo-
sures, industry and occupation information reported on death
certificates might not be the industry and occupation in which
workplace exposures actually occurred because guidelines for
reporting industry and occupation on death certificates****
instruct recorders to report decedent’s “usual” industry and
occupation (i.e., “the type of job the individual was engaged in
for most of his or her working life”). Fourth, no work history
was available to assess changes in employment. Retired and PMRs in certain groups might also be explained in part by
workers leaving employment in industries and occupations
with workplace exposures that exacerbate their asthma and
moving to jobs with fewer workplace exposures (10). Likewise,
retired, unemployed, and nonpaid workers might have left the
workforce because of workplace exposures. p
p
Differences in asthma mortality by industry and occupa-
tion underscore the need for identifying workplace exposures,
early diagnosis, and treatment and management of asthma
cases, especially among industries and occupations with
higher mortality. Pharmaceutical treatment of asthma related
to occupational exposures is similar to that for asthma that
is not work-related (3). Early identification and elimination
of exposures is the preferred means of primary prevention to
reduce asthma related to occupational exposures; however,
reduction of exposure might be considered when elimination
of exposures is not possible (4). Establishing an accurate diag-
nosis and recommending appropriate management for workers
with asthma related to occupational exposures is necessary to
improve outcomes and could prevent asthma deaths (4). MMWR / January 19, 2018 / Vol. 67 / No. 2 63 Morbidity and Mortality Weekly Report TABLE 2. (Continued) Industries and occupations with ≥25 asthma* deaths among persons aged 15–64 years, by sex — 26 states,† 1999, 2003,
2004, and 2007–2012
Characteristic
No. See table footnotes on page 64. ¶ Retired, unemployed, and unpaid (229 males and 687 females) and unknown or not reported (90 males and 78 females
volunteers, homemakers and unemployed (233 males and 688 females) and unknown or not reported (78 males and 6
PMR analyses ales and 687 females) and unknown or not reported (90 males and 78 females) workers in industries, and retired, students,
d (233 males and 688 females) and unknown or not reported (78 males and 68 females) occupations were excluded from y
** Statistically significant elevated PMR unemployed persons might have left the workforce because
of severe asthma in relation to work. Finally, information on
industry and occupation might not be nationally representative
because only selected states provided information on industry
and occupation, and only for certain years. asthma mortality among workers in certain industries and
occupations underscores the importance of optimal asthma
management, and identification and elimination or reduction
of potential workplace exposures (3,4,9). 7 / No. 2
US Department of Health and Human Services/Centers for Disease Control and Prevention See table footnotes on page 64. of deaths
PMR§,¶ (95% CI)
Occupation
Male (n = 1,087)
Office and administrative support occupations
62
1.25 (0.97–1.61)
Other production occupations, including supervisors
51
1.21 (0.91–1.61)
Sales and related occupations
89
1.17 (0.95–1.45)
Laborers and material movers, hand
92
1.09 (0.88–1.34)
Motor vehicle operators
74
1.07 (0.85–1.36)
Metal workers and plastic workers
35
0.95 (0.66–1.33)
Food preparation and serving related occupations
46
0.91 (0.66–1.21)
Construction trades workers
149
0.89 (0.76–1.05)
Management occupations, except agricultural
61
0.89 (0.69–1.15)
Building and grounds cleaning and maintenance occupations
54
0.88 (0.67–1.16)
Electrical equipment mechanics and other installation, maintenance, and repair workers
26
0.85 (0.56–1.25)
Vehicle and mobile equipment mechanics, installers, and repairers
32
0.82 (0.56–1.15)
All other occupations
316
—
Female (n = 1,239)
Community and social services occupations
36
1.46 (1.02–2.01)**
Laborers and material movers, hand
47
1.19 (0.88–1.59)
Healthcare support occupations
110
1.15 (0.95–1.39)
Food preparation and serving related occupations
100
1.12 (0.92–1.37)
Personal care and service occupations
75
1.09 (0.87–1.38)
Sales and related occupations
134
1.09 (0.92–1.30)
Health diagnosing and treating practitioners and technical occupations
59
1.00 (0.77–1.31)
Building and grounds cleaning and maintenance occupations
62
1.00 (0.78–1.30)
Management occupations, except agricultural
85
0.99 (0.80–1.24)
Business operations specialists
25
0.96 (0.62–1.42)
Education, training, and library occupations
70
0.93 (0.73–1.18)
Health technologists and technicians
28
0.91 (0.61–1.32)
Office and administrative support occupations
186
0.90 (0.77–1.04)
All other occupations
222
—
Abbreviations: CI = confidence interval; PMR = proportionate mortality ratio. * D
d
h
h d h I
i
l Cl
ifi
i
f Di
10 h R
i i
d
J45 (
h
)
J46 (
h
i
)
i
d
h
d
l i
f TABLE 2. (Continued) Industries and occupations with ≥25 asthma* deaths among persons aged 15–64 years, by sex — 26 states,† 1999, 2003,
2004, and 2007–2012 TABLE 2. (Continued) Industries and occupations with ≥25 asthma* deaths among persons aged 15–64 years, by sex — 26 states,† 1999, 2003,
2004, and 2007–2012
Characteristic
No. of deaths
PMR§,¶ (95% CI) Building and grounds cleaning and maintenance occupation Management occupations, except agricultural Education, training, and library occupations Health technologists and technicians Office and administrative support occupations
All other occupations Abbreviations: CI = confidence interval; PMR = proportionate mortality ratio. Summary What is already known about this topic? In 2015, a total of 3,396 asthma deaths were reported among
adults aged ≥18 years in the United States. An estimated
11%–21% of asthma deaths might be attributable to occupa-
tional exposures. Asthma deaths are preventable with proper
asthma management and rapid response to asthma attacks. Corresponding author: Jacek M. Mazurek, jmazurek1@cdc.gov, 304-285-5983. Corresponding author: Jacek M. Mazurek, jmazurek1@cdc.gov, 304-285-5983. Acknowledgments Effective asthma management tools are available from CDC
at https://www.cdc.gov/asthma/tools_for_control.htm, and
information on the evaluation and treatment of asthma is
available from the American Thoracic Society at https://www. thoracic.org/statements/allergy-asthma.php. Additional guid-
ance for diagnosing work-related asthma is available from the
Occupational Safety and Health Administration at https://
www.osha.gov/SLTC/occupationalasthma/. The elevated Eileen Storey, MD, Respiratory Health Division, National Institute
for Occupational Safety and Health, CDC; Carol Johnson, MPH,
Division of Environmental Hazards and Health Effects, National
Center for Environmental Health, CDC; Vital Statistics Offices
from the following 26 states providing industry and occupation
information: Colorado, Florida, Georgia, Hawaii, Idaho, Indiana,
Kansas, Kentucky, Louisiana, Michigan, Nebraska, Nevada, MMWR / January 19, 2018 / Vol. 67 / No. 2
US Department of Health and Human Services/Centers for Disease Control and Prevention 64 Morbidity and Mortality Weekly Report 1Association of Schools and Programs of Public Health/CDC Public Health
Fellowship Program; 2Respiratory Health Division, National Institute for
Occupational Safety and Health, CDC. References 1. CDC. Asthma: most recent asthma data. 2017. Atlanta, GA: US
Department of Health and Human Services, CDC; 2017. https://www. cdc.gov/asthma/most_recent_data.htm MMWR / January 19, 2018 / Vol. 67 / No. 2 No conflicts of interest were reported. US Department of Health and Human Services/Centers for Disease Control and Prevention What is added by this report? 2. Steenland K, Burnett C, Lalich N, Ward E, Hurrell J. Dying for work: the
magnitude of US mortality from selected causes of death associated with
occupation. Am J Ind Med 2003;43:461–82. https://doi.org/10.1002/
ajim.10216 Among U.S. adults aged 15–64 years, 33,307 deaths from
asthma occurred during 1999–2016, including an estimated
3,664–6,994 (approximately 204–389 annually) that could be
attributable to occupational exposures and were therefore
potentially preventable. The highest asthma death rates were
among adults aged 55–64 years, females, persons who were not
Hispanic or Latino, and blacks or African Americans. By industry,
asthma mortality was significantly elevated among males in
food, beverage, and tobacco products manufacturing, other
retail trade, and miscellaneous manufacturing, and among
females in social assistance. By occupation, asthma mortality
was significantly elevated among females in community and
social services. 3. National Asthma Education and Prevention Program. Expert panel
report 3 (EPR-3): guidelines for the diagnosis and management
of asthma—summary report 2007. J Allergy Clin Immunol
2007;120(Suppl):S94–138. https://doi.org/10.1016/j.jaci.2007.09.029 4. Heederik D, Henneberger PK, Redlich CA; ERS Task Force on the
Management of Work-related Asthma. Primary prevention: exposure
reduction, skin exposure and respiratory protection. Eur Respir Rev
2012;21:112–24. https://doi.org/10.1183/09059180.00005111 g
5. Moorman JE, Akinbami LJ, Bailey CM, et al. National surveillance of
asthma: United States, 2001–2010. Vital Health Stat 3 2012;35:1–58. 6. Calvert GM, Luckhaupt SE, Sussell A, Dahlhamer JM, Ward BW. The
prevalence of selected potentially hazardous workplace exposures in the
US: findings from the 2010 National Health Interview Survey. Am J
Ind Med 2013;56:635–46. https://doi.org/10.1002/ajim.22089 What are the implications for public health practice? Elevated asthma mortality among male and female workers in
certain industries and occupations highlights the importance of
optimal asthma management, and identification and preven-
tion of workplace exposures. 7. White GE, Seaman C, Filios MS, et al. Gender differences in work-related
asthma: surveillance data from California, Massachusetts, Michigan, and
New Jersey, 1993–2008. J Asthma 2014;51:691–702. https://doi.org/1
0.3109/02770903.2014.903968 8. Baur X, Bakehe P, Vellguth H. Bronchial asthma and COPD due to
irritants in the workplace - an evidence-based approach. J Occup Med
Toxicol 2012;7:19. https://doi.org/10.1186/1745-6673-7-19 New Hampshire, New Jersey, New Mexico, North Carolina, North
Dakota, Ohio, Rhode Island, South Carolina, Texas, Utah, Vermont,
Washington, West Virginia, and Wisconsin. 9. Tarlo SM, Balmes J, Balkissoon R, et al. Diagnosis and management of
work-related asthma: American College Of Chest Physicians consensus
statement. Chest 2008;134(Suppl):1S–41S. https://doi.org/10.1378/
chest.08-0201 Conflict of Interest Conflict of Interest No conflicts of interest were reported. 10. Le Moual N, Kauffmann F, Eisen EA, Kennedy SM. The healthy
worker effect in asthma: work may cause asthma, but asthma may also
influence work. Am J Respir Crit Care Med 2008;177:4–10. https://
doi.org/10.1164/rccm.200703-415PP MMWR / January 19, 2018 / Vol. 67 / No. 2 US Department of Health and Human Services/Centers for Disease Control and Prevention 65
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English
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Potent and selective inhibitors for M32 metallocarboxypeptidases identified from high-throughput screening of anti-kinetoplastid chemical boxes
|
PLoS neglected tropical diseases
| 2,019
|
cc-by
| 10,896
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Editor: Laura-Isobel McCall, University of
Oklahoma, UNITED STATES Editor: Laura-Isobel McCall, University of
Oklahoma, UNITED STATES
Received: March 6, 2019
Accepted: June 18, 2019
Published: July 22, 2019 Editor: Laura-Isobel McCall, University of
Oklahoma, UNITED STATES
Received: March 6, 2019
Accepted: June 18, 2019
Published: July 22, 2019 Copyright: © 2019 Salas-Sarduy et al. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. Funding: This study was supported by PICT 2014-
3510 from the Agencia Nacional de Promocio´n
Cientı´fica y Tecnolo´gica (ANPCyT) to GTN (https://
www.argentina.gob.ar/ciencia/agencia), PIP
11220130100303CO from Argentinian National
Research Council to GTN (https://www.conicet. gov.ar/), and PICTO-2013-0067 from ANPCyT to RESEARCH ARTICLE Emir Salas-Sarduy1, Lionel Ura´n LandaburuID1, Adriana K. CarmonaID2, Juan
Jose´ Cazzulo1, Ferna´n Agu¨ero1, Vanina E. Alvarez1, Gabriela T. NiemirowiczID1* Emir Salas-Sarduy1, Lionel Ura´n LandaburuID1, Adriana K. CarmonaID2, Juan
Jose´ Cazzulo1, Ferna´n Agu¨ero1, Vanina E. Alvarez1, Gabriela T. NiemirowiczID1* 1 Instituto de Investigaciones Biotecnolo´gicas “Dr. Rodolfo Ugalde”–Universidad Nacional de San Martı´n–
CONICET, San Martı´n, B1650HMP, Buenos Aires, Argentina, 2 Departamento de Biofı´sica, Universidade
Federal de São Paulo, São Paulo, Brazil a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 * gniemiro@iibintech.com.ar * gniemiro@iibintech.com.ar Abstract Enzymes of the M32 family are Zn-dependent metallocarboxypeptidases (MCPs) widely
distributed among prokaryotic organisms and just a few eukaryotes including Trypanosoma
brucei and Trypanosoma cruzi, the causative agents of sleeping sickness and Chagas dis-
ease, respectively. These enzymes are absent in humans and several functions have been
proposed for trypanosomatid M32 MCPs. However, no synthetic inhibitors have been
reported so far for these enzymes. Here, we present the identification of a set of inhibitors
for TcMCP-1 and TbMCP-1 (two trypanosomatid M32 enzymes sharing 71% protein
sequence identity) from the GlaxoSmithKline HAT and CHAGAS chemical boxes; two col-
lections grouping 404 compounds with high antiparasitic potency, drug-likeness, structural
diversity and scientific novelty. For this purpose, we adapted continuous fluorescent enzy-
matic assays to a medium-throughput format and carried out the screening of both collec-
tions, followed by the construction of dose-response curves for the most promising hits. As
a result, 30 micromolar-range inhibitors were discovered for one or both enzymes. The best
hit, TCMDC-143620, showed sub-micromolar affinity for TcMCP-1, inhibited TbMCP-1 in
the low micromolar range and was inactive against angiotensin I-converting enzyme (ACE),
a potential mammalian off-target structurally related to M32 MCPs. This is the first inhibitor
reported for this family of MCPs and considering its potency and specificity, TCMDC-
143620 seems to be a promissory starting point to develop more specific and potent chemi-
cal tools targeting M32 MCPs from trypanosomatid parasites. OPEN ACCESS Citation: Salas-Sarduy E, Landaburu LU, Carmona
AK, Cazzulo JJ, Agu¨ero F, Alvarez VE, et al. (2019)
Potent and selective inhibitors for M32
metallocarboxypeptidases identified from high-
throughput screening of anti-kinetoplastid chemical
boxes. PLoS Negl Trop Dis 13(7): e0007560. https://doi.org/10.1371/journal.pntd.0007560 Potent and selective inhibitors for M32
metallocarboxypeptidases identified from
high-throughput screening of anti-
kinetoplastid chemical boxes Emir Salas-Sarduy1, Lionel Ura´n LandaburuID1, Adriana K. CarmonaID2, Juan
Jose´ Cazzulo1, Ferna´n Agu¨ero1, Vanina E. Alvarez1, Gabriela T. NiemirowiczID1* Introduction Members of the Trypanosomatidae family comprise parasitic organisms that cause highly dis-
abling and often fatal diseases in humans and animals. The species that are responsible for
human infections are Trypanosoma brucei, which cause Human African trypanosomiasis
(HAT), Trypanosoma cruzi, the etiological agent of Chagas disease (American trypanosomia-
sis), and Leishmania spp., which cause different forms of leishmaniasis. Together, these vector-
borne diseases constitute a substantial public health problem for which there is not a satisfac-
tory treatment [1]. Major side-effects, and in some cases low effectiveness, are common prob-
lems associated with existing therapy. This situation makes imperative the development of
new chemotherapeutic options. In this context, new drugs based on unique aspects of parasite
biology and biochemistry are of great interest, particularly in the case of emerging resistance
to traditional treatments [2–4]. In this scenario, proteases have become popular targets as
these enzymes play key functions in parasite biology; namely nutrition, cell cycle progression,
invasion and pathogenesis, among others. The M32 family of metallocarboxypeptidases (MCPs) contains a group of hydrolases,
which although being broadly distributed among prokaryotic organisms, are only present in a
few eukaryotes including some green algae and trypanosomatids [5]. This unique phylogenetic
distribution, in particular the absence of M32 enzymes in metazoans, has been considered an
attractive trait due to the high specificity/selectivity potential of this family for drug target
development. Within the Trypanosomatidae family several conserved M32 MCPs have been
characterized [5–10]. Nonetheless, the cellular or biological functions of these proteins are cur-
rently unknown, as well as their essentiality status. In T. brucei, the genome-wide study by Als-
ford et al. (2011) reported no significant lost-of-fitness after induction of T. brucei MCP-1
(TbMCP-1) RNAi in bloodstream and procyclic stages, as well as in the differentiation from
procyclic to bloodstream forms [11]. More recently, however, it has been shown that TbMCP-
1 null mutant strains display extended doubling times in culture, suggesting that this enzyme
might contribute to the adaptive fitness of the bloodstream form [12]. On the basis of their bio-
chemical properties and stage-specific expression, the L. major M32 carboxypeptidase has
been implicated in the catabolism of peptides and proteins to single amino acids required for
protein synthesis [7]. The restricted substrate preference of T. Author summary In recent years, the pharmaceutical company GlaxoSmithKline announced the disclosure
of small collections of antiparasitic compounds to facilitate research and drug develop-
ment for three of the main Tropical Neglected Diseases- i.e. Human African 1 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560
July 22, 2019 Synthetic inhibitors for M32 metallocarboxypeptidases FA and VEA. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript. Trypanosomiasis, Leishmaniasis and Chagas Disease. These collections include new
chemical entities with potential novel mechanisms of action that are likely to be active
against a wide variety of targets. Taking advantage of these open access molecules, we suc-
cessfully set up medium-throughput screening assays to find the first inhibitors of two
metallocarboxypeptidases of the M32 family, a group of proteolytic enzymes proposed to
play several roles in the biology of trypanosomatids including peptide catabolism, mainte-
nance of parasite adaptive fitness and hydrolysis of bioactive peptides from the human
host. Competing interests: The authors have declared
that no competing interests exist. Development of continuous metallocarboxypeptidase assays To evaluate compounds in the HAT and CHAGAS chemical boxes, we devised a continuous
assay for each MCP, based on FRET (fluorescence resonance energy transfer) peptides. We
carried out the optimization process in 384 well plates, the same format used for the screening
of the compound collections. For the selection of the most suitable substrate for the HTS assay,
we initially assayed six FRET peptides against both enzymes. These were recently designed
considering subsite preferences (P1´-P4) of TcMCP-1 and TbMCP-1 [12]. However, because
no peptide was completely satisfactory for both enzymes, we selected independent substrates,
Abz-LKFK(Dnp)-OH and Abz-RFFK(Dnp)-OH, for TcMCP-1 and TbMCP-1 assays, respec-
tively. After substrate selection, a convenient enzyme concentration in the assay was deter-
mined through the activity of 2-fold dilutions of TcMCP-1 and TbMCP-1 at a fixed substrate
concentration (Fig 1A and 1B). Moreover, the Selwyn test [17] revealed no enzyme inactiva-
tion under the conditions tested (Fig 1C and 1D). Thus, for a wide range of enzyme concentra-
tions (for both MCPs), the V0 vs. [E]0 curves showed a linear behavior (Fig 1E and 1F). In
particular, for [TcMCP-1]0 < 0,34 nM and [TbMCP-1]0 < 1,53 nM, the rate of the substrate
hydrolysis remained constant for at least 40 minutes, a suitable time to perform the screening
(Fig 1A and 1B). The best balance between TcMCP-1 activity on Abz-LKFK(Dnp)-OH substrate (estimated
as dF/dt) and the time over which the reaction displayed linear kinetics was achieved at
[TcMCP-1]0 = 0,17 nM. Under these conditions, the enzyme showed the typical hyperbolic
behavior predicted by the Michaelis-Menten equation (Hill coefficient = 1,06) and an esti-
mated KM value of 2,23 ± 0,28 μM (Fig A in S1 Text). Similarly, when the TbMCP-1 concentra-
tion was fixed at 1,25 nM we obtained a KM value on Abz-RFFK(Dnp)-OH substrate of
0,37 ± 0,06 μM (Hill coefficient = 1,03) (Fig A in S1 Text). To afford the best opportunity to
find compounds with different inhibition modalities, we decided to employ balanced assay
conditions (i.e. KM/[S] = 1)[18]. Using these conditions, preliminary characterization experi-
ments of both optimized assays showed good general performance, with a dynamic range (μC
+—μC-) higher than 15 RFU/sec, a μC+/μC- ratio 50, good reproducibility (VC < 5%) and a Z
´ factor value in the range 0,6–0,8. Synthetic inhibitors for M32 metallocarboxypeptidases activity would be of great value to ask mechanistic and phenotypic questions in both biochemi-
cal and cell-based studies. However, no inhibitors have been reported to date for these
enzymes or other members of this family. Recently, a diverse collection of ~ 1.8 million compounds from the proprietary library of
GlaxoSmithKline (GSK) has been run through whole-cell phenotypic screens against L. dono-
vani, T. cruzi and T. brucei. As a result, three anti-kinetoplastid chemical boxes of ~200 com-
pounds each were assembled and open sourced [16]. The guiding design criteria for these
molecule sets were chosen to include structures from different chemical families that are likely
to be active against a wide variety of targets. By taking advantage of this diversity, we identified
the first inhibitors of the M32 family of MCPs within the GSK HAT and CHAGAS chemical
boxes. As model enzymes of the M32 family we employed TcMCP-1 and TbMCP-1, which
have similar basic amino acid preference at the P1´ position and share 71% of protein sequence
identity [5, 6]. Introduction cruzi MCP-1 (TcMCP-1), plus
its strong structural similarity to angiotensin I-converting enzyme (ACE), neurolysin and thi-
met oligopeptidase [8], have also pointed out a possible regulatory role of this family in the
metabolism of small peptides. In fact, it has been shown that TcMCP-1 can produce des-
Arg9-bradykinin [6], a peptide that promotes the process of cell invasion through B1 receptors
by the T. cruzi trypomastigotes [13]. In this sense, two reports have suggested that M32 pepti-
dases are secreted by trypanosomatids [14, 15], a fact that is in agreement with this hypothesis. In the current scenario, the availability of selective small-molecule modulators of M32 MCPs PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560
July 22, 2019 2 / 20 Primary screening of HAT and CHAGAS chemical boxes Using the same lot of substrate and enzyme, the 404 compounds present in the HAT and
CHAGAS chemical boxes were screened at a single fixed dose (25 μM). Each plate included 24 3 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560
July 22, 2019 Synthetic inhibitors for M32 metallocarboxypeptidases Fig 1. Continuous fluorogenic assays for recombinant MCPs. (A) Kinetic progression curves for different TcMCP-1 concentrations at a fixed Abz-LKFK(Dnp)-OH
dose (1,25 μM). (B) The activity of 2-fold dilutions of TbMCP-1 was analyzed with Abz-RFFK(Dnp)-OH as substrate (4,8 μM). For both enzymes, working dilutions
were selected from those that showed linear kinetics for more than 40 minutes. (C, D) Selwyn test for different TcMCP-1 and TbMCP-1 concentrations. In both cases,
the global fitting of experimental data from different enzyme dilutions to a unique curve was good, indicating that enzymes remained stable during the whole assay. To
facilitate observation, curves were slightly displaced from each other in the Y axis. (E, F) Curve of V0 vs. [E]0 for TcMCP-1 and TbMCP-1 respectively. In both cases, the
expected linear behavior was observed. https://doi.org/10.1371/journal.pntd.0007560.g001
Synthetic inhibitors for M32 metallocarboxypeptidases Fig 1. Continuous fluorogenic assays for recombinant MCPs. (A) Kinetic progression curves for different TcMCP-1 concentrations at a fixed Abz-LKFK(Dnp)-OH
dose (1,25 μM). (B) The activity of 2-fold dilutions of TbMCP-1 was analyzed with Abz-RFFK(Dnp)-OH as substrate (4,8 μM). For both enzymes, working dilutions
were selected from those that showed linear kinetics for more than 40 minutes. (C, D) Selwyn test for different TcMCP-1 and TbMCP-1 concentrations. In both cases,
the global fitting of experimental data from different enzyme dilutions to a unique curve was good, indicating that enzymes remained stable during the whole assay. To
facilitate observation, curves were slightly displaced from each other in the Y axis. (E, F) Curve of V0 vs. [E]0 for TcMCP-1 and TbMCP-1 respectively. In both cases, the
expected linear behavior was observed. https://doi.org/10.1371/journal.pntd.0007560.g001 positive and negative controls, plus 16 wells containing 31,25 mM EDTA (inhibition control)
alternately located in columns 11, 12, 23 and 24. In general, for each MCP, both plates pre-
sented highly similar Z´ scores although best values were obtained for the TbMCP-1 assay pre-
sumably due to the lower background signal of the Abz-RFFK(Dnp)-OH substrate. To avoid positive and negative controls, plus 16 wells containing 31,25 mM EDTA (inhibition control)
alternately located in columns 11, 12, 23 and 24. Synthetic inhibitors for M32 metallocarboxypeptidases Table 1. Statistics for the plates during primary screening. TcMCP-1
TbMCP-1
Plate 1
Plate 2
Plate 1
Plate 2
Compounds (n)
320
84
320
84
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Enzyme control (C+) (RFU/sec)
19,12
1,60
20,19
2,14
16,80
0,57
21,23
0,57
Substrate control (C-) (RFU/sec)
2,98
0,19
3,63
0,24
0,01
0,20
-0,12
0,38
EDTA control (RFU/sec)
1,28
0,24
2,32
0,34
0,13
0,20
0,39
0,11
Z´ factor
0,67
0,57
0,86
0,87
TbMCP-1 and TcMCP-1 activities were assayed fluorometrically with Abz-RFFK(Dnp)-OH and Abz-LKFK(Dnp)-OH substrates, respectively, in 100 mM MOPS pH 7,2
containing 0,01% Triton X-100 (C+). Final substrate concentration was set to a value KM /[S] ~ 1. Additionally, 24 negative or substrate controls (no enzyme added, C-)
plus 16 inhibition controls (EDTA final concentration 31,25 mM) were included in each plate. Z factor was calculated as in [19]. Table 1. Statistics for the plates during primary screening. TbMCP-1 and TcMCP-1 activities were assayed fluorometrically with Abz-RFFK(Dnp)-OH and Abz-LKFK(Dnp)-OH substrates, respectively, in 100 mM MOPS pH 7,2
containing 0,01% Triton X-100 (C+). Final substrate concentration was set to a value KM /[S] ~ 1. Additionally, 24 negative or substrate controls (no enzyme added, C-)
plus 16 inhibition controls (EDTA final concentration 31,25 mM) were included in each plate. Z factor was calculated as in [19]. TbMCP-1 and TcMCP-1 activities were assayed fluorometrically with Abz-RFFK(Dnp)-OH and Abz-LKFK(Dnp)-OH substrates, respectively, in 100 mM MOPS pH 7,2
containing 0,01% Triton X-100 (C+). Final substrate concentration was set to a value KM /[S] ~ 1. Additionally, 24 negative or substrate controls (no enzyme added, C-)
plus 16 inhibition controls (EDTA final concentration 31,25 mM) were included in each plate. Z factor was calculated as in [19]. https://doi.org/10.1371/journal.pntd.0007560.t001 the interference of highly fluorescent compounds, an auto-fluorescence cut-off value equal to
2x105 RFU was used to accept or discard a molecule from the subsequent analysis. Using this
limit, ~19% of the compounds were eliminated for TcMCP-1 and TbMCP-1 assays. Statistics
are summarized in Table 1. As shown in Table 2, if we consider a cut-off value 3 standard deviations from the control
mean (μc+ - 3σc+), 70 and 132 inhibitory molecules were retrieved for TcMCP-1 and TbMCP-
1, respectively. To reduce the number of resultant hits, we explored other two thresholds focus-
ing only in outliers: i) those compounds showing slopes >3σ standard deviations above the
average of all slopes in the plate (control independent) and ii) those compounds showing an
inhibition percentage >3σ standard deviations above the average for the plate (control depen-
dent). Interestingly, both criteria retrieved exactly the same list of compounds for TcMCP-1
(n = 5) while for TbMCP-1 the intersection between this two groups was lower (2 out of 4
compounds). Primary screening of HAT and CHAGAS chemical boxes In general, for each MCP, both plates pre-
sented highly similar Z´ scores although best values were obtained for the TbMCP-1 assay pre-
sumably due to the lower background signal of the Abz-RFFK(Dnp)-OH substrate. To avoid positive and negative controls, plus 16 wells containing 31,25 mM EDTA (inhibition control)
alternately located in columns 11, 12, 23 and 24. In general, for each MCP, both plates pre-
sented highly similar Z´ scores although best values were obtained for the TbMCP-1 assay pre-
sumably due to the lower background signal of the Abz-RFFK(Dnp)-OH substrate. To avoid 4 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560
July 22, 2019 https://doi.org/10.1371/journal.pntd.0007560.t002 () Different hit selection criteria were applied to both HAT and CHAGAS boxes. 1) Compounds showing an
inhibition three standard deviation from control mean. 2) Compounds showing slopes >3σ standard deviations
above the average of all slopes in the plate and 3) those compounds showing a percent inhibition >3σ standard
deviations above the average for the plate. https://doi.org/10.1371/journal.pntd.0007560.t001 () Highly fluorescent compounds were discarded from the analysis. Finally, another 7 molecules
performed better in the secondary screening than in the first round. Despite the observed
round to round discrepancies (Table A in S1 Text), we decided to continue curve analysis for
all the compounds, with the exception of the three that showed no inhibition at 31,5 μM dur-
ing secondary screening. recombinant MCPs using identical assay conditions as in the primary screening. Prior to the
analysis of the complete dataset, we examined whether there was a correlation between the
inhibition percentages in the primary (compound concentration 25 μM) and secondary
screening, using only the data corresponding to a compound concentration of 31,5 μM. This
was important to assess consistency of data, as both screening rounds were performed without
technical replicates due to limitation of compound stocks. For TcMCP-1, 9 compounds pre-
sented similar behavior in both screenings (correlation coefficient r2 = 0,9868; slope = 1,146)
(Fig 2A) whereas 7 molecules failed to reach 40% of inhibition threshold (n = 6) or displayed
no inhibition (n = 1) (correlation coefficient r2 = -0,518; slope = 0,2595). Additionally, 7 com-
pounds performed better in the secondary screening (correlation coefficient r2 = 0,5156;
slope = 1,2749). For the T. brucei enzyme, consistent results in both assays were achieved only
by 8 compounds (correlation coefficient r2 = 0,9349; slope = 1,080) (Fig 2B). About 45% of the
samples did not repeat the 40% of inhibition criterium (n = 10) or did not inhibit (n = 2)
TbMCP-1 (correlation coefficient r2 = 0,1163; slope = 0,3173). Finally, another 7 molecules
performed better in the secondary screening than in the first round. Despite the observed
round to round discrepancies (Table A in S1 Text), we decided to continue curve analysis for
all the compounds, with the exception of the three that showed no inhibition at 31,5 μM dur-
ing secondary screening. For TcMCP-1, five compounds (TCMDC-143620, TCMDC-143422, TCMDC-143456,
TCMDC-143209 and TCMDC-143385) showed an IC50 value 10 μM (Fig 3A and Table 3). In good agreement, the four more potent molecules (TCMDC-143620, TCMDC-143422,
TCMDC-143456 and TCMDC-143209) also inhibited the T. brucei enzyme (Table 3). Com-
pounds TCMDC-143385 and TCMDC-143172 (which display an IC50 ~10 μM for TcMCP-1)
did not reach the 40% inhibition threshold in the TbMCP-1 primary screening and were left
out from the secondary analysis. Other potent molecules, namely TCMDC-143409 and
TCMDC-143323 were specific inhibitors of T. Synthetic inhibitors for M32 metallocarboxypeptidases Fig 2. Correlation between the inhibition percentages in the primary and secondary screenings for the most reproducible compounds. As both screening
rounds were performed without technical replicates (see Material and Methods), we introduced this analysis to assess data consistency. The analysis was
performed using the 25μM and 31,5μM data points from the primary and secondary screenings, respectively. The main panel shows correlation of the most
reproducible hits, whereas the insets show the correlation analysis for all the hit compounds at indicated concentrations. (A) TcMCP-1 (B) TbMCP-1. https://doi org/10 1371/journal pntd 0007560 g002 Fig 2. Correlation between the inhibition percentages in the primary and secondary screenings for the most reproducible compounds. As both screening
rounds were performed without technical replicates (see Material and Methods), we introduced this analysis to assess data consistency. The analysis was
performed using the 25μM and 31,5μM data points from the primary and secondary screenings, respectively. The main panel shows correlation of the most
reproducible hits, whereas the insets show the correlation analysis for all the hit compounds at indicated concentrations. (A) TcMCP-1 (B) TbMCP-1. h
//d i
/10 13 1/j
l
d 000
60 002 https://doi.org/10.1371/journal.pntd.0007560.g002 recombinant MCPs using identical assay conditions as in the primary screening. Prior to the
analysis of the complete dataset, we examined whether there was a correlation between the
inhibition percentages in the primary (compound concentration 25 μM) and secondary
screening, using only the data corresponding to a compound concentration of 31,5 μM. This
was important to assess consistency of data, as both screening rounds were performed without
technical replicates due to limitation of compound stocks. For TcMCP-1, 9 compounds pre-
sented similar behavior in both screenings (correlation coefficient r2 = 0,9868; slope = 1,146)
(Fig 2A) whereas 7 molecules failed to reach 40% of inhibition threshold (n = 6) or displayed
no inhibition (n = 1) (correlation coefficient r2 = -0,518; slope = 0,2595). Additionally, 7 com-
pounds performed better in the secondary screening (correlation coefficient r2 = 0,5156;
slope = 1,2749). For the T. brucei enzyme, consistent results in both assays were achieved only
by 8 compounds (correlation coefficient r2 = 0,9349; slope = 1,080) (Fig 2B). About 45% of the
samples did not repeat the 40% of inhibition criterium (n = 10) or did not inhibit (n = 2)
TbMCP-1 (correlation coefficient r2 = 0,1163; slope = 0,3173). Secondary screening In the secondary screening we decided to include all compounds that showed 40% of inhibi-
tion (TcMCP-1: 23 compounds; TbMCP-1: 27 compounds). To estimate IC50 for the resulting
hits, two-fold serial dilutions, ranging from 7,5 pM to 62,5 μM, were analyzed against both Table 2. Primary screening results. TcMCP-1
TbMCP-1
Plate 1
Plate 2
Plate 1
Plate 2
Compounds (n)
320
84
320
84
Analyzed compounds (n)
256
72
254
71
1
51
19
92
40
2
4
1
2
2
3
4
1
0
2
40% inhibition
19
4
22
5
() Highly fluorescent compounds were discarded from the analysis. () Different hit selection criteria were applied to both HAT and CHAGAS boxes. 1) Compounds showing an
inhibition three standard deviation from control mean. 2) Compounds showing slopes >3σ standard deviations
above the average of all slopes in the plate and 3) those compounds showing a percent inhibition >3σ standard
deviations above the average for the plate. https://doi.org/10.1371/journal.pntd.0007560.t002 Table 2. Primary screening results. TcMCP-1
TbMCP-1
Plate 1
Plate 2
Plate 1
Plate 2
Compounds (n)
320
84
320
84
Analyzed compounds (n)
256
72
254
71
1
51
19
92
40
2
4
1
2
2
3
4
1
0
2
40% inhibition
19
4
22
5 () Highly fluorescent compounds were discarded from the analysis. () Highly fluorescent compounds were discarded from the analysis. () Highly fluorescent compounds were discarded from the analysis. () Different hit selection criteria were applied to both HAT and CHAGAS boxes. 1) Compounds showing an
inhibition three standard deviation from control mean. 2) Compounds showing slopes >3σ standard deviations
above the average of all slopes in the plate and 3) those compounds showing a percent inhibition >3σ standard
deviations above the average for the plate. 5 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560
July 22, 2019 Synthetic inhibitors for M32 metallocarboxypeptidases indicated in Material and Methods. For each compound, solid line represents the best fit of four-parameter Hill equation to experimental data (closed circles). (A)
Dose-response curves corresponding to the most potent TcMCP-1 inhibitors. (B) Equivalent analysis for the top-five TbMCP-1 inhibitors. (C) Structure and identifiers
corresponding to the most potent hit compounds identified for both enzymes. Subgroup (a) contains those molecules that inhibited both MCPs. Subgroup (b) is
formed by TCMDC-133485, which selectively acts on TcMCP-1 whereas subgroup (c) includes TbMCP-1 specific inhibitors. https://doi.org/10.1371/journal.pntd.0007560.g003 https://doi.org/10.1371/journal.pntd.0007560.g003 TcMCP-1 (< 30%) (Fig 3B and Table 3). The structure of the top-five inhibitors for each
enzyme is shown in Fig 3C. brucei enzyme or produced little inhibition on 6 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560
July 22, 2019 Synthetic inhibitors for M32 metallocarboxypeptidases Fig 3. Dose-response curves and structures of top-five inhibitors identified for each MCPs. To estimate the potency of the inhibitory activity, enzymes were
incubated with different concentrations (ranging from 7,5 pM to 62,5 μM) of the selected compounds and the inhibition percentages determined for each condition as
Synthetic inhibitors for M32 metallocarboxypeptidases Fig 3. Dose-response curves and structures of top-five inhibitors identified for eac
incubated with different concentrations (ranging from 7,5 pM to 62,5 μM) of the selec Fig 3. Dose-response curves and structures of top-five inhibitors identified for each MCPs. To estimate the potency of the inhibitory activity, enzymes were
incubated with different concentrations (ranging from 7,5 pM to 62,5 μM) of the selected compounds and the inhibition percentages determined for each condition as Fig 3. Dose-response curves and structures of top-five inhibitors identified for each MCPs. To estimate the potency of the inhibitory activity, enzymes were
incubated with different concentrations (ranging from 7,5 pM to 62,5 μM) of the selected compounds and the inhibition percentages determined for each condition as Fig 3. Dose-response curves and structures of top-five inhibitors identified for each MCPs. To estimate the potency of the
ncubated with different concentrations (ranging from 7,5 pM to 62,5 μM) of the selected compounds and the inhibition perce PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560
July 22, 2019 7 / 20 Compounds that presented similar behavior in both primary and secondary screening (<15% variation between both assays https://doi.org/10.1371/journal.pntd.0007560.t003 https://doi.org/10.1371/journal.pntd.0007560.t003 ted similar behavior in both primary and secondary screening (<15% variation between both assays) are highlighted in grey. Lead compounds have low structural redundancy Red squares for TbMCP-1, blue squares for TcMCP-1, grey squares for
non-active in TbMCP-1/TcMCP-1, accordingly. https://doi.org/10.1371/journal.pntd.0007560.g004 physicochemical properties (Fig 4). Whereas the Tanimoto clustering was expected to be
inconclusive based on the premises used to assemble the chemical boxes (one or two putative
chemotypes per box [16]); the clustering based on physicochemical properties also showed no
significant correlation between these properties and the observed IC50s. Similarly, MCS clus-
tering provided no insights into candidate substructures guiding the activity or specificity of
the compounds against each enzyme. In all three strategies, the clusters not only group up dis-
similar potencies, but also mix compounds with different enzyme specificity. Lead compounds have low structural redundancy To first assess the possibility that these lead compounds have shared structural features that
help explain their bioactivity profile, we performed three different clustering strategies: one
using Tanimoto similarity (Fig B in S1 Text), one based on shared substructures (overlap of
Maximum Common Subgraphs, MCS) (Fig C in S1 Text), and the third one based on shared Table 3. IC50 values and Hill slopes for identified hits. Table 3. IC50 values and Hill slopes for identified hits. Compound
Chemical Box
TcMCP-1
TbMCP-1
IC50 (μM)
Hill Slope
R square
IC50 (μM)
Hill Slope
R square
TCMDC-143620
CHAGAS
0,6939
-1,06
0,9821
4,989
-1,461
0,9674
TCMDC-143422
CHAGAS
1,52
-1,038
0,8644
10,46
-1,206
0,8407
TCMDC-143456
HAT
2,206
-0,9597
0,9609
14,74
-1,247
0,9121
TCMDC-143209
CHAGAS
4,182
-1,019
0,8086
28,34
-1,2405
0,9851
TCMDC-143385
CHAGAS
9,473
-0,9614
0,838
-
TCMDC-143172
HAT
11,21
-2,825
0,9751
-
TCMDC-143513
HAT
13,52
-0,9734
0,8486
28,33
-0,8371
0,9523
TCMDC-143551
HAT
13,84
-1,834
0,4295
34,51
-1,562
0,5659
TCMDC-143462
HAT
15,48
-0,9436
0,8296
-
TCMDC-143382
HAT
20,59
-0,7277
0,861
22,55
-0,7258
0,9649
TCMDC-143515
HAT
21,83
-1,149
0,7376
31,71
-0,8447
0,9624
TCMDC-143432
CHAGAS
26,26
-0,6129
0,9382
-
TCMDC-143242
HAT
26,5
-1,237
0,851
-
TCMDC-143592
CHAGAS
27,91
-0,8919
0,8208
-
TCMDC-143408
CHAGAS
32,86
-1,153
0,9473
-
TCMDC-143496
HAT
34,9
-0,822
0,6521
-
TCMDC-143071
CHAGAS
38,91
-1,235
0,8373
-
TCMDC-143263
HAT
40,74
-0,9946
0,8174
-
TCMDC-143543
HAT
>60
TCMDC-143323
HAT
-
3,938
-0,894
0,9353
TCMDC-143409
CHAGAS
-
10,42
-1,2409
0,9815
TCMDC-143191
CHAGAS
-
16,11
-1,267
0,977
TCMDC-143645
HAT
-
20,79
-1,127
0,9238
TCMDC-143143
CHAGAS
-
21,78
-1,466
0,9195
TCMDC-143332
CHAGAS
-
23,27
-1,063
0,9614
TCMDC-143158
HAT
-
27,64
-1,014
0,8757
TCMDC-143254
HAT
-
43,43
-0,8163
0,768
TCMDC-143265
HAT
-
44,2
-2,036
0,7989
TCMDC-143454
HAT
-
>60
TCMDC-143187
CHAGAS
-
>60 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560
July 22, 2019 8 / 20 Synthetic inhibitors for M32 metallocarboxypeptidases Fig 4. A dendrogram representing compound clustering using molecular features, and summarizing the activity distribution among tested MCPs. Squares
next to the names give an idea of IC50 observed: the brighter the color, the lower the IC50. Red squares for TbMCP-1, blue squares for TcMCP-1, grey squares for
non-active in TbMCP-1/TcMCP-1, accordingly. https://doi org/10 1371/journal pntd 0007560 g004 Fig 4. A dendrogram representing compound clustering using molecular features, and summarizing the activity distribution among tested MCPs. Squares
next to the names give an idea of IC50 observed: the brighter the color, the lower the IC50. Synthetic inhibitors for M32 metallocarboxypeptidases compounds), pyrazole (3 compounds), diol (1 compound) and hydrazide (1 compound). The
majority of compounds (24 out of 30) presented at least one ZBG in the structure. More specif-
ically, 15 with a single group and 9 with two groups were found. All compounds and their cor-
responding ZBGs have been summarized in Fig D in S1 Text. MCP inhibitors are specific Considering the abundance of ZBGs and heteroatom-containing moieties in the hits, we evalu-
ated the possibility of a nonspecific mechanism of inhibition (involving metal chelation) for
the top-five inhibitors identified in the screening for each enzyme. Because M32 MCPs show a
strong topological similarity with ACE [22], we chose this enzyme to estimate the IC50 value
for each molecule. As done for the MCPs essays, ACE activity was analyzed employing a FRET
substrate, Abz-FRK(Dnp)P-OH, at a concentration equal to the apparent KM of the enzyme
~3 μM [23]. Experiment set up is summarized in Figs E and F in S1 Text. For comparative pur-
poses, captopril, a potent competitive ACE inhibitor, was included in the analysis (IC50 ~1
nM) (Fig 5A). Under these conditions, no inhibition could be detected for any of the com-
pounds evaluated, thus suggesting that these molecules are not promiscuous metallocarboxy-
peptidase inhibitors (Fig 5B, 5C and 5D) but are instead specific inhibitors of M32 MCPs. Most compounds have at least one Zinc-biding group To determine the number and type of Zinc-binding groups (ZBGs) among the compound
leads, an MCS analysis was performed using an ad hoc curated [20, 21] database of ZBGs. From a total of 48 groups available in the database, only six of them were found among 24 of
the 30 lead compounds: pyridine (14 compounds), sulfonamide (7 compounds), imidazole (4 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560
July 22, 2019 9 / 20 Discussion M32 MCPs have an unusual phylogenetic distribution (with trypanosomatids being among
the few eukaryotic genomes encoding these enzymes). Hence M32 MCPs from parasites arose
naturally as interesting candidates for drug target development. Furthermore, the current lack
of knowledge about the cellular and/or physiological role(s) of these enzymes makes the identi-
fication of potent inhibitors a task of great significance, as these compounds may be used as
molecular probes to potentially identify natural substrates, to recognize the specific pathways
in which they are involved or, hopefully, to perform their chemical validation as drug targets. In this work, we describe the first drug-like inhibitors of TcMCP-1 and TbMCP-1, two closely
related MCPs from the human pathogens T. cruzi and T. brucei, respectively. Our starting
point were the GSK HAT and CHAGAS boxes, two small collections containing non-redun-
dant, chemically diverse and highly bioactive compounds [16], which could facilitate future
optimization efforts. Although we initially aimed for a common assay for both MCPs, we soon realized that the
use of different FRET substrates for each enzyme resulted in better general performance of the
individual assays (considering signal robustness, temporal duration of linear kinetics, dynamic
range, μC+/μC- ratio and Z´ factor). Surprisingly, the substrates that resulted most suitable for
the developed HTS assays were not, in any case, those that showed the best values of kcat, KM
and kcat/KM in their previous kinetic characterization [12]. Although different assays were
used to screen these collections, we were able to find specific inhibitors for both enzymes, and
perhaps more important, mutual inhibitors; suggesting the consistency of inter-assay results. Of note, specific inhibitors for each enzyme were distributed evenly among HAT and CHA-
GAS boxes with no apparent bias. This fact confirms the importance of not circumscribing the
search to just the pathogen-specific box, but instead to widen the search to all the boxes avail-
able, as previously observed for T. cruzi cysteine peptidase cruzipain [24]. Due to the limited amount of compound stocks, we decided to implement the screening of
chemical boxes in singlet, with primary evaluation of all compounds at a fixed dose and further
dose-response analysis of unconfirmed hits in a secondary screening. Synthetic inhibitors for M32 metallocarboxypeptidases Fig 5. Dose-response curves for ACE. (A) Purified rabbit lung ACE was assayed at 37˚C in 0,1 M Tris-HCl pH 7,0 buffer containing 50 mM NaCl,
10 mM ZnCl2 and 0,01% Triton X-100. Captopril, a potent competitive ACE inhibitor, was included as a positive inhibition control (IC50~ 1 nM). NI, no inhibitor added. (B, C) Data corresponding to TcMCP-1 and TbMCP-1 specific inhibitors, respectively. (D) Dose-response curves
corresponding to those molecules that inhibited both MCPs. In all cases, solid lines represent the best fit of four-parameter Hill equation to
experimental data (closed circles). Yellow, dark-green and orange colors were used for ACE, TcMCP-1 and TbMCP-1, respectively. https://doi.org/10.1371/journal.pntd.0007560.g005 comparison to secondary dose-response evaluation. These discrepancies are common and
may be due to a variety of factors [25]. Besides intrinsic compound-specific and experimental
data variability [26], these factors may include solubility issues (given that in primary and sec-
ondary screenings both the final concentration and serial-dilution protocol were different),
differential stability of compounds in stock (10 mM) and working (2 mM) solutions [27],
unintended absorption of the compounds to different containing materials during storage,
moderate dose-dependent quenching effects of compounds on fluorescence readouts, among
others [28]. In addition, although we included 0,01% Triton X-100 in assay buffer, compound-
specific aggregate formation was not tested and thus, cannot be dismissed. As mentioned, we identified in this work eight molecules able to inhibit both MCPs. These
mutual inhibitors came from both boxes in similar numbers, as previously noted for enzyme-
specific compounds. Interestingly, in all cases they were more potent inhibitors of TcMCP-1,
for reasons that are as yet unclear. Importantly, four of these compounds proved to be inactive
on ACE, a Zinc-dipeptidyl carboxypeptidase involved in various physiological and physiopath-
ological conditions in mammals [29] which shows significant structural similarity to M32
enzymes [22, 30]. This fact strongly suggests that despite the structural resemblance and the
small number of compounds tested here, the identification of inhibitors with high selectivity
for trypanosomatid M32 MCPs over ACE can be achieved, a point in favor to the specific
druggability of these enzymes. The identified inhibitors display high structural diversity, with many showing only mar-
ginal similarity to the other hits, hence representing different structural clusters and presum-
ably, different inhibitory scaffolds. Discussion As expected, given the
error-prone nature of the single-well (single dose, single replicate) measurements used in pri-
mary screening, significant discrepancies in inhibition were observed for some compounds in 10 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560
July 22, 2019 Synthetic inhibitors for M32 metallocarboxypeptidases Synthetic inhibitors for M32 metallocarboxypeptidases
glected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560
July 22, 2019
11 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560
July 22, 2019 11 / 20 https://doi.org/10.1371/journal.pntd.0007560.g005 Synthetic inhibitors for M32 metallocarboxypeptidases presumptive explanation can be put forward. As in the case of many other metallopeptidase
inhibitors, it is likely that inhibition of trypanosomatid M32 MCPs occurs throughout the per-
turbation of the coordination sphere of the catalytic metal ion (presumably Zn2+ in the case of
TcMCP-1 and TbMCP-1, by extension from other M32 enzymes [31]). Typically, synthetic
metallopeptidase inhibitors achieve preliminary affinity and target selectivity through the for-
mation of stabilizing interactions with specific residues within the active site; while a ZBG is
responsible for metal chelation, enhancing binding affinity, modulating selectivity and dis-
rupting catalytic activity [32]. For the majority of the inhibitors presented here, it was possible
to identify typical ZBG or at least, heteroatom-containing groups able to establish a coordi-
native bond with a Zn2+ ion (Fig D in S1 Text). For those compounds, an inhibition mecha-
nism like the one described above is possible. For other molecules not having a Zn-
coordinating group, the most plausible explanation is that inhibition occurs as a result of the
prevention of substrate binding by the partial occupancy or the deformation of the catalytic
cleft by the inhibitor molecule, as previously observed for Non-Zinc-Binding inhibitors of
other metallopeptidases [33]. The vast majority of the hits identified here inhibit one or both MCPs in the micromolar
range, with only a few of them showing potencies <10 μM. Outstandingly, TCMDC-143620
inhibits TcMCP-1 in the sub-micromolar range (it also inhibits TbMCP-1, but with potency
~7-fold lower). This is the most potent inhibitor described so far for an enzyme of the M32
family and seems a promising candidate for further structure-based optimization. The unusu-
ally high flexibility of the M32 MCPs around the active site [31, 34] prevented us to use a dock-
ing approach to get insights of the binding mode of this compound within TcMCP-1 and
TbMCP-1 catalytic clefts. However, the TCMDC-143620 molecule seems able to form a variety
of stabilizing interactions. These may include hydrophobic and electrostatic interactions,
hydrogen bonding and the coordination to the metal ion through the pyridine ring. In addi-
tion, the presence of a central sulfonamide group and a distal nitrile group add further interac-
tion possibilities to this molecule. For example, the sulfonamide group has been extensively
incorporated into metallopeptidase inhibitors due to its ability to improve the enzyme-inhibi-
tor binding by different mechanisms. In this regard, the presence of “unpaired” hits is not
surprising, considering that no more than two members of the same structural cluster were
included per box during collection assembly [16] and that “twin” compounds might well not
pass the activity or auto-fluorescence filters included in this work. Among the identified inhib-
itors, only TCMDC-143265 and TCMDC-143551 share similar core structures, thus probably
populating the same cluster and sharing a common active scaffold. A significant part of both
molecules is identical and adopts the same spatial conformation (Fig G in S1 Text), with the
largest differences located around the benzamide ring. Besides the obvious differences in the
length and position of sulfonamide substituents, the chlorine substitution in position 2
imposes a ~90˚ rotation of the benzamide ring in TCMDC-143265 compared to TCMDC-
143551, where all ring systems are almost coplanar. Interestingly, these structural differences
seem to dictate the selectivity toward TcMCP-1, as TCMDC-143551 inhibits both enzymes
whereas TCMDC-143265 is specific for TbMCP-1. Even for this pair of compounds, there is
no evident substructure responsible for M32 MCPs bioactivity; though this is probably a biased
observation due to the lack of well-defined structural features for M32 MCPs inhibitors. Although the crystallographic structure of TcMCP-1 has been determined [8] and subsite
specificity have been explored for both enzymes using FRET substrate libraries [12] and muta-
genesis [6, 8], little is yet known about how substrates are accommodated into the catalytic
groove, which residues are key determinants of subsite specificity and the significance of the
hinge-type movement between L and R domains in the stabilization of enzyme-substrate or
enzyme-inhibitor complexes. With all these gaps to fill, it seems risky to speculate about the
modes of interaction of these new inhibitors with TcMCP-1 and TbMCP-1. However, a 12 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560
July 22, 2019 Enzymes TcMCP-1 (MEROPS ID: M32.003) and TbMCP-1 were expressed as GST fusion proteins in E. coli BL21 (DE3) Codon Plus and purified as previously described [6, 8]. Reagents Triton X-100, MOPS (3-(N-morpholino)propanesulfonic acid), DMSO, EDTA and captopril
were purchased from Sigma-Aldrich. Substrates Abz-RFFK(Dnp)-OH and Abz-LKFK(Dnp)-
OH were from GenScript (Piscataway, NJ, USA). Black solid bottom polystyrene Corning NBS
384-well plates were from Sigma-Aldrich (CLS3654-100EA). Anti-kinetoplastid chemical boxes The HAT and CHAGAS chemical boxes [16] were provided by GlaxoSmithKline. The collec-
tion comprised 404 compounds, prepared as 10 mM stock solutions in DMSO (10 μL each) and
dispensed in 96 well plates. For primary screening, a working solution (final concentration of 2
mM) for each compound was prepared by 1/5 dilution in DMSO while 1 μL of the 10 mM stock
solution was used for secondary screening of selected compounds, as previously described [24]. The final concentration of compounds tested in primary screening was 25 μM, while the com-
pound concentrations assayed in secondary screening ranged from 7,5 pM to 62,5 μM. Synthetic inhibitors for M32 metallocarboxypeptidases its reported anti-T. cruzi activity. This might also explain, at least partially, the moderate cyto-
toxic and anti-T. brucei and L. donovani activities reported for this compound, considering the
global similarities of enzymes within CYP51 family [40, 41]. Although involved in other stud-
ies as part of the GSK CHAGAS Box [42], no further information is currently available from
the evaluation of TCMDC-143620 against other molecular targets, except for our previous cru-
zipain study [24] where it was found to be inactive (~7,5% of cruzipain inhibition at 25 μM). A
complete profile of the off-target activity of TCMDC-143620 would be critical for future opti-
mization efforts in order to achieve a suitable M32 MCPs probe from this compound. In summary, 30 micromolar-range inhibitors, presenting both high structural diversity and
novelty, have been discovered for TcMCP-1 and/or TbMCP-1 by using continuous, fluores-
cent-based and HTS-capable enzymatic assays. The best hit shows sub-micromolar affinity for
TcMCP-1, inhibits TbMCP-1 in the low micromolar range and, like other potent hits, is inac-
tive on ACE. Considering its potency and specificity, this molecule seems to be a promissory
starting point to develop more specific and potent tools to expand our understanding of the
biochemistry and biological role(s) of M32 MCPs from trypanosomatid parasites and, hope-
fully, to assess in a near future their value as drug targets. These mechanisms include: i) direct formation of hydro-
gen bonds to the enzyme backbone, ii) properly redirection of bulky groups into enzyme
pockets by inducing a twist in the structure of the inhibitor molecule and iii) even cooperate
with other chelating groups in the coordination of the catalytic metal ion [35]. Similarly, the
nitrile group in TCMDC-143620 can establish polar interactions, hydrogen bonds or react
with serine or cysteine side chains to form covalent adducts which would greatly stabilize
inhibitor binding [36]. Interestingly, the nitrile group is also able to form coordinative bonds
with a variety of metal ions including Co2+, Mn2+, Fe3+, Cu2+ and Zn2+ [37]. Thus, a possible
role of this group in the direct coordination of the catalytic metal ion cannot be discarded at
present. The determination of the crystallographic structure of TcMCP-1 or TbMCP-1 in com-
plex with TCMDC-143620 would provide a definitive answer to these questions as well as
important clues to undertake the future lead-optimization of this hit. A preliminary analysis of the bioactivity profile of TCMDC-143620 (https://pubchem.ncbi. nlm.nih.gov/compound/91800813) indicates that it shows potent activity against T. cruzi in
culture and only moderate but measurable activity on T. brucei and L. donovani. Also, this
compound exhibits moderate cytotoxicity on mammalian cell NIH 3T3 (IC50 = 13 μM) but
resulted inactive on HepG2 (IC50 > 100 μM). Considering target-specific assays; this com-
pound has a single bioactivity report. TCMDC-143620 was found to be a potent inhibitor
(IC50 = 79 nM) of T. cruzi sterol 14-α demethylase (CYP51) enzyme, which is involved in the
ergosterol biosynthesis pathway and was considered until recent years as a promissory thera-
peutic target for Chagas disease [38, 39]. The inhibition of this target is probably the cause of 13 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560
July 22, 2019 Synthetic inhibitors for M32 metallocarboxypeptidases (iii) display linear kinetics for enough time to perform several reading cycles (at least 8 cycles,
minimum time between cycles: 264 sec) through the 384-wells. In all cases, EDTA (final con-
centration 31,25 mM) was used as positive inhibition control. Primary screening To perform the primary screening, 1 μL of each compound (2 mM in DMSO, final concentra-
tion in the assay: 25 μM), EDTA (500 mM, final concentration in the assay: 31,25 mM) were
dispensed into 384-well Corning black solid-bottom assay plates. Then, 40 μL of 100 mM
MOPS, 0,01% Triton X-100 pH 7,2 containing TbMCP-1 (2,50 nM) or TcMCP-1 (0,34 nM)
were added to each well, plates were homogenized (30 seg, orbital, medium intensity) and
each well subjected to a single autofluorescence read (ex/em = 320/420 nm). Plates were incu-
bated in darkness for 15 min at 30 ˚C and then 40 μL of Abz-RFFK(Dnp)-OH (4 μM) or Abz-
LKFK(Dnp)-OH (0,8 μM) in assay buffer were added to each well to start the reaction. After
homogenization (30 seg, orbital, medium intensity), the fluorescence of the Abz group (ortho-
aminobenzoic acid) (ex/em = 320/420 nm) was acquired kinetically for each well (8 read
cycles, one cycle every 300 seconds). Considering our previous experiences, the auto-fluores-
cent cut-off was arbitrarily set at 2x105 RFU to discard highly interfering compounds. All com-
pounds were assayed in singlet (without replicates) due to the limited availability of stocks. Raw screening measurements were used to determine the slope (dF/dt) of progression
curves by linear regression for control and non-interfering compound wells. In the case of
control-dependent hit selection criteria, percent inhibition percentage (%Inh) was calculated
for each compound according to the following equation: Inh ¼ 100
1 dF
dt
WELL mC
ðmCþ mC Þ
2
4
3
5
ð1Þ ð1Þ where dF/dtWELL represents the slope of each compound well and μC+ and μC−the average of
MCP (no-inhibition) and substrate (no-enzyme) controls, respectively. where dF/dtWELL represents the slope of each compound well and μC+ and μC−the average of
MCP (no-inhibition) and substrate (no-enzyme) controls, respectively. MCPs assays TbMCP-1 and TcMCP-1 activities were assayed fluorometrically with Abz-RFFK(Dnp)-OH
and Abz-LKFK(Dnp)-OH substrates, respectively, in 100 mM MOPS pH 7,2 containing 0,01%
Triton X-100. Assays were performed in solid black 384-well plates (final reaction volume ~80
μL) and the hydrolysis of the K(Dnp)-OH group was monitored continuously at 30 ˚C with a
Beckman Coulter DTX 880 Multimode Reader (Radnor, Pennsylvania, USA) using standard
320 nm excitation and 420 nm emission filter set. For each MCP, final substrate concentration was set to a value KM /[S] ~ 1. Optimal enzyme
concentration was selected from 2-fold serial dilutions to match three criteria: (i) being linearly
proportional to V0, (ii) display robust signal evolution at substrate concentration chosen and PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560
July 22, 2019 14 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560
July 22, 2019 Synthetic inhibitors for M32 metallocarboxypeptidases Table 4. Molecular features used to build compound clustering dendrogram. NAME
MW
HBD
HBA
RB
RINGS
XLOGP
TCMDC-143071
416,3
1
3
6
4
3,18
TCMDC-143143
242,2
1
3
2
2
4,36
TCMDC-143158
443,3
1
3
6
6
1,46
TCMDC-143172
299,1
1
2
3
3
4,6
TCMDC-143187
463,8
3
4
7
5
3,67
TCMDC-143191
354,3
0
3
3
5
3,04
TCMDC-143209
378,2
2
4
8
3
3,44
TCMDC-143242
374,3
1
2
6
5
2,78
TCMDC-143254
415,3
3
3
7
4
3,99
TCMDC-143263
302,2
2
3
6
3
2,71
TCMDC-143265
407,8
1
5
6
3
2,53
TCMDC-143323
386,3
3
5
8
4
-0,04
TCMDC-143332
430,3
1
4
6
4
2,37
TCMDC-143382
378,3
3
4
5
4
2,08
TCMDC-143385
443,3
1
4
7
4
2,68
TCMDC-143408
340,3
1
4
5
4
1,81
TCMDC-143409
375,1
0
3
3
4
2,5
TCMDC-143422
279,2
0
1
2
4
3,31
TCMDC-143432
350,3
0
1
6
3
3,74
TCMDC-143454
259,6
2
2
3
3
3,38
TCMDC-143456
318,2
1
4
6
3
1,58
TCMDC-143462
434,3
2
5
7
4
3,11
TCMDC-143496
464,4
1
4
8
4
2,9
TCMDC-143513
461,8
2
4
8
5
2,4
TCMDC-143515
459,8
2
2
7
5
4,55
TCMDC-143543
460,4
0
2
6
6
4,23
TCMDC-143551
436,4
1
5
8
4
2,3
TCMDC-143592
351,7
1
1
6
3
3,9
TCMDC-143620
396,3
0
4
5
4
2,65
TCMDC-143645
363,3
3
1
7
3
4,32
MW: Molecular Weight; HBD: Hydrogen Bond donor; HBA: Hydrogen Bond Acceptor; RB: Rotable Bonds; RINGS: Number of Rings; XLOGP: XlogP (Partition Table 4. Molecular features used to build compound clustering dendrogram. MW: Molecular Weight; HBD: Hydrogen Bond donor; HBA: Hydrogen Bond Acceptor; RB: Rotable Bonds; RINGS: Number of Rings; XLOGP: XlogP (Partition
coefficient score) ogen Bond donor; HBA: Hydrogen Bond Acceptor; RB: Rotable Bonds; RINGS: Number of Rings; XLOGP: XlogP (Partition MW: Molecular Weight; HBD: Hydrogen Bond donor; HBA: Hydrogen Bond Acceptor; RB: Rotable Bonds; RINGS: Numb
coefficient score). where dF/dtWELL represents the slope of each compound well and μC+ and μC−the average of
MCP (no-inhibition) and substrate (no-enzyme) controls, respectively. The IC50 and Hill
slope parameters for each compound were estimated by fitting the four-parameter Hill equa-
tion to experimental data from dose-response curves using the GraphPad Prism program (ver-
sion 5.03). Secondary assay Compounds selected from primary screening were re-tested in a dose-response manner (final
concentration ranging from 7,5 to 62,5 μM) using identical assay conditions. To avoid any
positional and/or association bias, we randomly defined the row position for each compound. One μL of compounds stock (10 mM in DMSO) and EDTA (31,25 mM) were added to the
first well of column 1, followed by addition of 40 μL of 100 mM MOPS, 0,01% Triton X-100
pH 7,2 buffer. After addition of 20 μL of the same buffer to subsequent wells of the plate, 22
serial 2-fold dilutions were made horizontally. The last two positions of every row were used,
alternatively, for C+ and C−controls to reduce any positional and/or association bias. Then,
20 μL of activity buffer containing TbMCP-1 or TcMCP-1 were added to each well, except for
those corresponding to C−; completed with 20 μL of activity buffer. After homogenization, 15
minutes of incubation at 30˚C and autofluorescence measurement, the substrate (in activity
buffer) was added to the previous mix. Data collection and processing were performed exactly
as described above. Percentage of M32 MCPs residual activity was calculated for each condi-
tion according to the following equation: %Res:ActMCP ¼ 100
dF
dt
WELL mC
ðmCþ mC Þ
2
4
3
5
ð2Þ ð2Þ 15 / 20 PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560
July 22, 2019 https://doi.org/10.1371/journal.pntd.0007560.t004 Supporting information S1 Text. Supplementary information. (PDF) Compound clustering Three separate compound clustering routines were used. One of them derived from calculated
or predicted molecular features, and the other two directly inferred from different distance
metrics between compounds: one using Tanimoto similarity and another one using the over-
lap score calculated in a MCS (Maximum Common Subgraph) pipeline. The Tanimoto dis-
tance compound clustering was performed to rapidly find compound pairs, if available, within
the leads. OpenBabel 2.4.1 [43] was used to export molecule MDLs from SMILES format,
available from GSK chembox summary. For Tanimoto clustering, the indexes were calculated using ChemFP 1.3 [44] with ob2fps
bindings and simsearch -NxN as parameter. ChemFP results were parsed and analyzed using
an ad hoc perl script, setting the distance (D) between compounds as D = 1—Tindex. The dis-
tance matrix was built using melt and acast from R Data table package [45]. To assess the MCS clustering, all compounds were imported into a R script using Chemmi-
ner [46] and further analyzed using fmcsR [47] for batch MCS calculations. For the molecular feature clustering, a perl script was built to run XlogP3 v3.2.2 [48]
through all lead compounds. Features used to build distance matrix, along with their corre-
sponding values, can be found in Table 4. All clustering plots were achieved using the R base
hierarchical clustering tool, hclust. Synthetic inhibitors for M32 metallocarboxypeptidases Acknowledgments ESS, GTN, JJC, VEA and FA are members of the research career of the National Research
Council (CONICET, Argentina), and LUL has a doctoral fellowship from CONICET. Zinc-binding group assessment among lead compounds To find ZBGs among lead compounds, a curated database of such chemotypes was first created
(Table B in S1 Text). Structures were drawn using Marvin Sketcher (Chemaxon) and exported
to SMILES format. This database was then imported to R and processed similarly to the MCS
clustering, though instead of calculating overlapping scores between compounds, the overlap-
ping score was determined for each compound against all ZBGs in the database. Only those
compound-ZBG pairs where overlap was complete (score = 1 and, hence, ZBG completely
contained in the lead compound) were counted as a match. ACE assay Purified rabbit lung ACE (EC 3.4.15.1) was purchased from Sigma-Aldrich. Enzyme activity
was assayed fluorimetrically with Abz-FRK(Dnp)P-OH (ex/em = 320/420 nm) as substrate in
buffer 0,1 M Tris-HCl, 50 mM NaCl, 10 mM ZnCl2, pH 7.0 containing 0,01% Triton X-100 as
indicated in [23]. Selected compounds were tested in a dose-response manner (final concen-
tration ranging from 7,5 pM to 62,5 μM) using identical assay conditions employed with both
MCPs. Captopril (15 pM—125 μM) was used as inhibition control. PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007560
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21363968; PubMed Central PMCID: PMC3106324. 12. Frasch AP, Bouvier LA, Oppenheimer FM, Juliano MA, Juliano L, Carmona AK, et al. Synthetic inhibitors for M32 metallocarboxypeptidases Resources: Adriana K. Carmona, Juan Jose´ Cazzulo. Software: Lionel Ura´n Landaburu. Supervision: Emir Salas-Sarduy, Juan Jose´ Cazzulo, Ferna´n Agu¨ero, Vanina E. Alvarez. Validation: Emir Salas-Sarduy, Gabriela T. Niemirowicz. Visualization: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Gabriela T. Niemirowicz. Writing – original draft: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Ferna´n Agu¨ero, Vanina
E. Alvarez, Gabriela T. Niemirowicz. Writing – review & editing: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Adriana K. Car-
mona, Juan Jose´ Cazzulo, Ferna´n Agu¨ero, Vanina E. Alvarez, Gabriela T. Niemirowicz. Supervision: Emir Salas-Sarduy, Juan Jose´ Cazzulo, Ferna´n Agu¨ero, Vanina E. Alvarez. Validation: Emir Salas-Sarduy, Gabriela T. Niemirowicz. Visualization: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Gabriela T. Niemirowicz. Writing – original draft: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Ferna´n Agu¨ero, Vanina
E. Alvarez, Gabriela T. Niemirowicz. Writing – original draft: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Ferna´n Agu¨ero, Vanina
E. Alvarez, Gabriela T. Niemirowicz. Writing – review & editing: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Adriana K. Car-
mona, Juan Jose´ Cazzulo, Ferna´n Agu¨ero, Vanina E. Alvarez, Gabriela T. Niemirowicz. Author Contributions Conceptualization: Emir Salas-Sarduy, Lionel Ura´n Landaburu. Data curation: Emir Salas-Sarduy, Lionel Ura´n Landaburu. Formal analysis: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Gabriela T. Niemirowicz. Formal analysis: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Gabriela T. Niemirowicz. unding acquisition: Ferna´n Agu¨ero, Vanina E. Alvarez, Gabriela T. Niemirowicz. Funding acquisition: Ferna´n Agu¨ero, Vanina E. Alvarez, Gabriela T. Niemirowicz. Investigation: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Ferna´n Agu¨ero, Vanina E. Alvarez,
Gabriela T. Niemirowicz. Investigation: Emir Salas-Sarduy, Lionel Ura´n Landaburu, Ferna´n Agu¨ero, Vanina E. Alvarez,
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PyRhO: a virtual optogenetics laboratory
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Acknowledgements
Thi
k g
This work was supported by the UK BBSRC grant BB/L018268/1. From a minimal set of photocurrent data, PyRhO will
fit and parameterize the Three [1], Four [1] and Six-state
[2] rhodopsin models to capture the underlying biophysi-
cal photocycle which defines their kinetics. These models
are then used to accurately compute the photocurrents
across a range of flux, voltage and other experimental
conditions for the given rhodopsin. After selecting a sui-
table model based on the desired balance between simu-
lation accuracy and speed, the artificial rhodopsin can be
seamlessly inserted into software such as NEURON and
Brian for use in simulations from the cellular to the net-
work level. We demonstrate the use of PyRhO in fitting
models to channelrhodopsin-2 (ChR2) [3] data and pre-
sent results for typical illumination strategies and experi-
mental protocols designed to tease apart the effects of
key model parameters. Published: 18 December 2015 Published: 18 December 2015 Evans et al. BMC Neuroscience 2015, 16(Suppl 1):P178
http://www.biomedcentral.com/1471-2202/16/S1/P178 Open Access PyRhO: a virtual optogenetics laboratory
Benjamin D Evans1*, Sarah Jarvis2, Simon R Schultz2, Konstantin Nikolic1
From 24th Annual Computational Neuroscience Meeting: CNS*2015
Prague, Czech Republic. 18-23 July 2015 PyRhO: a virtual optogenetics laboratory
Benjamin D Evans1*, Sarah Jarvis2, Simon R Schultz2, Konstantin Nikolic1
From 24th Annual Computational Neuroscience Meeting: CNS*2015
Prague, Czech Republic. 18-23 July 2015 open-source modules and software. An accompanying
GUI running in IPython [4] has also been developed to
facilitate more interactive exploration of the models for
both experimental and didactic purposes. Furthermore,
IPython has been identified as a particularly promising
medium for sharing models and reproducing results in
computational neuroscience [5]. Simulations based on
these virtual opsins will enable neuroscientists to gain
insight into their behaviour and rapidly identify the most
suitable variant for application in a particular biological
system, not only guiding choice, but potentially also rho-
dopsin development. In this way, we expect PyRhO will
help to significantly improve the effectiveness of optoge-
netics as a tool for transforming biological sciences. Optogenetics has become a key tool for understanding
the function of neural circuits and controlling their beha-
viour. An array of opsins have been genetically isolated
from several families of organism, including algae and
bacteria, with a wide range of temporal and spectral
properties. In an effort to develop more effective and tai-
lored opsins, hybrids and genetic mutants are continually
being created. Experimentally characterizing these new
variants is a lengthy process requiring substantial effort
before they can be harnessed to address questions in neu-
roscience. Experimentally testing each combination of
opsin and target cell type of interest is practically impos-
sible, effectively limiting the use of optogenetics as a tool. To aid in this effort we propose PyRhO; an integrated
suite of open-source, multi-scale computational tools to
characterize rhodopsins, then rapidly develop and con-
duct virtual experiments with them in silico. © 2015 Evans et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited. The 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: benjamin.evans@imperial.ac.uk
1Institute of Biomedical Engineering, Department of Electrical & Electronic
Engineering, Imperial College London, London, UK
Full list of author information is available at the end of the article Authors’ details
1I
i
f Bi 1Institute of Biomedical Engineering, Department of Electrical & Electronic
Engineering, Imperial College London, London, UK. 2Department of
Bioengineering, Electrical Engineering, Imperial College London, London, UK. References 1. Nikolic K, Grossman N, Grubb MS, Burrone J, Toumazou C, Degenaar P:
Photocycles of Channelrhodopsin-2. Photochemistry and Photobiology
2009, 85:400-411. 2. Grossman N, Simiaki V, Martinet C, Toumazou C, Schultz SR, Nikolic K: The
spatial pattern of light determines the kinetics and modulates
backpropagation of optogenetic action potentials. Journal of
Computational Neuroscience 2013, 34(3):477-488. 3. Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K: Millisecond-
timescale, genetically targeted optical control of neural activity. Nature
Neuroscience 2005, 8(9):1263-1268. 4. Perez F, Granger BE: IPython: a System for Interactive Scientific
Computing. Computing in Science and Engineering 2007, 9(3):21-29. The tools are written in Python for easy scripting of
experiments and compatibility with a large array of p
g
p
g
g
g
5. Topalidou M, Leblois A, Boraud T, Rougier NP: A Long Journey into
Reproducible Computational Neuroscience. Frontiers in Computational
Neuroscience 2015, 9(30):1-2. doi:10.1186/1471-2202-16-S1-P178
Cite this article as: Evans et al.: PyRhO: a virtual optogenetics
laboratory. BMC Neuroscience 2015 16(Suppl 1):P178. doi:10.1186/1471-2202-16-S1-P178
Cite this article as: Evans et al.: PyRhO: a virtual optogenetics
laboratory. BMC Neuroscience 2015 16(Suppl 1):P178. © 2015 Evans et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://
creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited. The 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.
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Author Correction: The lengths of trachea and main bronchus in Chinese Shanghai population
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www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Author Correction: The lengths
of trachea and main bronchus
in Chinese Shanghai population
OPEN Xiahui Ge, Haidong Huang, Chong Bai, Xuejun Guo, Christoforos Kosmidis,
Konstantinos Sapalidis, Sofia Baka, Kosmas Tsakiridis, Wolfgang Hohenforst‑Schmidt,
Lutz Freitag, Anastasios Vagionas, Konstantinos Drevelegas & Paul Zarogoulidis Xiahui Ge, Haidong Huang, Chong Bai, Xuejun Guo, Christoforos Kosmidis,
Konstantinos Sapalidis, Sofia Baka, Kosmas Tsakiridis, Wolfgang Hohenforst‑Schmidt,
Lutz Freitag, Anastasios Vagionas, Konstantinos Drevelegas & Paul Zarogoulidis Correction to: Scientific Reports https://doi.org/10.1038/s41598-021-81744-0, published online 26 January 2021 The original version of this Article contained errors in the Material and Methods section under subheading
‘Study population’, “Therefore, 153 adult patients were eligible for evaluation in the study, including 71 patients with unilateral mild
pneumonia, 60 patients with pulmonary small nodule (major diameter ≤ 1 cm) and 21 patients with no lesion
in chest CT scan but chronic cough or occasional bloody sputum.” now reads: “Therefore, 153 adult patients were eligible for evaluation in the study, including 72 patients with unilateral mild
pneumonia, 60 patients with pulmonary small nodule (major diameter ≤ 1 cm) and 21 patients with no lesion
in chest CT scan but chronic cough or occasional bloody sputum.” “Therefore, 153 adult patients were eligible for evaluation in the study, including 72 patients with unilateral mild
pneumonia, 60 patients with pulmonary small nodule (major diameter ≤ 1 cm) and 21 patients with no lesion
in chest CT scan but chronic cough or occasional bloody sputum.” Furthermore, in the Material and Methods section under subheading ‘Measurements.’, urthermore, in the Material and Methods section under subheading ‘Measurements.’, “Similarly, the length of right main bronchi was to calculate the distance from left carina 2 to the carina and the
length of left main bronchi was to measure the distance from right carina 1 to the carina.” “Similarly, the length of right main bronchi was to calculate the distance from left carina 2 to the carina and the
length of left main bronchi was to measure the distance from right carina 1 to the carina.” “Similarly, the length of right main bronchi was to calculate the distance from right carina 1 to the carina and
the length of left main bronchi was to measure the distance from left carina 2 to the carina.” “Similarly, the length of right main bronchi was to calculate the distance from right carina 1 to the carina and
the length of left main bronchi was to measure the distance from left carina 2 to the carina.” “However, height between men and women was significantly different either.”
now reads: “However, height between men and women was significantly different.” The original Article has been corrected. | https://doi.org/10.1038/s41598-021-98467-x Scientific Reports | (2021) 11:19917 re.com/scientificreports/
Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons 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. now reads: 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/. © Th A h
( ) 2021 www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons 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 Author(s) 2021 https://doi.org/10.1038/s41598-021-98467-x Scientific Reports | (2021) 11:19917 |
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Implementing the World Health Organization Pandemic Influenza Severity Assessment framework—Singapore's experience
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Influenza and other respiratory viruses
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O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E Received: 1 April 2019 | Revised: 12 August 2019 | Accepted: 4 September 2019 Received: 1 April 2019 | Revised: 12 August 2019 | Accepted: 4 September 2019 Received: 1 April 2019 | Revised: 12 August 2019 | Accepted: 4 September 2019
DOI: 10.1111/irv.12680 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited. Correspondence Methods: We assessed the severity of influenza by monitoring indicators of influenza
transmissibility, seriousness of disease and impact on healthcare resource utilisation. Indicators were described by various parameters collected weekly from eight gov-
ernment hospitals, 20 government and 30 private primary care clinics, and the na-
tional public health laboratory. Transmissibility and seriousness of disease indicators
were each represented by multiple parameters, and alert thresholds were set at the
70th and 90th percentile of a parameter's past 2‐year surveillance data. We derived a
collective measure for each indicator using the average percentile rank of the related
parameters. Alert thresholds for the single impact parameter were set at predefined
values and evaluated for its sensitivity, specificity and positive predictive value. Results: For the transmissibility and seriousness of disease parameters, calculation of
the percentile rank was simple and independent of a parameter's underlying distribu-
tion. For the impact parameter, predefined alert thresholds had high sensitivity and
specificity (>80%) but low positive predictive value (15%‐30%). Assessment scales
were used to qualitatively classify the activity of an indicator as low, moderate or
high together with a confidence level. Conclusion: We applied different methods for threshold setting depending on the
attributes of each parameter and indicator. For indicators represented by multiple
parameters, an aggregated assessment of the indicator's level of activity and confi-
dence level of the assessment was needed for effective reporting. | 3
wileyonlinelibrary.com/journal/irv This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2019 The Authors Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd. Implementing the World Health Organization Pandemic
Influenza Severity Assessment framework—Singapore's
experience Rachael Pung
| Vernon Jian Ming Lee Communicable Diseases Division, Ministry
of Health, Singapore, Singapore Communicable Diseases Division, Ministry
of Health, Singapore, Singapore Abstract
Background: We report our experience in evaluating the severity of local influ-
enza epidemics using the World Health Organization Pandemic Influenza Severity
Assessment framework. Correspondence
Vernon Jian Ming Lee, Communicable
Diseases Division, Ministry of Health,
College of Medicine Building, 16 College
Road, Singapore 169854, Singapore. Email: Vernon_Lee@moh.gov.sg K E Y W O R D S This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited. © 2019 The Authors Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd. | 3
wileyonlinelibrary.com/journal/irv | 3
wileyonlinelibrary.com/journal/irv Influenza Other Respi Viruses. 2020;14:3–10. 4 |
1 | BACKGROUND PUNG and LEE recommended indicators for monitoring severity were the transmis-
sibility of the influenza virus, the seriousness of the disease and the
impact of influenza on healthcare resource utilisation (referred to
as transmissibility, seriousness of disease and impact, in the subse-
quent sections). By assessing severity from multiple dimensions, this
encourages countries to establish surveillance at different levels of
the healthcare system to create a holistic picture of an influenza ep-
idemic or pandemic. Early severity assessment of pandemic influenza is helpful for guid-
ing pandemic response actions. However, during the 2009 H1N1
pandemic, severity assessment was not standardised across coun-
tries, making it difficult to evaluate the local or global situation as
the pandemic evolved.1 The lack of a consistent measure of severity
also posed a challenge to calibrate pandemic response, which is de-
pendent on geographical spread, clinical severity and public interest,
among other factors.1 Using virological and surveillance data from different sources,
the severity of each indicator can be represented by more than one
type of data, or parameter. The choice of parameters may vary across
countries due to different data availability, of which some require
substantial resource to collect. While the challenge of data com-
parison remains, PISA plays an essential role—to promote enhanced Through the lessons learnt from the 2009 H1N1 pandemic, the
World Health Organization (WHO) has developed a framework for
pandemic influenza severity assessment (PISA).2 PISA is a struc-
tured way of tracking influenza epidemics or pandemics. The three TA B LE 1 Parameters considered for assessing severity of influenza TA B LE 1 Parameters considered for assessing severity of influenza TA B LE 1 Parameters considered for assessing severity of influenza
Indicator
Singapore parameters
considered
Data source
WHO recommended parameters
Transmissibility
How many people in a population
get sick from influenza on a weekly
basis
Average daily attendance
for ARI
20 government primary care
clinics
Weekly ILI or MAARI cases as a propor-
tion of total visits or incidence rates. Weekly percentage of respiratory
pathogen samples testing positive for
influenza. Abbreviations: ARI: acute respiratory infection; ED: emergency department of a government hospital; ICU: intensive care unit of a government hospi-
tal; ILI: influenza‐like illness; MAARI: medically attended acute respiratory illness; SARI: severe acute respiratory infection. 8 government acute hospitals SARI/ARI or ILI ratio
Cumulative death: hospitalisation ratio
(ideally for confirmed influenza)
Cumulative ICU: hospitalisation ratio (ide-
ally for confirmed influenza) Weekly or monthly number or proportion
of SARI cases with percentage flu‐posi-
tive among SARI cases
Weekly excess pneumonia & influenza
(P&I) or all‐cause mortality stratified by
age.
Weekly number of confirmed influenza
cases admitted to ICU, or weekly number
of confirmed influenza cases admitted
to hospital. 1.1 | Influenza surveillance in Singapore Singapore, a city‐state in South East Asia, is a major global travel hub
with over 18 million tourist arrivals3 and a population of over 5.6
million in 2018.4 It has a high population density of over 8000 peo-
ple per square kilometre, which may facilitate the spread of contact
transmissible and airborne diseases such as influenza. Locally, influenza A (H1N1) pdm09, A (H3N2) and influenza B vi-
ruses circulate year‐round. Following the 2009 influenza pandemic,
we expanded our influenza surveillance network and encouraged
government and private primary care clinics to participate in the
National Influenza Surveillance Programme. To date, 20 government
primary care clinics, providing about 20% of primary healthcare ser-
vices in the population,5 and 30 sentinel clinics spread across the
country out of 1400 private primary care clinics are enrolled in the
programme. Our influenza surveillance network also comprises eight
acute government hospitals, providing about 80% of all acute care
hospital services in the population5 and the National Public Health
Laboratory (NPHL). A time series plot of each parameter was used to illustrate the
parameter's variability during each seasonal epidemic and surveil-
lance artefacts arising from reporting changes. These two factors
were considered in the final selection of parameters used for PISA
reporting. In this paper, we document Singapore's experience in developing
and evaluating the PISA indicators and parameters, and this would
provide other countries with suggestions that they can use in devel-
oping their own indicators. 2.1 | Data sources For a parameter, we calculated the percentile rank or the percen-
tile of an observed value with respect to the previous 2‐year histori-
cal data (eg the percentage of data from January 2016 to December
2017 that were equal or lower than a weekly parameter data col-
lected in 2018). We limited the comparison to 2‐year historical data
due to recent changes in data extraction methods. Let xw denote the
observed value of a parameter and pw denote the percentile of that
observed value in week w of a year. Also, let h denote the historical
data in the previous 2 years. A wide range of parameters were reported weekly to the Ministry of
Health (MOH) and considered for PISA (Table 1). Influenza transmis-
sion in the community was monitored using the average daily at-
tendance for acute respiratory infection (ARI) and the average daily
attendance for influenza‐like illness (ILI) at the government primary
care clinics. An ARI diagnosis was made when a case had at least
one acute respiratory symptom such as cough, sore throat and co-
ryza, while an ILI diagnosis was made when a case had a fever of
≥38.0°C and cough, with onset within the last 10 days. The average
daily attendance for ARI and average daily attendance for ILI at the
government primary care clinics were used, instead of the weekly
attendances, to offset the effect of public holidays and clinic closure
on weekends. pw
⎧
⎪
⎪
⎨
⎪
⎪⎩
= 0 if xw <min (h)
= CF+0.5f
n
=100 if xw >max (h) Consent was sought for the collection of respiratory sam-
ples from all patients if they received outpatient consultation at
a government or private primary care clinics that are enrolled in
the National Influenza Surveillance Programme and presented
with ILI. These samples were routinely submitted to the NPHL
and tested using the FilmArray Respiratory Panel and/or real‐time
reverse transcription‐polymerase chain reaction (RT‐PCR) to de-
tect respiratory viruses. The weekly number of samples was small
as not all identified patients participated in the surveillance, and where CF is the number of values in h that is below xw (ie cumulative
frequency). f is the number of values in h that is equal to xw (ie fre-
quency). n is the number of values in h. surveillance and increase information sharing among public health
officials during an influenza epidemic or pandemic. hence, we pooled the results across four weeks and monitored
the proportion of respiratory samples positive for influenza over
a 4‐weekly moving interval. As not all ILI attendances at the government primary care clin-
ics were attributed to influenza, we explored using the product of
the average daily attendance for ILI and weekly proportion of respi-
ratory samples positive for influenza to estimate the average daily
number of influenza‐positive ILI cases at the government primary
care clinics. We also collect parameters from the eight acute govern-
ment hospitals comprising of the weekly number of ARI Emergency
Department (ED) attendances and admissions, the weekly number of
pneumonia ED attendances and admissions, and the weekly number
of laboratory‐confirmed influenza cases admitted to the intensive
care unit (ICU) or died. The former two parameters were collected
through MOH’s healthcare utilisation database while the latter was
compiled by a team of healthcare professionals in each hospital and
forwarded to MOH. 2.2 | Assessing the transmissibility and
seriousness of disease indicators’ level of activity As the transmissibility and seriousness of disease indicators were
represented by more than one parameter, an overall measure of each
indicator's level of activity and the confidence of the indicator was
necessary for weekly reporting. 4 |
1 | BACKGROUND Composite (product) of weekly ILI or
MAARI and weekly percentage positivity
rates for influenza
Average daily attendance
for ILI
Proportion of respira-
tory samples positive for
influenza over a 4‐weekly
moving interval
20 government and 30 pri-
vate primary care clinics
Estimated average daily
number of influenza‐posi-
tive ILI cases
Seriousness of disease
How severely sick an individual gets
when infected with the influenza
virus
Weekly number of ARI ED
attendances
8 government acute hospitals
SARI/ARI or ILI ratio
Cumulative death: hospitalisation ratio
(ideally for confirmed influenza)
Cumulative ICU: hospitalisation ratio (ide-
ally for confirmed influenza)
Weekly number of ARI ED
admissions
Weekly proportion of ARI
ED attendances resulting
in admission
Weekly number of pneu-
monia ED attendances
Weekly number of pneu-
monia ED admissions
Weekly proportion of
pneumonia ED at-
tendances resulting in
admission
Impact
How the influenza epidemic or
pandemic affects the healthcare
system (and society)
Weekly number of labora-
tory‐confirmed influenza
cases admitted to ICU or
died
8 government acute hospitals
Weekly or monthly number or proportion
of SARI cases with percentage flu‐posi-
tive among SARI cases
Weekly excess pneumonia & influenza
(
) Seriousness of disease
How severely sick an individual gets
when infected with the influenza
virus SARI/ARI or ILI ratio
Cumulative death: hospitalisation ratio
(ideally for confirmed influenza)
Cumulative ICU: hospitalisation ratio (ide-
ally for confirmed influenza) Weekly or monthly number or proportion
of SARI cases with percentage flu‐posi-
tive among SARI cases
Weekly excess pneumonia & influenza
(P&I) or all‐cause mortality stratified by
age. Weekly number of confirmed influenza
cases admitted to ICU, or weekly number
of confirmed influenza cases admitted
to hospital. Weekly or monthly number or proportion
of SARI cases with percentage flu‐posi-
tive among SARI cases
Weekly excess pneumonia & influenza
(P&I) or all‐cause mortality stratified by
age. Weekly number of confirmed influenza
cases admitted to ICU, or weekly number
of confirmed influenza cases admitted
to hospital. Impact
How the influenza epidemic or
pandemic affects the healthcare
system (and society) Abbreviations: ARI: acute respiratory infection; ED: emergency department of a government hospital; ICU: intensive care unit of a government hospi-
tal; ILI: influenza‐like illness; MAARI: medically attended acute respiratory illness; SARI: severe acute respiratory infection. 5 PUNG and LEE 2.1 | Data sources To quantify an indicator's level of activity, we calculated the av-
erage percentile of all the parameters of an indicator. On a scale from
zero to 100, percentile values of 70 and 90 were used as cut‐offs 6 |
PUNG and LEE
(ie alert thresholds) to provide three classifications of an indicator's
level of activity depending on where the average percentile value
lies on the scale (low: [0, 70]; moderate: [70, 90]; high: [90, 100]). Furthermore, the distance of the average percentile value from the
cut‐offs percentiles provided a measure of confidence—the further
away, the average percentile is from an alert threshold, the greater
the confidence in the assessment of an indicator's level of activity
and vice versa. 2.3 | Assessing the impact indicator's
level of activity
The weekly number of laboratory‐confirmed influenza cases who
were admitted to the intensive care unit (ICU) or died is the only im-
pact parameter, and we used data from January 2011 to December
2017 for threshold setting due to the absence of reporting artefacts
over the years. The discrete data had a small range of observed
values, and hence, we used a different approach to set the alert
thresholds and to ensure that alert thresholds had integer values. A sustained high (moderate) influenza activity is said to occur when
the impact parameter values remain above the high (moderate) alert
thresholds for 2 weeks after the first alert week. We set alert thresh-
olds at predefined values and tested two different scenarios. In the
first scenario, the moderate and high alert thresholds were set at
three and six, respectively. In the second scenario, they were revised
to four and six, respectively. We evaluated key performance met-
rics of sensitivity, specificity and positive predictive value (PPV) of
a threshold to assess the threshold's ability to provide early warn-
ing prior to the peak of an influenza season.6 The sensitivity was
the proportion of sustained high influenza activity with a moderate
alert raised in at least one of the 2 weeks prior to crossing the high
alert threshold. The specificity was the proportion of weeks with no
alerts during the baseline influenza periods. The PPV for high (mod-
erate) influenza activity was the proportion of true high (moderate)
alerts among all high (moderate) alerts. 2.1 | Data sources 3 | RESULTS
3.1 | Parameters selected for PISA reporting
Time series plots of the parameters in Table 1 are shown in Figure 1. The average daily attendance for ARI at the government primary
care clinics (Figure 1A) exhibits a multimodal distribution as it is in-
fluenced by the activity of other respiratory viruses, and the sea-
sonal peaks of these viruses might not be in sync with the influenza
seasons. While the average daily attendance for ARI at government
primary care clinics is less representative of the local influenza trans-
missibility as compared to its ILI counterpart, it is still important to
track it as influenza with low clinical severity may appear more fre-
quently as ARI
From 2011 to 2015, the average daily attendance for ILI at the
government primary care clinics declined (Figure 1B) and this could
be attributed to gradual underreporting after the 2009 H1N1 pan-
demic. In 2016, ILI case definition was reiterated to all government
primary care clinics leading to a rise in the measure. The estimated
average daily number of influenza‐positive ILI cases at the gov-
ernment primary care clinics was not chosen for as a parameter
for transmissibility eventually as it is a repeated representation of
its individual components and any variations caused by reporting
artefacts will affect its interpretation. Additionally, the proportion
of total visits attributed to ARI or ILI, as recommended by WHO,
was not monitored as it represented the burden of influenza com-
pared to other diseases instead of the transmissibility of the virus. The proportion of respiratory samples positive for influenza over
a 4‐weekly moving interval was the only laboratory‐confirmed in-
fluenza parameter for the transmissibility indicator. Higher local in-
fluenza activity was observed from May to July and from November
to January, and generally coincides with the winter in the Southern
and Northern Hemisphere, respectively (Figure 1C). Seasonal fluctu-
ations were observed in the weekly attendances and admissions at
the emergency department (ED) of acute government hospitals for
both ARI and pneumonia (Figure 1E‐J). During the 2009 influenza
pandemic, the weekly number of ARI ED attendances (Figure 1E) and
admissions (Figure 1F) and, consequently, the weekly proportion of
ARI ED attendances resulting in admission (Figure 1G) indicated a
clear spike. One‐off adjustment in 2016 was also observed in the
pneumonia parameters (Figure 1H‐J) due to change in the disease
classification and coding systems of some hospitals. 2.1 | Data sources The weekly
proportion of ARI or pneumonia ED attendances resulting in admis-
sion (Figure 1G,J) was selected as parameters for the seriousness of
disease as it indicated the extent to which individual gets sick and
required hospital care. The weekly number of laboratory‐confirmed influenza cases who
were admitted to the ICU or died described the impact of influenza
on healthcare resource utilisation and was the only parameter for
the impact indicator. Influenza mortality was a component of the pa-
rameter as the management of critically ill patients in general wards
could also be resource intensive (eg manpower needed for frequent
monitoring a patient's progress and calibration of treatment). Sharp
peaks in this parameter were typically observed during May to July,
coinciding with winter in the Southern Hemisphere (Figure 1K). 3.2 | Performance of the impact parameter
alert thresholds
The weekly number of laboratory‐confirmed influenza cases that
were admitted to ICU or died ranged from 0 to 24 (Figure 1K). When
the moderate and high alert thresholds were predefined at an inte-
ger value of 3 and 6, respectively, 29 moderate alerts and 19 high
alerts were raised from 2011 to 2017 Of these alerts nine moder
(A)
(B)
(C)
(D)
(E)
(F)
(G)
(H) PUNG and LEE |
(ie alert thresholds) to provide three classifications of an indicator's
level of activity depending on where the average percentile value
lies on the scale (low: [0, 70]; moderate: [70, 90]; high: [90, 100]). Furthermore, the distance of the average percentile value from the
cut‐offs percentiles provided a measure of confidence—the further
away, the average percentile is from an alert threshold, the greater
the confidence in the assessment of an indicator's level of activity
and vice versa. 2.3 | Assessing the impact indicator's
level of activity
(A) (ie alert thresholds) to provide three classifications of an indicator's
level of activity depending on where the average percentile value
lies on the scale (low: [0, 70]; moderate: [70, 90]; high: [90, 100]). Furthermore, the distance of the average percentile value from the
cut‐offs percentiles provided a measure of confidence—the further
away, the average percentile is from an alert threshold, the greater
the confidence in the assessment of an indicator's level of activity
and vice versa. 2.1 | Data sources Seasonal fluctu-
ations were observed in the weekly attendances and admissions at
the emergency department (ED) of acute government hospitals for
both ARI and pneumonia (Figure 1E‐J). During the 2009 influenza
pandemic, the weekly number of ARI ED attendances (Figure 1E) and
admissions (Figure 1F) and, consequently, the weekly proportion of
ARI ED attendances resulting in admission (Figure 1G) indicated a
clear spike. One‐off adjustment in 2016 was also observed in the
pneumonia parameters (Figure 1H‐J) due to change in the disease
classification and coding systems of some hospitals. The weekly
proportion of ARI or pneumonia ED attendances resulting in admis-
sion (Figure 1G,J) was selected as parameters for the seriousness of
disease as it indicated the extent to which individual gets sick and
required hospital care. The weekly number of laboratory confirmed influenza cases who
(A)
(B)
(C)
(D)
(E)
(F) pared to other diseases instead of the transmissibility of the virus
The proportion of respiratory samples positive for influenza ove
a 4‐weekly moving interval was the only laboratory‐confirmed in
fluenza parameter for the transmissibility indicator. Higher local in
fluenza activity was observed from May to July and from Novembe
to January, and generally coincides with the winter in the Southern
and Northern Hemisphere, respectively (Figure 1C). Seasonal fluctu
ations were observed in the weekly attendances and admissions at
the emergency department (ED) of acute government hospitals fo
both ARI and pneumonia (Figure 1E‐J). During the 2009 influenza
pandemic, the weekly number of ARI ED attendances (Figure 1E) and
admissions (Figure 1F) and, consequently, the weekly proportion o
ARI ED attendances resulting in admission (Figure 1G) indicated a
(D) The weekly number of laboratory‐confirmed influenza cases who
were admitted to the intensive care unit (ICU) or died is the only im-
pact parameter, and we used data from January 2011 to December
2017 for threshold setting due to the absence of reporting artefacts
over the years. The discrete data had a small range of observed
values, and hence, we used a different approach to set the alert
thresholds and to ensure that alert thresholds had integer values. A sustained high (moderate) influenza activity is said to occur when
the impact parameter values remain above the high (moderate) alert
thresholds for 2 weeks after the first alert week. We set alert thresh-
olds at predefined values and tested two different scenarios. 2.1 | Data sources In the
first scenario, the moderate and high alert thresholds were set at
three and six, respectively. In the second scenario, they were revised
(C) d to
The p
(D) ed to o
The pr
(D) clear spike. One‐off adjustment in 2016 was also observed in the
pneumonia parameters (Figure 1H‐J) due to change in the disease
classification and coding systems of some hospitals. The weekly
proportion of ARI or pneumonia ED attendances resulting in admis-
sion (Figure 1G,J) was selected as parameters for the seriousness of
disease as it indicated the extent to which individual gets sick and
required hospital care. The weekly number of laboratory‐confirmed influenza cases who
were admitted to the ICU or died described the impact of influenza
on healthcare resource utilisation and was the only parameter for
the impact indicator. Influenza mortality was a component of the pa-
rameter as the management of critically ill patients in general wards
could also be resource intensive (eg manpower needed for frequent
monitoring a patient's progress and calibration of treatment) Sharp
(F) to four and six, respectively. We evaluated key performance met-
rics of sensitivity, specificity and positive predictive value (PPV) of
a threshold to assess the threshold's ability to provide early warn-
ing prior to the peak of an influenza season.6 The sensitivity was
the proportion of sustained high influenza activity with a moderate
alert raised in at least one of the 2 weeks prior to crossing the high
alert threshold. The specificity was the proportion of weeks with no
alerts during the baseline influenza periods. The PPV for high (mod-
erate) influenza activity was the proportion of true high (moderate)
alerts among all high (moderate) alerts. 3 | RESULTS
(E) rics o
a thr
ing p
the p
alert
alert
alert
erate
alert
3 | f sens
eshold
(E) sio
dise
req
we
on
the
ram
cou monitoring a patient s progress and calibration of treatment). Sharp
peaks in this parameter were typically observed during May to July
coinciding with winter in the Southern Hemisphere (Figure 1K). 3.2 | Performance of the impact parameter
alert thresholds
The weekly number of laboratory‐confirmed influenza cases tha
were admitted to ICU or died ranged from 0 to 24 (Figure 1K). 2.1 | Data sources 2.3 | Assessing the impact indicator's
level of activity
From 2011 to 2015, the average daily attendance for ILI at the
government primary care clinics declined (Figure 1B) and this could
be attributed to gradual underreporting after the 2009 H1N1 pan-
demic. In 2016, ILI case definition was reiterated to all government
primary care clinics leading to a rise in the measure. The estimated
average daily number of influenza‐positive ILI cases at the gov-
ernment primary care clinics was not chosen for as a parameter
for transmissibility eventually as it is a repeated representation of
its individual components and any variations caused by reporting
artefacts will affect its interpretation. Additionally, the proportion
of total visits attributed to ARI or ILI, as recommended by WHO,
was not monitored as it represented the burden of influenza com-
(A)
(B) From 2011 to 2015, the average daily attendance for ILI at t
government primary care clinics declined (Figure 1B) and this cou
be attributed to gradual underreporting after the 2009 H1N1 pa
demic. In 2016, ILI case definition was reiterated to all governme
primary care clinics leading to a rise in the measure. The estimate
average daily number of influenza‐positive ILI cases at the go
ernment primary care clinics was not chosen for as a paramet
for transmissibility eventually as it is a repeated representation
its individual components and any variations caused by reporti
artefacts will affect its interpretation. Additionally, the proportio
of total visits attributed to ARI or ILI, as recommended by WH
was not monitored as it represented the burden of influenza com
(B) From 2
(B) (ie alert thresholds) to provide three classifications of an indicator's
level of activity depending on where the average percentile value
lies on the scale (low: [0, 70]; moderate: [70, 90]; high: [90, 100]). Furthermore, the distance of the average percentile value from the
cut‐offs percentiles provided a measure of confidence—the further
away, the average percentile is from an alert threshold, the greater
the confidence in the assessment of an indicator's level of activity
and vice versa. 2.3 | Assessing the impact indicator's
level of activity
The weekly number of laboratory‐confirmed influenza cases who
were admitted to the intensive care unit (ICU) or died is the only im-
pact parameter, and we used data from January 2011 to December
2017 for threshold setting due to the absence of reporting artefacts
over the years. 2.1 | Data sources The discrete data had a small range of observed
values, and hence, we used a different approach to set the alert
thresholds and to ensure that alert thresholds had integer values. A sustained high (moderate) influenza activity is said to occur when
the impact parameter values remain above the high (moderate) alert
thresholds for 2 weeks after the first alert week. We set alert thresh-
olds at predefined values and tested two different scenarios. In the
first scenario, the moderate and high alert thresholds were set at
three and six, respectively. In the second scenario, they were revised
to four and six, respectively. We evaluated key performance met-
rics of sensitivity, specificity and positive predictive value (PPV) of
a threshold to assess the threshold's ability to provide early warn-
ing prior to the peak of an influenza season.6 The sensitivity was
the proportion of sustained high influenza activity with a moderate
alert raised in at least one of the 2 weeks prior to crossing the high
alert threshold. The specificity was the proportion of weeks with no
alerts during the baseline influenza periods The PPV for high (mod
From 2011 to 2015, the average daily attendance for ILI at the
government primary care clinics declined (Figure 1B) and this could
be attributed to gradual underreporting after the 2009 H1N1 pan-
demic. In 2016, ILI case definition was reiterated to all government
primary care clinics leading to a rise in the measure. The estimated
average daily number of influenza‐positive ILI cases at the gov-
ernment primary care clinics was not chosen for as a parameter
for transmissibility eventually as it is a repeated representation of
its individual components and any variations caused by reporting
artefacts will affect its interpretation. Additionally, the proportion
of total visits attributed to ARI or ILI, as recommended by WHO,
was not monitored as it represented the burden of influenza com-
pared to other diseases instead of the transmissibility of the virus. The proportion of respiratory samples positive for influenza over
a 4‐weekly moving interval was the only laboratory‐confirmed in-
fluenza parameter for the transmissibility indicator. Higher local in-
fluenza activity was observed from May to July and from November
to January, and generally coincides with the winter in the Southern
and Northern Hemisphere, respectively (Figure 1C). 2.1 | Data sources When
the moderate and high alert thresholds were predefined at an inte
ger value of 3 and 6, respectively, 29 moderate alerts and 19 high
alerts were raised from 2011 to 2017. Of these alerts, nine moder
ate alerts and four high alerts preceded sustained moderate or high
(H) 3.1 | Parameters selected for PISA reporting
Time series plots of the parameters in Table 1 are shown in Figure 1. The average daily attendance for ARI at the government primary
care clinics (Figure 1A) exhibits a multimodal distribution as it is in-
fluenced by the activity of other respiratory viruses, and the sea-
sonal peaks of these viruses might not be in sync with the influenza
seasons. While the average daily attendance for ARI at government
primary care clinics is less representative of the local influenza trans-
missibility as compared to its ILI counterpart, it is still important to
track it as influenza with low clinical severity may appear more fre-
quently as ARI. (G)
FI G U R E 1 ks in t
ciding
(H) FI G U R E 1 | 7 | 7 PUNG and LEE |
(I)
(K)
(J)
FI G U R E 1 Time series plots of (A) average daily attendance for ARI at government primary care clinics, (B) average daily attendance
for ILI at government primary care clinics, (C) proportion of respiratory samples positive for influenza over a 4‐weekly moving interval, (D)
estimated average daily number of influenza‐positive ILI cases, (E) weekly number of ARI ED attendances, (F) weekly number of ARI ED
admissions, (G) weekly proportion of ARI ED attendances resulting in admission, (H) weekly number of pneumonia ED attendances. 4 | DISCUSSION Influenza surveillance in Singapore spans all acute government hos-
pitals, all government and some private primary care clinics. PISA
indicators representing the transmissibility of influenza virus, seri-
ousness of disease or the impact of influenza on healthcare resource
utilisation highlight different aspects of influenza activity. This pro-
vides comprehensive surveillance of the severity of a current in-
fluenza season and allows the ministry to determine the extent of
public health responses required to manage the transmission and to
protect vulnerable populations. Influenza surveillance in Singapore spans all acute government hos-
pitals, all government and some private primary care clinics. PISA
indicators representing the transmissibility of influenza virus, seri-
ousness of disease or the impact of influenza on healthcare resource
utilisation highlight different aspects of influenza activity. This pro-
vides comprehensive surveillance of the severity of a current in-
fluenza season and allows the ministry to determine the extent of
public health responses required to manage the transmission and to
protect vulnerable populations. 2.1 | Data sources cont'd (I)
weekly number of pneumonia ED admissions, (J) weekly proportion of pneumonia attendances at the ED resulting in admission, (K) weekly
number of laboratory‐confirmed influenza cases admitted to ICU or died (I)
(J) (J) (I) (I) (K) FI G U R E 1 Time series plots of (A) average daily attendance for ARI at government primary care clinics, (B) average daily attendance
for ILI at government primary care clinics, (C) proportion of respiratory samples positive for influenza over a 4‐weekly moving interval, (D)
estimated average daily number of influenza‐positive ILI cases, (E) weekly number of ARI ED attendances, (F) weekly number of ARI ED
admissions, (G) weekly proportion of ARI ED attendances resulting in admission, (H) weekly number of pneumonia ED attendances. cont'd (I)
weekly number of pneumonia ED admissions, (J) weekly proportion of pneumonia attendances at the ED resulting in admission, (K) weekly
number of laboratory‐confirmed influenza cases admitted to ICU or died influenza activity (ie 31.0% and 21.1% PPV for moderate and high
alert threshold, respectively). In all four of the observed sustained
high influenza activity, a moderate alert was made known at least
one week prior to trigger of the high alert (ie 100% sensitivity). No
alerts were made in 83 of the 103 weeks of baseline influenza activ-
ity (ie specificity of 80.6%). meaningful cut‐offs, and hence, a separate assessment scale was
created (Figure 2B) based on the results in the previous section,
Performance Matrices for the Impact Parameter Alert Threshold. Table 2 illustrates the weekly PISA results from E‐week 1 to 10
of 2018. In the second scenario, the moderate alert threshold was in-
creased to a value of 4, and 20 moderate alerts were raised from
2011 to 2017. Of these alerts, three resulted in sustained mod-
erate influenza activity (ie PPV of moderate alert threshold of
15.0%). The sensitivity remained at 100%. No alerts were made in
138 of the 156 weeks of baseline influenza activity (ie specificity
of 88.5%). 3.3 | Assessment scale for indicators Figure 2A shows the assessment scale used to qualitatively clas-
sify the level of activity of the transmissibility and seriousness of
disease indicators. The coloured scale showed gradual transition
from dark green to dark red signifying increasing levels of activ-
ity of an indicator. The small range of discrete values observed in
the single impact parameter limits our ability to provide multiple, Of the three indicators, transmissibility has the widest variety
of parameters customised for each country's setting. In Singapore,
data on the ARI and ILI attendance at government primary care clin-
ics are conveniently extracted from various healthcare surveillance PUNG and LEE 8 8 FI G U R E 2 Assessment scale for (A) transmissibility and seriousness of disease indicators, (B) impact indicator. Severity of an indicator is
classified as L: low, M: moderate, H: high. Confidence level of an indicator is classified as 1: low, 2: medium, 3: high FI G U R E 2 Assessment scale for (A) transmissibility and seriousness of disease indicators, (B) impact indicator. Severity of an indicator is
classified as L: low, M: moderate, H: high. Confidence level of an indicator is classified as 1: low, 2: medium, 3: high technological and manpower capability, critical care could be pro-
vided in general wards. An improved measure would be the ratio of
cases fulfilling the definitions of complicated or severe influenza11 to
the number of influenza‐positive admission. However, the feasibility
of measuring this is dependent on the healthcare system's ability to
integrate laboratory and epidemiological data. platforms for weekly reporting. In other countries, the number of
callers to public health hotline reporting ILI7,8 or prescription re-
cords9,10 were also explored as means to characterise the extend
of spread when complemented with data sources from healthcare
institutions. Key challenges remain in achieving a representative indicator for
seriousness of disease in Singapore. The weekly proportion of ARI or
pneumonia ED attendances that were hospitalised were chosen to
illustrate the severity of each condition, but the absence of hospital
laboratory surveillance data limits our ability to verify the infection
status of each patient. Spikes in the weekly proportion of ARI ED at-
tendances that were hospitalised (Figure 1G) could be attributed to
changes in health‐seeking behaviour, reporting habits of physicians
and higher tendency to admit a patient during a pandemic, though
extent of influence has yet to be studied. integer values. The PPV of the thresholds was poor and implied that
in many occasions, there was no sustained moderate or high influ-
enza activity occurring after a moderate or high alert was triggered. The moderate threshold was eventually set at four as about 70% of
the historical data was below this value, and a moderate alert was
triggered before the onset of all sustain high influenza activity. abnormalities needs to be highlighted and the interpretation of
the average percentile under such conditions should be done with
caution. Rachael Pung
https://orcid.org/0000-0002-1188-8231 Rachael Pung
https://orcid.org/0000-0002-1188-8231 Rachael Pung
https://orcid.org/0000-0002-1188-8231 The quantification of an indicator's level of activity is achieved
by averaging the percentile rank of all the parameters representing
an indicator with the assumption that all parameters were equally
informative. However, ARI parameters can be influenced by the ac-
tivity of other respiratory viruses. As such, there may be occasions
where the average daily attendance for ARI was high but the same
was not observed for ILI surveillance data. However, it is still import-
ant to track the ARI attendances at the government acute hospitals
and primary care clinics as it potentially informs us of any changes
in the clinical representation of influenza cases. One possible way
of overcoming this challenge is to assign weights to each parameter
based on its importance in assessing the local influenza situation. The weighted average percentile rank could be computed to repre-
sent an indicator's level of activity. 5 | CONCLUSION In this paper, we also presented an assessment scale, which pro-
vides a combined measure of an indicator's level of activity and the
confidence level of the assessment. With more than one parame-
ter serving as proxies for an indicator, the method of providing an
aggregated assessment for an indicator remains undocumented in
PISA. Furthermore, the confidence of an indicator's assessment is
part of PISA reporting, but its interpretation is multifaceted. It is
dependent on, but not limited to, reporting biases, timeliness and
agreement between the parameters. The first two factors are re-
lated to the reliability of the information provided at various sen-
tinel sites and can be improved with a structured data collection
process. On the contrary, the agreement between the parameters
is intrinsic to the influenza activity of a season. Each parameter is a
unique proxy of an indicator and might be influenced by the activity
of other respiratory viruses. Thus, a high agreement between the
parameters provides greater certainty to the measure of an indica-
tor's level of activity. We share Singapore's practices in the weekly assessment of PISA
indicators. For indicators represented by multiple parameters,
a collective assessment of the indicator's level of activity and the
confidence level of this assessment were necessary. Here, we have
introduced an assessment scale to accomplish both objectives. We
placed priority in creating a simple collective assessment for a com-
plex indicator. The choice of parameters, sampling criteria and case
definitions were regularly reviewed and updated to ensure consist-
ent performance of our surveillance system. Our method of PISA
reporting could be applied in other countries, with parameters cho-
sen based on the resources of the country, and the assessment scale
customised to the local setting. 3.3 | Assessment scale for indicators Severity assessment has been largely focused on developing
different methods to establish alert thresholds that signal the start
or the end of an influenza season. Based on the characteristics of
a parameter,12 a variety of methods such as the Moving Epidemic
Method (MEM) or cumulative sum control charts (CUSUM) have
been developed for early epidemic detection. For Singapore, thresh-
olds setting methods were chosen based on the data characteristics. Regular review and enhancement of data extraction methods helps
to improve accuracy of the parameters but inevitably creates arte-
facts in the historical surveillance data and limits the feasibility of
using methods that require long history of surveillance data. Hence,
for transmissibility and seriousness of disease parameters, the mod-
erate and high alert thresholds of a year were set using the 70th and
90th percentiles of the past 2‐year data. For the impact parameter,
the moderate and high alert thresholds were set using predefined The cumulative number of patients tested positive for influenza
admitted to ICU is a component to some WHO recommended pa-
rameters in Table 1. This component is limited by the number of ICU
beds, and ICU admission of a severe influenza case is subjected to
competing requirements of other non‐influenza‐positive patients
depending on severity. Furthermore, depending on a hospital's TA B LE 2 Weekly PISA results
PISA reporting:
Transmissibility
Seriousness of disease
Impact
Year
E‐week
Risk
Confidence
Risk
Confidence
Risk
Confidence
2018
1
Moderate
Low
Low
Medium
High
Medium
2018
2
Moderate
Low
Low
Medium
High
Low
2018
3
High
Low
Moderate
High
Moderate
Low
2018
4
High
Low
Low
High
Low
High
2018
5
High
Low
Low
High
Low
High
2018
6
High
Low
Low
High
High
Low
2018
7
Moderate
Low
Low
Low
Low
High
2018
8
Moderate
High
Moderate
Low
Moderate
Low
2018
9
Moderate
Low
Low
Low
Low
High
2018
10
Low
Low
Moderate
Medium
Moderate
Low | 9 PUNG and LEE REFERENCES 1. World Health Organization. Strengthening response to pandem-
ics and other public‐health emergencies: report of the Review
Committee on the Functioning of the International Health
Regulations (2005) and on Pandemic Influenza (H1N1) 2009. https
://apps.who.int/iris/bitstream/handle/10665/75235/9789241564
335_eng.pdf;jsessionxml:id=6C57B890718C3C665CC1B36C7
0BC3A15?sequence=1 Accessed 15 July, 2019. 2. World
Health
Organization. Pandemic
Influenza
Severity
Assessment (PISA): a WHO guide to assess the severity of influenza
epidemics and pandemics. https://apps.who.int/iris/bitstream/
handle/10665/259392/WHO-WHE-IHM-GIP-2017.2-eng.pdf?se-
quence=1 Accessed January 5, 2019. 3. Singapore Tourism Board. International visitor arrival statistics. https://www.stb.gov.sg/content/dam/stb/documents/statistics-
marketing-insights/international-visitor-arrivals/pdf/visitor-arriv
als-2018.pdf Accessed February 20, 2019. In addition, when a parameter is higher (or lower) than the histor-
ical maximum (or minimum), the percentile of that parameter's data
was capped at 100 (or zero). Taking the average percentile values of
all parameters of an indicator then helps to ensure that the extreme
results of one parameter would not dominate the measure of an in-
dicator but allows it to skew the measure towards a higher (or lower)
classification of the indicator's level of activity. 4. Singapore Department of Statistics. Population and population
structure. https://www.singstat.gov.sg/find-data/search-by-
theme/population/population-and-population-structure/lat-
est-data Accessed February 20, 2019. 5. Lee CE, Satkunanantham K. Singapore’s Health Care System: What
50 Years Have Achieved. Singapore: World Scientific Publishing Co.,
Pte. Ltd.; 2016:18. The confidence assessment did not consider the number of
parameters used to represent an indicator. It is possible for an in-
dicator's parameter to reflect a very different level of activity com-
pared to the rest of the parameters. In situations where there are
few parameters representing an indicator, the extreme parameter
is likely to skew an indicator's level of activity towards an extreme. The effect of the extreme parameter on the indicator's level of
activity would attenuate when the number of parameters repre-
senting an indicator increases. Also, any sustained occurrence of 6. Hashimoto S, Murakami Y, Taniguchi K, Nagai M. Detection of epi-
demics in their early stage through infectious disease surveillance. Int J Epidemiol. 2000;29(5):905‐910. 7. Vette K, Bareja C, Clark R, Lal A. Establishing thresholds and pa-
rameters for pandemic influenza severity assessment, Australia. Bull World Health Organ. 2018;96(8):558‐567. 8. Public Health England. Surveillance of influenza and other re-
spiratory viruses in the UK: winter 2017 to 2018. https://as-
sets.publishing.service.gov.uk/government/uploads/system/
uploads/attachment_data/file/740606/Surveillance_of_influ 8. Public Health England. Surveillance of influenza and other re-
spiratory viruses in the UK: winter 2017 to 2018. https://as-
sets.publishing.service.gov.uk/government/uploads/system/
uploads/attachment_data/file/740606/Surveillance_of_influ 10 PUNG and LEE 10 | 12. World Health Organization. Global epidemiological surveillance
standards for influenza. https://www.who.int/influenza/resources/
documents/WHO_Epidemiological_Influenza_Surveillance_Stand
ards_2014.pdf Accessed January 5, 2019. enza_and_other_respiratory_viruses_in_the_UK_2017_to_2018. pdf Accessed January 5, 2019. 9. Sugawara T, Ohkusa Y, Ibuka Y, Kawanohara H, Taniguchi K, Okabe
N. Real‐time prescription surveillance and its application to mon-
itoring seasonal influenza activity in, Japan. J Med Internet Res. 2012;14(1):e14. How to cite this article: Pung R, Lee VJM. Implementing the
World Health Organization Pandemic Influenza Severity
Assessment framework—Singapore's experience. Influenza
Other Respi Viruses. 2020;14:3–10. https://doi.org/10.1111/
irv.12680 10. Chen JH, Schmit K, Chang H, Herlihy E, Miller J, Smith P. Use of
medicaid prescription data for syndromic surveillance –‐ New York. MMWR Morb Mortal Wkly Rep. 2005;54(Suppl):31‐34. How to cite this article: Pung R, Lee VJM. Implementing the
World Health Organization Pandemic Influenza Severity
Assessment framework—Singapore's experience. Influenza
Other Respi Viruses. 2020;14:3–10. https://doi.org/10.1111/
irv.12680 11. World Health Organization. WHO guidelines for pharmacologi-
cal management of pandemic influenza A(H1N1) 2009 and other
influenza viruses. https://www.who.int/csr/resources/publicatio
ns/swineflu/h1n1_guidelines_pharmaceutical_mngt.pdf Accessed
January 5, 2019.
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88
David Lorenz: Die umkämpfte Dublin-Verordnung
Damit verschob sich der Schwerpunkt in der Anwendung Dublins weg von der Verteilung der Verantwortung für Asylverfahren zwischen den Mitgliedstaaten hin zur
Verhinderung mehrfacher Asylverfahren. Das bedeutet auch, dass die mit Dublin
intendierte Umverteilung der Zuständigkeit für Asylverfahren an die Grenzstaaten
im Zeitraum dieser Untersuchung der Kommission nicht realisiert wurde:
»Contrary to a widely shared supposition that the majority of transfers are directed towards the Member States located at an external border, it appears that
the overall allocation between border and non border countries is actually rather
balanced.« (Ebd., S. 51)
Für das Jahr 2005 veröffentlichte die Europäische Kommission (ebd., S. 50–52)
umfassende Zahlen zu Überstellungen (siehe Tabelle 3 im Anhang). Deutschland ist
weiterhin der in Bezug auf Überstellungen aktivste Mitgliedstaat. Allerdings halten
sich im Unterschied zu den Daten von 1998 – 1999 die Überstellungen nach und von
Deutschland fast die Waage.
Der Umstand, dass Mitgliedstaaten sowohl ausgehende als auch ankommende
Überstellungen haben, führt zu einer neuen statistischen Größe: den Netto-Überstellungen beziehungsweise der Überstellungsbilanz. Netto-Überstellungen und
die Überstellungsbilanz bezeichnen die Differenz aus ausgehenden und ankommenden Überstellungen. Netto-Überstellungen geben Auskunft darüber, welche
Mitgliedstaaten durch Dublin-Überstellungen mehr Asylverfahren durchführen
müssen und welche weniger. Während Deutschland zwar mit 2.748 ausgehenden
und 2.716 ankommenden Überstellungen der bezogen auf Überstellungen aktivste
Mitgliedstaat ist, machen die 32 zusätzlichen Überstellungen, die aus der Differenz resultieren, kaum einen Unterschied für die Gesamtzahl der Asylverfahren in
Deutschland. Eine positive Zahl an Netto-Überstellungen bedeutet, die ausgehenden Überstellungen sind in der Überzahl, eine negative Zahl steht für eine Überzahl
der ankommenden Überstellungen. Durch die Osterweiterung der EU 2004 hat
Polen Deutschland als das Land abgelöst, in das in der Bilanz mit -1.048 die meisten
Asylsuchenden überstellt werden. Polen hat nicht nur die meisten ankommenden Netto-Überstellungen, sondern auch mit fast 20 % einen relativ hohen Anteil
der Überstellungen an den gestellten Asylanträgen. Das Vereinigte Königreich
überstellte in der Bilanz die meisten Asylsuchenden an andere Mitgliedstaaten.
5.3.1 Start der Verhandlungen um Dublin III
Mit der Evaluation von Dublin II durch die Kommission startete der politische
Prozess um die Überarbeitung der Dublin-Verordnung zu Dublin III. Wie beschrieben war die Kommission trotz aller Probleme in ihrer Evaluation der Meinung,
dass Dublin II »zufriedenstellend angewandt« (Europäische Kommission 2007a,
5 Kämpfe um Gestaltung: Geschichte Dublins
S. 6) wurde und die Durchführung der Dublin-Verfahren »funktioniert« (ebd.,
S. 6). Diesem positiven Urteil widersprach das Europäische Parlament: Im Ausschuss für bürgerliche Freiheiten, Justiz und Inneres, dem sogenannten LIBEAusschuss, wurde am 25. Juni 2008 mit 49 Ja-Stimmen, einer Nein-Stimme und
einer Enthaltung (vgl. Europäisches Parlament 2008, S. 16) ein Bericht angenommen, der das Dublin System in seiner damaligen Form als unbefriedigend unter
technischen und humanitären Gesichtspunkten und sowohl für Asylsuchende als
auch Mitgliedstaaten ungerecht beschreibt (vgl. ebd., S. 6). Neben verschiedenen
Forderungen, die im Wesentlichen auf eine verstärkte »Lastenverteilung« zwischen
den Mitgliedstaaten und einen verbesserten Schutz der betroffenen Asylsuchenden
ausgerichtet sind, enthält der Bericht des Parlaments die Forderung nach einem
Aussetzungsmechanismus für Überstellungen in Dublin:
»[Das Europäische Parlament] ersucht die Kommission, verbindliche Mechanismen vorzusehen, die die Überstellung von Asylbewerbern in Mitgliedstaaten verhindern, welche keine umfassende und faire Bearbeitung ihres Antrags garantieren, und systematisch Maßnahmen gegen diese Staaten zu ergreifen;« (Ebd., S. 7)
Diese Forderung des Parlaments steht im Kontext einer beginnenden europaweiten Kampagne verschiedener NGOs gegen Überstellungen von Asylsuchenden nach
Griechenland (siehe Kapitel 5.3.2).
Der eigentliche Gesetzgebungsprozess startete mit dem Entwurf der Kommission für Dublin III im Dezember 2008 (Europäische Kommission 2008). Im Vorfeld konsultierte die Kommission neben Repräsentant*innen der Mitgliedstaaten
auch Sachverständige des UNHCR, von NGOs, sowie Rechtsanwält*innen, Richter*innen und Parlamentarier*innen des Europaparlaments. Die Kommission stellt
die Ergebnisse dieser Konsultationen folgendermaßen dar: Die Mehrzahl der Mitgliedstaaten würden die Grundprinzipien der Verordnung schätzen, wollten allerdings die Leistungsfähigkeit des Systems verbessern. Der größte Teil der NGOs und
der UNHCR würden die Grundprinzipien des aktuellen Systems ablehnen und ein
System bevorzugen, welches die Zuständigkeit auf der Basis des Ortes des ersten
Asylantrags verteilt. In Anbetracht der für diese Forderung ungünstigen politischen
Kräfteverhältnisse würden diese Akteur*innen dazu aufrufen, den Schutz der Asylsuchenden in der neuen Verordnung zu stärken. Letzterer Forderung würde sich das
Europäische Parlament anschließen. Ihr eigener Entwurf für Dublin III trage laut
Kommission all diesen verschiedenen Forderungen Rechnung – an der Grundstruktur von Dublin II solle festgehalten werden und gleichzeitig solle die Leistungsfähig
des Systems und der Schutz der Asylsuchenden verstärkt werden. (vgl. ebd., S. 5)
Wie vom Europaparlament gefordert schlägt die Kommission ein Verfahren vor,
mit dem Überstellungen in einen Mitgliedstaat zeitweise ausgesetzt werden können:
89
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David Lorenz: Die umkämpfte Dublin-Verordnung
»Um zu vermeiden, dass Mitgliedstaaten mit begrenzten Aufnahme- und Absorptionskapazitäten, die einem besonderen Druck ausgesetzt sind, durch
Überstellungen im Rahmen des Dublin-Systems zusätzlich belastet werden, wird
ein neues Verfahren eingeführt, mit dem Überstellungen in den zuständigen Mitgliedstaat auf der Grundlage des Dublin-Verfahrens ausgesetzt werden können.
Von diesem Verfahren kann auch in Fällen Gebrauch gemacht werden, in denen
Bedenken bestehen, dass die Überstellung zur Folge hat, dass einem Antragsteller im zuständigen Mitgliedstaat kein angemessenes Schutzniveau, insbesondere
im Hinblick auf Aufnahmebedingungen und Zugang zum Asylverfahren, geboten
wird.« (Ebd., S. 9)
Artikel 31 des Entwurfs für Dublin III regelte den Aussetzungsmechanismus. Er sah
vor, dass Mitgliedstaaten in Notsituationen einen Antrag auf temporäre Aussetzung
der Überstellungen an die Kommission stellen können. Im Falle einer Aussetzung
auf der Grundlage eines nicht angemessenen Schutzniveaus für Asylsuchende hätte entweder ein anderer Mitgliedstaat einen Antrag an die Kommission richten oder
die Kommission eigeninitiativ tätig werden können. Die Kommission würde daraufhin eine Entscheidung bezüglich der Aussetzung der Überstellungen treffen, die
innerhalb eines Monats durch eine anderslautende Entscheidung mit qualifizierter
Mehrheit des Rates korrigiert werden könnte. Die Dauer der Aussetzung der Überstellungen hätte einen Zeitraum von sechs Monaten nicht überschreiten sollen und
hätte um weitere sechs Monate verlängert werden können. Die Zuständigkeit für
die betroffenen Asylverfahren wäre dann auf die Mitgliedsstaaten übergegangen,
in denen sich die entsprechenden Asylsuchenden aufhalten. (vgl. ebd., S. 50–52)
Auf den Entwurf der Kommission reagierte das Parlament am 29.04.2009 mit
einem Bericht, der den Entwurf der Kommission unterstützte. Es fügte allerdings
im Entwurf für Artikel 31, den Aussetzungsmechanismus, einen neuen Absatz ein.
In diesem wird ein System zur Umverteilung anerkannter Flüchtlinge zwischen den
Mitgliedstaaten gefordert:
»ein System zur Umverteilung von Personen, die Anspruch auf internationalen
Schutz haben, von Mitgliedstaaten, die einer spezifischen und unverhältnismäßigen Belastung ausgesetzt sind, an andere Mitgliedstaaten im Benehmen mit dem
Amt des Hohen Flüchtlingskommissars der Vereinten Nationen, wobei gewährleistet wird, dass die Umverteilung nach nichtdiskriminierenden, transparenten
und eindeutigen Regeln erfolgt.« (Europäisches Parlament 2009, S. 26–27)
Ein Umverteilungsmechanismus für anerkannte Asylsuchende war bis dahin nicht
in der Dublin-Verordnung geregelt worden. Damit konkretisierte das Parlament
seine Forderung aus dem Bericht von 2008 (Europäisches Parlament 2008) nach
einer »Lastenteilung« unter den Mitgliedstaaten und ergänzte damit den »Aussetzungsmechanismus« um einen sogenannten »Solidaritätsmechanismus«. Die
5 Kämpfe um Gestaltung: Geschichte Dublins
Stimmung im Parlament im Hinblick auf die Dublin-Verordnung beschrieb eine
für das Parlament an den Verhandlungen beteiligte Person als kritisch:
»I think if you would have a vote in the parliament on whether or not to have the
Dublin system or to go for something else, I think you would have a majority for
something else to stop the Dublin system because it is very, very criticised in the
parliament. I’m not sure there would be a majority but it would be almost fiftyfifty. But the Dublin system is very criticised within the parliament.« (Interview
mit einem Mitglied des Europaparlaments, Verhandlungsgruppe Dublin III, 2012)
Das ist insofern erstaunlich, als im Europaparlament nach der Europawahl 2004
die rechtskonservativen Fraktionen stärker vertreten waren als linke. Die christlichkonservative EVP war mit 268 von insgesamt 732 Sitzen mit Abstand die stärkste
Fraktion und gleichzeitig diejenige Fraktion, deren Verhandlungsposition der konservativen Position des Rates am nächsten kam. Die Verhandlungsposition der rechten Parteien war durch eine Spaltung geschwächt: Die Positionen der konservativen
Abgeordneten der Zielstaaten und derjenigen der Kernstaaten waren entgegengesetzt – griechische EVP Abgeordnete hatten zum Beispiel eine sehr andere Position zu Dublin als deutsche EVP Abgeordnete. Den einflussreichen Posten des Schattenberichterstatters der EVP Fraktion für die Verhandlungen um Dublin III besetzte ein griechischer Abgeordneter. Dies hatte Folgen für die Dynamik innerhalb der
Gruppe der Berichterstatterin und Schattenberichterstatter*innen:
»For example, on Dublin we had a Greek shadow from the EVP group, and his positions where not very much in line with those of the German delegation within the
EVP, that is the big delegation. So it is up to the shadow to control, sort of speak
on behalf of the group and not just for his own interests. But there you could see
some conflicts sometimes. The fact that he was the shadow made the group of
shadows coming closer together, because his position was closer to the one of the
Liberals, the SPE, the Greens and the GUE because were in favour of this solidarity system. And he of course – because of the Greek perspective, they are having
these problems. But that made it a bit difficult for him to get the rest of his group
on board. And I think in many of the other asylum files we have a German EVP
shadow and they can see that it is really a difference between the EVP group on
the one hand and the other groups on the other hand.« (Ebd.)
Abgesehen von dieser Spaltung der konservativen Fraktion wurden in Hintergrundgesprächen oder Interviews weitere Erklärungsansätze angedeutet. So wurde in einem Interview betont, dass die Parlamentarier*innen im Europaparlament im Gegensatz zu den Akteur*innen des Rates eine eher europäische als nationalstaatliche
Perspektive einnehmen und sich damit auch sehr weit von den Positionen ihrer Kolleg*innen in den nationalen Parlamenten oder Regierungen entfernen können (vgl.
91
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David Lorenz: Die umkämpfte Dublin-Verordnung
ebd.). Zweitens wurde der zuständige Innenausschuss des Parlaments als – für die
Verhältnisse des Parlaments – linksliberal geprägt beschrieben (vgl. Interview mit
einem Mitglied des Europaparlaments, LIBE-Ausschuss, 2012). Drittens wurde in
einem Hintergrundgespräch mit einer Mitarbeiterin einer Parlamentarierin der liberalen Parlamentsfraktion die Rolle von Expert*innen für die Arbeit der Parlamentarier*innen betont. Während der Rat auf die Expertise der Beamt*innen der nationalen Verwaltungen zurückgreifen könne, sei das Parlament auf die Expertise externer Expert*innen angewiesen – und diese Rolle wurde im Fall der Dublin Verhandlung in erster Linie durch die Expert*innen und Lobbyist*innen der linksliberalen
NGOs im Asylbereich ausgefüllt, nicht nur für die linken Fraktionen des Europaparlaments. Vor diesem Hintergrund lässt sich vielleicht auch die ungewöhnliche
Einigkeit verstehen, die in Bezug auf Dublin zwischen den Fraktionen der Liberalen, Sozialdemokrat*innen, Grünen und Linken (vgl. Interview mit einem Mitglied
des Europaparlaments, Verhandlungsgruppe Dublin III, 2012) bestand.
Während das Parlament den Vorschlag der Kommission unterstützte war der
Rat skeptisch. Eine für die deutsche Delegation an den Verhandlungen im Rat beteiligte Person sagte dazu auf meine Frage, welche Punkte der Rat abgelehnt habe:
»Ein Punkt war der sogenannte Aussetzungsmechanismus. […] Die große Mehrheit der Mitgliedsstaaten hat diesen Vorschlag nicht mitgetragen, und es war auch
einer der Punkte, die verschiedentlich auf politischer Ebene von den Ministern
selber isoliert verhandelt wurden. Das EP hat dagegen den KOM-Vorschlag mehrheitlich unterstützt, das EP hatte relativ schnell seine Stellungnahme abgegeben,
so dass erkennbar war, wie die Positionierung des EP ist. Aber der Rat hat den Aussetzungsmechanismus abgelehnt und dann musste dann ausgelotet werden, wie
es weitergeht.« (Interview mit einem Mitarbeiter des Bundesinnenministeriums
(BMI), deutsche Verhandlungsdelegation zu Dublin III im Rat der EU, 2012)
In Bezug auf Dublin war der Rat gespalten. Auf der einen Seite standen die Delegationen der eher reicheren Staaten im Kern der EU und an den westlichen und
nördlichen Rändern, die tendenziell das Ziel von Binnenmigration in der EU waren. Zu dieser Gruppe zählten laut Interviews und Verhandlungsdokumenten zumindest die Delegationen aus Österreich, Belgien, Deutschland, Frankreich, Großbritannien, den Niederlanden, Dänemark, Finnland und Schweden (vgl. Interview
mit einem Mitglied des Europaparlaments, LIBE-Ausschuss, 2012) (BMI 2012). Auf
der anderen Seite standen die Delegationen einiger Transitstaaten an den südlichen
Grenzen, die sich nicht mit der Rolle als Türsteher*innen des Schengenraums zufrieden geben wollten. Dies waren die Delegationen aus zumindest Zypern, Griechenland, Italien, Spanien und Malta (vgl. Interview mit einem Mitglied des Europaparlaments, LIBE-Ausschuss, 2012) (vgl. Innenminister*innen von Zypern, Griechenland, Italien und Malta 2009; Innenminister*innen von Zypern, Griechenland,
5 Kämpfe um Gestaltung: Geschichte Dublins
Italien, Malta und Spanien 2011). Die Zielstaaten konnten sich in allen zentralen
Punkten gegen die Transitstaaten durchsetzen und damit die Verhandlungsposition des Rates prägen. Das ohnehin bestehende Machtgefälle zwischen diesen beiden
Staatengruppen wurde durch die parallel stattfindenden Eurokrisenprozesse verschärft. Vier der fünf Transitstaaten waren deshalb von Krediten und Bürgschaften
der anderen Mitgliedstaaten, der EU und des IWF abhängig.
Der Rat entschied im Fall der Verhandlungen um Dublin III mit qualifizierter
Mehrheit: Für eine Annahme brauchte es mindestens eine einfache Mehrheit der
Mitgliedstaaten und gleichzeitig eine Mehrheit von mindestens 255 der insgesamt
345 Stimmen im Rat. Keine der beiden Gruppen konnte eine einfache Mehrheit
der Mitgliedstaaten verhindern. Die Gruppe der Zielstaaten, die gegen den Aussetzungsmechanismus verhandelten, hatte allerdings insgesamt 132 Stimmen –
Österreich 10, Belgien 12, Deutschland 29, Frankreich 29, Großbritannien 29, die
Niederlande 13 und Schweden 10. Die kleinere Gruppe der oppositionellen Transitstaaten kam dagegen gemeinsam nur auf 75 Stimmen – Italien 29, Spanien 27,
Griechenland 12, Zypern 4 und Malta 3. In den Verhandlungen hätte dementsprechend die Gruppe der Zielstaaten eine qualifizierte Mehrheit verhindern können,
die Gruppe der Transitstaaten nicht.10
Dazu kam, dass die Zielstaaten die Grundzüge des Status Quo verteidigten, also im Zweifelsfall den Aussetzungsmechanismus auch durch ein Scheitern der Verhandlungen und eine fortgesetzte Anwendung von Dublin II hätten verhindern können. Demgegenüber waren die Transitstaaten für die Durchsetzung ihrer Verhandlungsziele auf die Zustimmung einer Reihe von anderen Staaten und auf einen erfolgreichen Abschluss der Verhandlungen angewiesen.11
In dieser Konstellation, in der auf der einen Seite die Kommission mit dem Parlament für einen Aussetzungsmechanismus und der Rat, trotz seiner inneren Spaltung, gegen den Aussetzungsmechanismus verhandelten, zogen sich die Verhandlungen bis Ende 2010 hin, ohne dass die verschiedenen Positionen sich annäherten
oder eine Einigung in Sicht gewesen wäre. Etwa zwei Jahre nach dem Entwurf der
Kommission waren die Verhandlungen festgefahren. Oder, in den Worten eines Interviewpartners:
»In dem Fall bedeutete das tatsächlich eine Änderung an dem Dublin-System
vorzunehmen, indem man diesen Notfall-Mechanismus einbauen wollte, […]
mit dem man dann die Transfers aussetzen kann. Das war von der Kommission von Anfang an unterstützt worden eigentlich. Das Parlament hat das auch
10
11
Diese Stimmgewichtung entspricht dem Entscheidungsverfahren nach qualifizierter Mehrheit vor 2017, also auf Basis des Vertrags von Nizza in der EU-27.
Für eine ausführlichere Analyse der politischen Konstellation im Rat siehe Lorenz (2013,
S. 71–82).
93
94
David Lorenz: Die umkämpfte Dublin-Verordnung
unterstützt, der Rat hatte da ein Problem und das ist natürlich der Kernpunkt
gewesen und auch von vorneherein der Punkt, wo die Verhandlungen gestartet
sind, aufeinandergeprallt und eingefroren.« (Interview mit einem Mitglied des
Europaparlaments, LIBE-Ausschuss, 2012)
5.3.2 Rechtskämpfe um Überstellungen nach Griechenland
Der im Kapitel zum Dubliner Übereinkommen geschilderte Rechtsstreit um effektiven Rechtsschutz gegen Überstellungen spitzte sich in Deutschland unter Dublin
II wegen der Situation in Griechenland zu. Die griechische Verwaltungspraxis bot
viele Ansatzpunkte für Klagen gegen Dublin-Bescheide. So wurde 2005 ein Bericht
über die Auswirkungen eines griechischen Gesetzes veröffentlicht, welches es den
Behörden erlaubte, ein Asylverfahren einzustellen, wenn die asylsuchende Person
eigenmächtig den ihr zugewiesenen Wohnort verlässt (Papadimitriou und Papageorgiou 2005, S. 308). Diese Regelung wurde von den griechischen Asylbehörden
dazu genutzt, in Griechenland eröffnete Asylverfahren von Personen einzustellen,
die nach der Eröffnung des Verfahrens in andere Dublin-Staaten reisten. Nach der
Überstellung zurück nach Griechenland hatten diese Personen dann keinen legalen
Aufenthaltstitel und waren von einer Abschiebung in ihren Herkunftsstaat bedroht,
ohne dass ihr Asylverfahren in einem der Mitgliedstaaten jemals geprüft worden
wäre (ebd., S. 310).
Die Situation des griechischen Asylsystems verschärfte sich in den darauf folgenden Jahren immer weiter. Während der ersten Jahre der globalen Finanzkrise
ab 2007 stieg die ohnehin schon hohe Staatsverschuldung Griechenlands und der
griechischen Staat stürzte in eine Staatsschuldenkrise. Um einen Staatsbankrott
abzuwenden beantragte der damalige griechische Ministerpräsident Papandreou
im April 2010 Finanzhilfe. Diese wurden einen Monat später nach Verhandlungen
mit der Troika genannten Trias aus IWF, EU und EZB unter der Auflage einer strengen Austeritätspolitik gewährt. Zeitgleich zu dieser Staatsschuldenkrise, in der die
Politik Griechenlands in weiten Teilen gemäß den Interessen und entsprechend den
Forderungen der Gläubigerinstitutionen gestaltet wurde (vgl. Kotzias 2016), entwickelte sich Griechenland zum Tor nach Europa für illegalisierte Migration.
Die Haupteinreiserouten in die EU verschoben sich aufgrund der grenzpolitischen Kooperationen von Spanien mit Senegal und Mauretanien sowie von Italien
mit Libyen vom zentralen Mittelmeer zum östlichen Mittelmeer. Die neue Haupteinreiseroute in die EU führte von der Türkei nach Griechenland (vgl. Frontex 2010,
S. 12). 2009 wurden 75 % der »irregulären Grenzübertritte« in die EU an den griechischen Grenzen festgestellt, Ende 2010 waren es 90 % (vgl. European Union Agency
for Fundamental Rights 2011, S. 12). 2010 reisten Asylsuchende, die ohne die Möglichkeit der legalen Einreise auf dem Weg in das Territorium der EU waren, fast immer über Griechenland. Von dort setzten sie ihre Reise laut Analysen von Frontex
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Fotografias como um recurso de pesquisa em marketing: o uso de métodos visuais no estudo de organizações de serviços
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RESUMO Argumenta-se que a adoção de métodos visuais de coleta e análise de dados em pesquisas de
marketing de serviços oferece riqueza de informações aos estudos na área, pois o uso de fotos (ou
vídeos) pode proporcionar objetividade e credibilidade, na medida em que uma imagem fornece
prova tangível da existência de determinado fenômeno. Para ilustrar o uso dos métodos visuais,
foram selecionadas e analisadas fotos de um hospital e de um hotel, extraídas dos sites das empresas
com o objetivo de verificar como o gerenciamento de impressões leva um hospital a usar estratégias
para intencionalmente se parecer com um hotel. Para dar respaldo ao trabalho, utilizou-se como
base teórica os métodos visuais na pesquisa, o papel do cenário nos encontros de serviços, o
gerenciamento de impressões e o marketing de serviços. E, visando demonstrar a possibilidade de
uso da metodologia em discussão, apesar das possíveis dificuldades da transposição dos métodos
visuais, que são adotados na Sociologia e na Antropologia, acredita-se que a discussão metodológica
e a ilustração do uso das técnicas de coleta e análise de dados irão contribuir para o enriquecimento
do debate e para a busca do aprimoramento das ferramentas metodológicas em administração e,
especificamente, em marketing. Palavras-chave: pesquisa; métodos visuais; marketing de serviços; cenários de serviços. Fotografias Como um Recurso de Pesquisa em
Marketing: o Uso de Métodos Visuais no Estudo de
Organizaçőes de Serviços Fotografias Como um Recurso de Pesquisa em
Marketing: o Uso de Métodos Visuais no Estudo de
Organizaçőes de Serviços J. Ricardo C. de Mendonça
Maria de Lourdes de Azevedo Barbosa
André Falcão Durão ABSTRACT It is argued that the visual methods of data gathering and analysis in service marketing research yield
a wealth of valuable information, as the use of pictures (or videos) imparts objectivity and credibility
to the researchers’ findings. In this article, pictures of a hospital and of a hotel were taken from their
official web sites, and subsequently analyzed, to show how the recourse to impression management
by both the hospital and the hotel has led the former to intentionally reproduce some features of the
latter. The theoretical basis of the paper lies on the use of the visual methods; on the emphasis on
the role of the scenery in service production, and on the literature on impression management and
service marketing. In spite of the possible difficulties involved in the use of visual methods in
marketing research, (and not only in Sociology and Anthropology, as is usually done), the contention
is made that this article contributes to the enrichment of the debate, and for the quest for improvement
of methodological tools in management and, specifically, marketing. Key words: research; visual methods; service marketing; service set. 57 RAC, v. 11, n. 3, Jul./Set. 2007: 57-81 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão INTRODUÇĂO A administração, desde os seus primórdios, vem beneficiando-se de perspectivas
e metodologias de outras áreas da ciência como, por exemplo, a sociologia, a
antropologia, a ciência política, a psicologia. Entretanto várias abordagens e métodos
dessas disciplinas permanecem desconhecidos ou pouco utilizados nos estudos
em administração. Uma dentre essas abordagens utiliza os métodos visuais
para coleta e análise de dados da vida social. Os métodos visuais têm suas origens na Sociologia e na Antropologia. Harper
(1988) coloca que a abordagem da Sociologia Visual contribuiu para muitos estudos
nas décadas iniciais da sociologia americana, mas desapareceu e ressurgiu durante
a década de sessenta. Para o autor “é justo dizer que de 1920 a 1960 não existiu
Sociologia Visual” (p. 58). Segundo Flick (2004), a discussão sobre uma sociologia visual, centrada em
torno de fotografias e de filmes, é recente. Entretanto para a Antropologia a
inclusão de fotos e filmes em estudos é mais significativa (Harper, 1988). Tanto é
que Lindlof e Taylor (2002) chamam a atenção para o fato de que fotografias,
filmes e vídeos têm sido utilizados como ferramentas comuns na coleta de dados
na Antropologia há mais de 70 anos, o que é confirmado por Gagliardi (2002),
quando coloca que a antropologia visual faz uso etnográfico do filme ou das
fotografias para registrar, por meio de imagens, fenômenos culturais. A abordagem visual é um enfoque metodológico em que o pesquisador ao estudar
um fenômeno social, utiliza as imagens como fonte de dados, sejam elas estáticas
(fotografias) ou em movimento (filmes e vídeos). Como método de pesquisa, a
abordagem visual leva em consideração aspectos como a subjetividade do indivíduo
que registra as imagens, bem como aquilo que ele escolheu incluir ou excluir da
fotografia que foi capturada. Harper (1988) salienta o crescente reconhecimento
do potencial dos métodos visuais para estudos tanto em laboratório, quanto em
ambientes naturais (estudos de campo). Caulfield (1996) também ressalta o
interesse dos cientistas sociais pelo método visual e a diversidade das imagens
visuais. De acordo com Caulfield (1996, p. 56) os dois principais interesses da
abordagem visual são “imagens produzidas pelo pesquisador (ou seus sujeitos) no
curso de um estudo de campo (...) e imagens produzidas pelos atores sociais no
contexto da vida cotidiana”. A capacidade da imagem fotográfica de conter a informação de maneira 58 RAC, v. 11, n. 3, Jul./Set. INTRODUÇĂO 2007 Fotografias Como um Recurso de Pesquisa em Marketing econômica e confiável torna possível uma catalogação mais eficiente de dados
nas pesquisas. Porém os métodos visuais são pouco adotados nos estudos em
administração e raramente têm sido usados em pesquisas qualitativas em
marketing. A pouca utilização do método nos estudos em administração pode ser constatada
por meio da colocação de Gagliardi (2002, p. 128), que afirma: “O estudo dos
artefatos e dos espaços coorporativos emergiu recentemente como uma das mais
interessantes correntes dentro da abordagem geral das organizações entendidas
como cultura”. Seguindo essa perspectiva, o marketing de serviços,
especificamente, pode valer-se do registro das evidências físicas para o envio de
mensagens fortes que ajudam a estabelecer a imagem(1) da empresa e a afetar
as expectativas dos consumidores (Hutton & Richardson, 1995). Nesse caso, o cenário aparece como um dos elementos que influenciam a
percepção do público em relação aos encontros de serviços, aspecto que é
referendado por Hutton e Richardson (1995), quando apontam que os elementos
tangíveis costumam ser citados pelos consumidores com muito mais freqüência
do que os demais atributos determinantes da satisfação e da qualidade do serviço
prestado. A adoção de métodos visuais de coleta e análise de dados em pesquisas de
marketing de serviços, bem como a inclusão das imagens coletadas nos resultados
dos estudos oferece grande riqueza de informações, pois se acredita que pode
proporcionar objetividade e credibilidade, na medida em que uma imagem fornece
a prova tangível da existência de determinado fenômeno. Assim, neste ensaio,
são abordados os métodos visuais, a importância do ambiente físico nos encontros
de serviços e mostra-se um caso-exemplo de como se pode adotar este método
na pesquisa de marketing. Para tanto são utilizadas quatro fotos (capturadas via
internet), duas de um hospital e duas de um hotel, que retratam os mesmos espaços
(apartamento e fachada) encontrados nos dois tipos de empresa. OS MÉTODOS VISUAIS DE PESQUISA A pesquisa qualitativa, onde se enquadram os métodos visuais, apesar de ser
adotada de forma regular e legítima por sociólogos e antropólogos há várias
décadas, só começou a ganhar espaço na área da administração a partir de 1970
(Godoy, 1995). O termo ‘pesquisa qualitativa’ é conceito amplo, que cobre várias
formas de investigação, porém o pressuposto filosófico-chave é que todos os
tipos de pesquisa qualitativa se baseiam em uma visão da realidade como 59 RAC, v. 11, n. 3, Jul./Set. 2007 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão socialmente construída pelos indivíduos em suas interações. Assim, as pesquisas
qualitativas procuram entender e explicar o significado dos fenômenos sociais,
causando o menor distúrbio possível no ambiente natural (Merriam, 1998). Nesse sentido, Collier (1973) defende que o registro fotográfico contribui como
fator de controle para a observação visual direta e acrescenta: “As fotografias
são registros preciosos da realidade material” (p.7). Em complementação, Caulfield
(1996) afirma que as imagens apresentam interesse sociológico pelo menos por
três razões: 1) as imagens refletem o mundo vivo e as relações sociais entre os
participantes; 2) fotografias são, freqüentemente, elementos formativos da vida
social; e 3) as imagens podem reter informações documentais sobre os seus
sujeitos. Ao discutir as fotografias como instrumento e objeto de pesquisa, Flick (2004, p
161) destaca que “as fotografias, os filmes e o vídeo são cada vez mais utilizados
como formas genuínas e fontes de dados”. Porém vale ressaltar a discussão de
Harper (1988) sobre as razões que levam a Sociologia Visual e os métodos visuais
a serem discriminados por muitos pesquisadores. Conforme o autor, isso ocorre
precisamente porque qualquer um pode tirar fotografias; “os métodos sociológicos,
as pessoas tendem a acreditar, devem ser misteriosos e difíceis. (...) É inaceitável
para muitos que algo tão simples possa ser útil como uma ferramenta sociológica”
(Harper, 1988, p. 60). Para Harper (1988) os sociólogos visuais sabem que tirar fotografias com
importância teórica não é mais fácil do que realizar uma observação ou identificar
variáveis significativas. Lindlof e Taylor (2002) afirmam que existem muitas
características da mídia visual que apóiam o seu uso em pesquisas científicas. De
acordo com Collier (1973), os seres humanos, por diversas razões, não são bons
observadores. Assim, a grande capacidade de penetração da câmera fotográfica
pode ajudar a ver mais e de forma mais acurada. OS MÉTODOS VISUAIS DE PESQUISA Com base em Flick (2004), algumas vantagens no uso de câmeras na pesquisa
social podem ser apontadas: 1) as câmeras permitem registros (gravações)
detalhados de fatos; 2) proporcionam apresentação mais abrangente e holística
de estilos de vida e de condições sociais; 3) possibilitam o transporte de artefatos
e a apresentação destes como retratos; 4) permitem a transgressão de limites de
tempo e espaço; 5) podem capturar fatos e processos que sejam muito rápidos ou
complexos para o olho humano; 6) permitem registros (gravações) não-reativas
das observações; 7) são menos seletivas que as observações; e 8) as fotografias
ficam à disposição de outras pessoas para serem reanalisadas. Collier (1973, p. 6) destaca que a câmera é um instrumento; “ela documenta
mecanicamente, mas a sua mecânica não limita, necessariamente, a sensibilidade 60 RAC, v. 11, n. 3, Jul./Set. 2007 Fotografias Como um Recurso de Pesquisa em Marketing do observador humano; ela é um instrumento que exige extrema seletividade”. Para Duffield (1998), os métodos visuais de pesquisa têm potencial para
cristalizarem dados e alcançarem audiências, de um modo que nenhum outro
método pode, pois praticamente todos os aspectos da vida humana usam o sentido
da visão, ou seja, a capacidade de ver. Apesar da maior credibilidade conferida
às imagens, o seu significado é negociável e subjetivo. Harper (2000) ressalta
que, apesar de as imagens representarem dados empíricos, elas não representam
“a verdade”, pois o próprio ato da observação escolhe um ponto de vista(2). Em pesquisas em que este método de coleta de dados é adotado, pode-se fazer
uso de imagens preexistentes ou o pesquisador pode ele mesmo produzir as imagens
que serão analisadas. Segundo Harper (1988) a Sociologia Visual apresenta várias
abordagens, em que os pesquisadores fazem uso de fotografias para descrever
ou analisar fenômenos sociais. Essas abordagens se subdividem em duas áreas
principais: a) a primeira que utiliza a fotografia no sentido convencional de coleta
de dados, sendo os métodos visuais normalmente adotados em trabalhos
relacionados a um problema de pesquisa e a uma teoria específica; b) e a segunda,
em que os sociólogos visuais utilizam fotos produzidas, ou seja, preexistentes na
cultura estudada (Harper, 1988). Harper (1988) descreve ainda, uma categorização de modos para a utilização
de métodos visuais em pesquisas: o modo científico; o modo narrativo; o modo
reflexivo; e o modo fenomenológico. No modo científico, as fotos podem
ser usadas em pesquisas empíricas de várias formas. OS MÉTODOS VISUAIS DE PESQUISA Diversas características de
um fenômeno observável podem ser mais bem entendidas se “congeladas” em
imagem fotográfica. As fotografias podem ser “lidas” no sentido de entender
matizes da interação, da apresentação do self e as relações entre as pessoas e
seu ambiente material. Além disso, como as imagens fotográficas isolam um
momento no tempo, podem-se avaliar mudanças por meio de novas fotografias
do mesmo fenômeno social ou de fenômeno similar. Ainda de acordo com Harper
(1988), nas narrativas visuais(3), que são utilizadas no modo narrativo da
abordagem visual, as fotos estáticas assumem papel similar às narrativas
etnográficas em filme ou de etnografias, que são construídas com base na vida
social como ela naturalmente se desenvolve; já o modo reflexivo exibe uma
relação diferente entre o pesquisador, a imagem e o sujeito da pesquisa. Nas
palavras de Harper (1988, p. 65): No método fotográfico reflexivo, o sujeito compartilha a definição dos
significados; deste modo as definições são “refletidas de volta(4)” a partir do
sujeito. Apesar disso ser difícil de fazer em uma pesquisa convencional, é mais
fácil fazê-lo por meio de métodos fotográficos. O pesquisador pode começar
da mesma maneira, ou seja, fotografando um sujeito ou um ambiente; mas 61 RAC, v. 11, n. 3, Jul./Set. 2007 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão então ele retorna ao sujeito para a definição do significado da imagem. Nesse
processo, que tem sido chamada de “elicitação fotográfica”(5), os papéis do
pesquisador e do sujeitos são alterados. Por fim, o modo fenomenológico, sugere que fotografias podem expressar
equivalência espiritual ou psicológica. Para Harper (1988, p. 66) fotografias podem literalmente descrever, mas nos deixam imóveis; outras
imagens podem inspirar nossas emoções, mas não ser úteis sociologicamente
(ou até falsas). Algumas fotos, entretanto, fazem o oposto; ou seja, comunicam
insights sociológicos de uma maneira artisticamente estimulante. Segundo o autor a abordagem fenomenológica é, em essência, a elicitação do
próprio conhecimento de alguém por meio da análise de fotografias que tenham
significado pessoal. Além dos aspectos mencionados, deve-se considerar também, ao se discutir
atualmente o uso dos métodos visuais, a tecnologia digital. E para ilustrar as
vantagens tecnológicas do uso das modernas câmeras digitais, Lindlof e Taylor
(2002, p. OS MÉTODOS VISUAIS DE PESQUISA 115) destacam que as máquinas fotográficas e câmeras de vídeo digitais
apresentam vantagens em relação às máquinas fotográficas tradicionais, pois
são extremamente compactas e leves; funcionam silenciosamente; fotografam
em situações de baixa iluminação e não requerem processos caros; são facilmente
carregadas em arquivos de computador, onde podem ser editadas e manipuladas
das mais variadas formas, combinadas com outras mídias para colocação em
sites na Web e eletronicamente compartilhadas com usuários distantes. A ilustração da utilização do método visual neste artigo é composta por fotos de
duas organizações, retiradas de sites na web - um Hospital e um Hotel - que
são caracterizados por Zeithaml e Bitner (2003) como organizações de serviços
interpessoais, em que o uso do cenário de serviço ocorre tanto por parte dos
clientes, quanto por parte dos funcionários. Em ambos os tipos de organização, a
complexidade do cenário de serviço é considerada elaborada. Nesse contexto,
acredita-se ser necessária e apropriada uma maior discussão sobre o cenário de
serviços para o atendimento do objetivo proposto neste ensaio. O PAPEL DO CENÁRIO NOS ENCONTROS DE SERVIÇOS Um encontro de serviço é um período de tempo durante o qual os empregados
e clientes de uma organização interagem diretamente (Lovelock & Wright, 2001). Vale destacar que esta interação não se limita à dimensão interpessoal, mas engloba 62 RAC, v. 11, n. 3, Jul./Set. 2007 Fotografias Como um Recurso de Pesquisa em Marketing todos os aspectos da empresa com os quais o consumidor pode relacionar-se,
incluindo, além do fator pessoal, as facilidades físicas e outros elementos visíveis
(Grove & Fisk, 1989). Reafirmando que os encontros de serviços não se limitam às relações entre
pessoas, Gagliardi (2002, p. 135) coloca que “em uma organização, fins são
perseguidos, energias são investidas e idéias são concretizadas em máquinas,
produtos e lugares”. O autor evidencia ainda que nas organizações contemporâneas
os espaços físicos estão vinculados a artefatos, pois as organizações, em geral,
são receptivas aos recursos que possam melhorar a sua eficiência. Para alguns
tipos de serviços, a evidência física(6) ou o cenário é parte vital do sistema de
prestação do serviço, porque, dentre todas as variáveis à disposição da organização,
os elementos tangíveis do serviço são potencialmente aqueles que podem ser
mais prontamente controlados pelos gestores (Hutton & Richardson, 1995). Ao discutirem as evidências físicas dos serviços, Zeithaml e Bitner (2003, p. 233) apontam que “os elementos genéricos de evidência física incluem todos os
aspectos das instalações físicas da organização (cenários), bem como outras
formas de comunicação tangíveis, quer sejam atributos exteriores quer sejam
atributos interiores”. Bitner (1992) apresenta contribuições importantes para a
compreensão dos sistemas de prestação de serviços, explorando o conceito de
Servicescape, que diz respeito aos ambientes sociais e físicos que envolvem
experiência, transação ou evento de serviço. Inicialmente, o modelo Servicescape
baseou-se exclusivamente na noção de ambiente físico. Contudo, considerando-
se que as pessoas em um ambiente podem influenciar as noções relacionadas ao
espaço, o ambiente social foi inserido no modelo (Zeithaml & Bitner, 2003). Analisando as dimensões ambientais do Servicescape, os fatores que constituem
o ambiente físico referem-se às condições ambientais (temperatura, barulho, odor,
música, dentre outros), espaço/funcionalidade (layout, equipamentos, mobília etc.)
e sinais, símbolos e artefatos: sinalização interna, decoração, uniforme dos
funcionários etc. Já os elementos do ambiente social envolvem as interações
sociais que ocorrem entre funcionários da empresa, clientes e qualquer outra
pessoa que esteja presente no mesmo ambiente físico. O PAPEL DO CENÁRIO NOS ENCONTROS DE SERVIÇOS O conjunto de elementos
ambientais irá determinar as respostas ou reações cognitivas, emocionais e físicas,
tanto de clientes quanto de funcionários da empresa. Aqui, cabe destacar a noção
de que as dimensões do ambiente de uma organização afetam tanto os
comportamentos relacionados aos clientes, quanto aqueles relacionados aos
funcionários da organização (Bitner, 1992). Gagliardi (2002) aponta a importância do estudo dos artefatos para o estudo
das organizações como sistemas de significados simbólicos. Segundo o autor, os
artefatos, constituem fenômeno cultural primário que influencia a vida 63 RAC, v. 11, n. 3, Jul./Set. 2007 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão organizacional de duas formas: os artefatos tornam materialmente possível
favorecer, obstruir ou mesmo prescrever a ação organizacional; e os artefatos,
de forma mais geral, podem influenciar a percepção da realidade, chegando até
a delinear crenças, normas e valores culturais. Além disso, “símbolos são
concretizações dos sentidos e as coisas constituem seu domicílio mais usual e
natural” (Gagliardi, 2002, p. 132). O cenário de serviço afeta a percepção e o comportamento dos clientes e,
conseqüentemente, sua satisfação e avaliação da qualidade; por isso é importante
conhecer quais as características do ambiente de serviços que podem influenciar
a percepção dos consumidores. Nesse sentido, Zeithaml e Bitner (2003)
identificaram quatro tipos de papéis desempenhados pelos cenários de serviços:
embalagem; facilitador; socializador; e diferenciador. A embalagem do cenário
é aquilo que uma organização mostra para o exterior e, por isso, pode ser
fundamental na formação das impressões(7) iniciais ou para a construção das
expectativas dos clientes (...)” (ênfase adicionada), fornecendo às organizações
a oportunidade de transmitir determinada imagem para seu público-alvo por meio
da configuração do cenário apresentado. A forma como o cenário é projetado, segundo Zeithaml e Bitner (2003) (layout
e arquitetura), pode ser um aspecto facilitador do processo de serviço e da
interação do prestador de serviço como consumidor. Portanto o cenário deve ser
planejado de acordo com o tipo de serviço a ser prestado e com as operações a
serem executadas e podem ser melhorados quando o cenário de serviços é projetado
com tal enfoque. Nas palavras de Zeithaml e Bitner (2003, p. 237), “o projeto do cenário de
serviços auxilia na socialização tanto de funcionários quanto de clientes, no sentido
de que colabora para formar papéis, comportamentos e relacionamentos
desejados”. O PAPEL DO CENÁRIO NOS ENCONTROS DE SERVIÇOS Os aspectos físicos do palco onde será desempenhado o serviço
proporcionam ‘pistas’ para os atores envolvidos no encontro de serviço
(funcionários e clientes) de como eles devem comportar-se nas interações. Por fim, o cenário também pode ser caracterizado como tendo papel
diferenciador para a organização de serviços, quando o seu projeto apresenta
características distintas daquelas de seus concorrentes. Além disso, o cenário
pode comunicar exatamente qual o segmento de mercado para o qual o serviço
está direcionado, bem como para reposicionar a organização de serviços, buscando
novos nichos de mercado (Zeithaml & Bitner, 2003). Berry (1981), Gronroos (1984) e Lovelock e Wright (2001) consideram tanto o
contexto social quanto o contexto físico das trocas no serviço como aspectos
fundamentais. Reforçando esse pensamento, Parasuraman, Zeithaml e Berry 64 RAC, v. 11, n. 3, Jul./Set. 2007 Fotografias Como um Recurso de Pesquisa em Marketing (1988) evidenciam que das 10 dimensões identificadas por eles como determinantes
de qualidade de serviços, a maioria relaciona-se a aspectos ligados ao cenário
social e ao cenário físico do serviço. (1988) evidenciam que das 10 dimensões identificadas por eles como determinantes
de qualidade de serviços, a maioria relaciona-se a aspectos ligados ao cenário
social e ao cenário físico do serviço. Os aspectos do cenário, quando bem gerenciados, podem potenciar todo o
processo de serviço, agregando valores à sua execução, os quais afetarão de
forma favorável o comportamento dos empregados e dos consumidores. Assim,
acredita-se que os métodos visuais podem oferecer importantes insights para o
estudo dos cenários em serviços. Nesse artigo, serão considerados para a análise os elementos das instalações
interiores e exteriores dos cenários de serviço, especificamente aqueles que
podem comunicar aspectos do serviço por meio da observação dos dados
visuais selecionados. Vergara, Carvalho e Gomes (2004, p. 16), ao discutirem
a espacialidade do teatro das organizações, argumentam que: “nos palcos
teatrais, o desenvolvimento de iluminação cenográfica consolidou o controle
do olhar do espectador. Analogamente, nas organizações, as ‘luzes’ se acendem
para a produção, canalizando a atenção do indivíduo para os resultados
organizacionais”. Observa-se que, ao discutir as interações dos funcionários e seus clientes na
organização de serviços como um teatro, parte-se para um enfoque dramatúrgico,
que é aceito na literatura especializada em marketing de serviços. Essa perspectiva
leva à discussão sobre a relação entre o gerenciamento de impressões (GI), o
marketing de serviços e a metáfora dramatúrgica. O Gerenciamento de Impressőes e o Marketing de Serviços Percebe-se crescente utilização, na literatura de marketing de serviços, da
metáfora teatral, no sentido de entender e explicar os encontros de serviços. Um
dos principais focos de análise da dramaturgia é como as pessoas criam e gerenciam
impressões diante de uma audiência (Grove & Fisk, 1989). A partir do enfoque
de Goffman (1959), autores como Grove e Fisk (1989) e Grove, Fisk e Dorsch
(1998) apontam que o teatro (os elementos da dramaturgia) passa a ser utilizado
como ferramenta analítica para se estudarem os encontros de serviços. Sob esta
perspectiva, os prestadores de serviços, como os atores em uma peça de teatro,
apresentam elementos comuns no desempenho de seus papéis. Tanto atores,
quanto os prestadores de serviços devem procurar criar e manter impressões(8)
favoráveis nas suas audiências, movimentando-se por cenário que é montado
em palco, contando com ambiente de bastidores para lhes dar suporte e buscando,
tal como o elenco de um espetáculo de teatro, o ‘aplauso’ (a satisfação) do seu
público (Carvalho, 1998). 65 RAC, v. 11, n. 3, Jul./Set. 2007 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão A perspectiva de dramaturgia se baseia na visão do comportamento como
metáfora dramática e retrata as interações sociais como teatrais, oferecendo o
meio de analisar e descrever os comportamentos. Um dos principais focos de
análise da dramaturgia é como as pessoas criam e gerenciam impressões diante
de uma audiência (Grove & Fisk, 1989). Conforme Goffman (1959), o gerenciamento de impressões (GI) envolve a
tentativa de estabelecer o significado ou o propósito de interações sociais, que
dirigirão as ações das pessoas, ajudando a projetar as expectativas de papéis, ou
seja, o que se espera de cada um. De acordo com Alvesson (1990), o objetivo do ‘gerenciamento da imagem’ é
produzir um ‘retrato’ (uma ‘descrição’), com certo apelo, de uma organização
para vários públicos - empregados, consumidores, acionistas, governo etc. - e
posicioná-lo de um modo positivo. A imagem é escolhida por alguém dentro de
um conceito específico, definindo alvo e significado para a ação, de forma a
marcar o ‘emissor’ que tenta projetar determinada impressão para audiência
(Alvesson, 1990). Mendonça (2003), de forma mais ampla, define o GI como
todas as atividades que um ator social (indivíduo, grupo ou organização) desenvolve
no sentido de influenciar o modo como outros o vêem, ou seja, a impressão ou a
imagem que desenvolvem ao seu respeito. O Gerenciamento de Impressőes e o Marketing de Serviços O estudo do GI, de acordo com Ornstein (1989), tem se focado principalmente
em comportamentos verbais; entretanto a exploração de comportamentos não-
verbais - tais como aqueles exibidos por meio da decoração de ambientes físicos
- devem ser considerados. Grove, Fisk e Bitner (1992, como citado em Grove,
Fisk, & Dorsch, 1998), apresentam um modelo da experiência do serviço como
drama, incorporando quatro componentes teatrais: (1) os atores, ou o pessoal do
serviço cujos comportamentos e presença ajudam a determinar o serviço; (2) a
audiência, ou os consumidores que são os receptores do serviço; (3) o cenário,
ou o ambiente físico em que o serviço ocorre; e (4) a performance, ou a entrega
do serviço. Para Goffman (1959, p.15) a performance é “toda a atividade de um
determinado participante, em uma dada ocasião, que serve para influenciar, de
algum modo, quaisquer dos outros participantes”. Conforme Gardner e Martinko
(1988), no processo de gerenciamento de impressões, o ambiente proporciona o
cenário e o contexto em que os atores realizarão suas performances para suas
audiências. RAC, v. 11, n. 3, Jul./Set. 2007 66 Fotografias Como um Recurso de Pesquisa em Marketing ILUSTRAÇĂO DO USO DE MÉTODOS VISUAIS NO ESTUDO EM
MARKETING DE SERVIÇOS ILUSTRAÇĂO DO USO DE MÉTODOS VISUAIS NO ESTUDO EM
MARKETING DE SERVIÇOS Na realização desta análise de imagem com o objetivo de identificar o seu
significado dentro de uma representação social, precisou-se estar atento para as
condições que permitiram a imagem representar o objeto, porque, antes de proceder
à análise de uma foto, é necessário, verificar os vários sentidos que ela é capaz
de propiciar. Essas várias leituras, conhecidas como o caráter polissêmico da imagem, não
caracteriza que uma foto possui vários sentidos, mas que seu sentido explícito
cria classes de correspondências que permitem múltiplas interpretações. Essa
característica é fruto da relação existente entre a imagem, o objeto e o
observador. Assim é que se procurou o sentido e a interpretação dos
pesquisadores envolvidos neste estudo, para verificar como o gerenciamento
de impressões leva o hospital, intencionalmente, a se parecer com o hotel,
investindo nos elementos físicos de seus cenários de serviços para criar apelo
visual atrativo para os consumidores. Considera-se neste trabalho que a fotografia é a interpretação de determinado
recorte, sendo um relato da empresa sobre alguns cenários de serviços
(apartamento e fachada) e fruto das representações de um indivíduo ou de um
grupo. Foram selecionadas para esse caso-exemplo duas fotos de hospital e duas de
hotel, extraídas dos sites das empresas, que foram escolhidas por apresentarem
similaridades de posicionamento de produto/serviço e por estarem na mesma
categoria, considerada luxo ou ‘cinco estrelas’, estimando-se as duas como
empresas do ramo da hospitalidade. Para ilustrar os procedimentos adotados na análise de imagens paradas foram
escolhidas fotos de dois cenários físicos comuns às duas empresas: apartamento
(ambiente interno) e fachada (ambiente externo). A partir da análise das
fotos, procurou-se verificar os aspectos comuns e diferenciados existentes nas
imagens dos ambientes selecionados, tomando-se por base os itens constantes da
Tabela 1 (4), e identificar em quais deles o hospital está teatralizando sua oferta
de forma a se parecer com hotel. RAC, v. 11, n. 3, Jul./Set. 2007 67 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão Tabela 1 (4): Critérios Adotados Para a Análise das Condições
Ambientais
Fonte: baseado em Zeithaml e Bitner (2000), Hoffman e Bateson (2003). Fonte: baseado em Zeithaml e Bitner (2000), Hoffman e Bateson (2003). A análise foi realizada por três avaliadores com perfis diferenciados para oferecer
maior consistência aos dados. ILUSTRAÇĂO DO USO DE MÉTODOS VISUAIS NO ESTUDO EM
MARKETING DE SERVIÇOS Um com experiência profissional na área de
hospitalidade, outro especialista na área de organizações e um terceiro pesquisador
da área de marketing. O processo de análise constou de três etapas. 1) Primeira etapa: cada
pesquisador, isoladamente, realizou a análise das fotos; 2) Segunda etapa: os
três pesquisadores, em conjunto, estabeleceram comparações entre os elementos
identificados por cada um deles na etapa anterior, formando o quadro básico que
contemplou os aspectos comuns e diferenciados encontrados; 3) Terceira etapa:
partiu-se para uma análise confirmatória das informações obtidas por meio das
imagens, realizada de forma conjunta, a partir do quadro extraído da segunda
etapa da análise, para verificar quais eram os elementos que permitiam identificar
aspectos do cenário físico do hospital que possuíam uma identidade com os dos
hotéis para, finalmente, levantar proposições sobre elementos do gerenciamento
de impressões que vem sendo utilizado pelos hospitais como estratégia
mercadológica para atrair e reter clientes. A Análise Grande parte da literatura recente tem analisado a mudança do eixo competitivo
relacionado ao advento dos serviços agregados aos produtos e à melhoria da
imagem da empresa na busca da criação de valor junto aos seus clientes. É nessa
direção que os hospitais parecem estar redesenhando estratégias que vêm sendo
empregadas para torná-los mais atrativos aos olhos dos clientes e do mercado. Uma das bases desse novo desenho indica uma ligação entre a gestão de saúde
e a gestão hoteleira, resultando em um campo mais amplo denominado de 68 RAC, v. 11, n. 3, Jul./Set. 2007 Fotografias Como um Recurso de Pesquisa em Marketing hospitalidade. Assim sendo, um estudo comparativo entre imagens pareceu
adequado e interessante para captar esses aspectos. As fotos foram analisadas considerando-se as semelhanças com o seu
referente, em que a imagem adquire uma relação como o objeto de sua
representação por meio dos diversos sentidos incorporados. Assim é que, para
efeito do caso ilustrativo desenvolvido neste estudo, se optou por abordar a
imagem no plano da representação (o que ela mostra), no plano de conteúdo (o
que ela significa) e no plano do significante (a realidade exterior a que ela faz
referência). Ou seja, buscou-se com este caso-exemplo identificar as
semelhanças ou diferenças com a realidade exterior a que a imagem remete. A tentativa de gerenciar impressões por meio do cenário nos hospitais, com
a intenção de fazê-los parecer-se com hotéis, está em função das relações
de serviços em um hospital ocorrerem nas fases que se desenvolve um
tratamento médico, que incluem diversos relacionamentos entre setores e
entre pessoas, caracterizando, principalmente, um contato pessoal de alta
complexidade que inclui a relação paciente/médico, bem como do pessoal de
enfermagem, fisioterapeutas, nutricionistas, psicólogos e outros funcionários,
como porteiros e recepcionistas, até o pessoal de limpeza e higiene. Além
desses aspectos, mais do que em qualquer outra organização, os funcionários
de contato se defrontam com pessoas fragilizadas pela doença e em processo
de regressão psicológica, bem como familiares sofridos e desgastados
(Gonçalves, 1998). Na tentativa de criar ambiente mais atrativo para gerenciar os efeitos negativos
dos aspectos anteriormente citados, os hospitais estão buscando apoio na indústria
hoteleira. Ou seja, algumas estratégias de gerenciamento de impressões começam
a ser incorporadas aos serviços hospitalares. Tal estratégia mercadológica já
vinha sendo empregada pelos hotéis, na medida em que sua função foi sempre a
de vender ‘sonhos’ e ‘experiências agradáveis’. A Análise Feitas essas ressalvas, cada um, dentre os critérios estabelecidos na Tabela 1
(4), mereceu algumas considerações, de modo que a análise levou aos seguintes
resultados. Ambiente Interno São analisados neste tópico os ambientes internos das organizações (conforme
as Figuras 1.4 e 2.4) e de acordo com quadro de referência constante na Tabela
1.4 e a teoria que dá suporte a este estudo. 69 RAC, v. 11, n. 3, Jul./Set. 2007 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão Apartamento ra 1(4): Quarto do Hospital - http://www.hospitalaleinstein.com.b
Figura 2(4): Quarto do Hotel - http://www.novotel.com.br Figura 1(4): Quarto do Hospital - http://www.hospitalaleinstein.com.br Figura 2(4): Quarto do Hotel - http://www.novotel.com.br Arquitetura Interna: Apartamento Arquitetura Interna: Apartamento Arquitetura Interna: Apartamento Os aspectos relacionados à arquitetura interna do apartamento permitem ao
cliente comparar o que é ofertado pelas empresas. Na medida em que a
competição aumenta em uma determinada área do setor de serviços, oportunidades
para diferenciar a empresa tornam-se cada vez mais difíceis de encontrar (Bateson
& Hoffman, 2001). A evidência física e o tratamento dado aos encontros de
serviços podem exercer impacto direto sobre os consumidores, estabelecendo
uma diferenciação no serviço prestado. Verificando-se o apartamento do hospital, percebe-se uma transformação de
sua concepção. Antes frios e brancos; hoje com cortinas coloridas para dar vida
ao ambiente; as paredes em tons pastéis para dar tranqüilidade e sensação de
aconchego, o que faz com que os apartamentos do hotel e do hospital sejam
bastante semelhantes. Esse aspecto refere-se ao papel de embalagem do cenário,
Zeithaml e Bitner (2003, p. 236), ou seja, o cenário de serviços (apartamento) é
aquilo que o hospital e o hotel mostram para o exterior e, por isso, é fundamental
na formação das impressões(9) iniciais ou para a construção das expectativas
dos clientes. Os elementos da evidência física do serviço permitem transmitir
determinada imagem para seu público-alvo por meio da configuração do cenário
apresentado. Analisando a arquitetura interna dos apartamentos (do hospital e do hotel),
observa-se um traço específico que caracteriza a diferença entre os serviços de 70 RAC, v. 11, n. 3, Jul./Set. 2007 Fotografias Como um Recurso de Pesquisa em Marketing saúde e os serviços hoteleiros, a cama hospitalar, que tem design específico para
esse tipo de atendimento. Essa característica, entretanto, não impede que a
aparência geral dos dois apartamentos seja semelhante, pois os espaços, as cores
e o restante do mobiliário apresentam aspectos similares. Até mesmo as cortinas
já estão sendo utilizadas em hospitais, o que constituía restrição comum em alguns
deles por questões de higiene e saúde. Verifica-se então, uma aproximação entre
os aspectos físicos do apartamento de hospital na direção do ambiente encontrado
no quarto de hotel. Layout: Apartamento Em virtude de que os ambientes de serviços são constituídos para atender às
necessidades e interesses específicos dos clientes, o layout do espaço tem
importância fundamental. A relevância do layout das instalações é especialmente
evidente nos cenários de serviços, o que pode influenciar a satisfação dos clientes,
o desempenho da empresa e o comportamento de busca de alternativa pelo
consumidor (Zeithaml & Bitner, 2003). Exemplos nesse sentido são encontrados,
no caso dos hotéis, em que investimentos em apartamentos para deficientes físicos
ou estruturas específicas para clientes da terceira idade, enquanto os hospitais
apresentam layout próprio para a movimentação de pacientes, médicos e
enfermeiros, mas com respeito e valorização do aspecto estético e aparência
geral do apartamento. Existe um layout padrão nos apartamentos, em que os móveis (camas e
acessórios) estão dispostos de modo a oferecer espaço amplo e adequado à
categoria dos produtos/serviços ofertados, permitindo no hospital e no hotel uma
boa circulação de pessoas amplitude e conforto, até mesmo no caso de haver
acompanhante, em ambos os casos. Iluminação: Apartamento Comparando-se o espaço do teatro com o das organizações, verifica-se que no
teatro a iluminação do cenário é capaz de controlar o olhar do espectador. O
mesmo pode ocorrer nas organizações, cujas luzes podem ser trabalhadas a favor
da produção do serviço, focando a atenção do consumidor para os resultados dos
encontros de serviços (Vergara, Carvalho, & Gomes, 2004). Os apartamentos, ambos com iluminação direta e indireta, possuem finalidades
específicas. No caso do hospital, o uso de iluminação indireta próxima à cama
para possibilitar cuidados com o paciente durante a noite, sem o incômodo da luz
direta. Para o hotel esse tipo de iluminação facilita a leitura individual ou alguma
outra necessidade do hóspede. A composição da luz na montagem dos dois
cenários é semelhante na concepção. Móveis e Equipamentos: Apartamento A evidência física é parte vital do sistema de prestação do serviço, tanto
que Hutton e Richardson (1995) colocam que, dentre todas as variáveis à
disposição da organização, os elementos tangíveis do serviço são
potencialmente aqueles que podem ser mais prontamente controlados pelos
gestores. Considerando-se também que os espaços físicos estão vinculados a
artefatos nas organizações contemporâneas, essas são receptivas aos recursos
que possam melhorar a sua eficiência (Gagliardi, 2002) e a escolha dos móveis
e equipamentos, bem assim a sua disposição no ambiente podem influenciar o
resultado dos serviços. Os móveis guardam semelhanças na qualidade percebida das peças, design e 71 RAC, v. 11, n. 3, Jul./Set. 2007 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão tipo de material, harmonia da localização em função da necessidade de cada
espaço e das cores, em combinação com o conjunto da decoração. As imagens
transmitem bem-estar, conforto, requinte, beleza, harmonia de espaços e cores
apropriadas, com a finalidade de tentar criar uma imagem de qualidade e cuidado
com os clientes. Cores: Apartamento As cores, suas tonalidades e combinações geram reações emocionais e
comportamentos, às vezes inesperados, afetando, por exemplo, o humor das
pessoas (Zeithaml & Bitner, 2003). No caso do apartamento do hospital, percebe-
se uma transformação de concepção, com a inclusão de cortinas coloridas, para
dar vida ao ambiente e as paredes em tons pastéis para dar tranqüilidade e sensação
agradável, o que deve colaborar na recuperação do paciente. No caso do hotel,
os tons claros nas paredes e detalhes de decoração com cores diferenciadas
(quadros e colcha) parecem indicar harmonia e beleza. Esse tipo de mudança caracteriza o que Thomas (2002) definiu como movimento
holístico da saúde, que estabelece o importante papel do meio ambiente no processo
terapêutico. Essa perspectiva leva a constatar a adoção, por algumas empresas
de saúde, de conceitos trazidos da área de hotelaria. Sinais, Símbolos e Objetos: Apartamento Sinais, Símbolos e Objetos: Apartamento O estudo dos artefatos é importante para as organizações como sistemas de
significados simbólicos, pois estes constituem fenômeno cultural primário que 72 RAC, v. 11, n. 3, Jul./Set. 2007 Fotografias Como um Recurso de Pesquisa em Marketing influencia a vida organizacional de duas formas: tornam materialmente possível
favorecer, obstruir ou mesmo prescrever a ação organizacional; e, de forma mais
geral, podem influenciar a percepção da realidade, chegando até a delinear crenças,
normas e valores culturais. Além disso, “símbolos são concretizações dos sentidos
e as coisas constituem seu domicílio mais usual e natural” (Gagliardi, 2002, p. 132). Nesse sentido, os sinais expostos no interior de uma estrutura funcionam como
comunicador explícito. Podem ser usados como etiquetas (ex. nome da empresa,
nome do departamento), para orientação (ex. saída, entrada, toilletes) e para
comunicar as regras de comportamento (ex. não fumar, silêncio). Entretanto os
símbolos e objetos do ambiente também têm papel fundamental para os usuários
dos serviços de saúde e de hotelaria, pois fornecem indicativos explícitos aos
usuários acerca do significado do local e as normas e expectativas sobre o
comportamento nesse local. Materiais de qualidade usados na construção, obras
de arte, tapetes e certificados expostos podem comunicar significados simbólicos
e criar uma impressão estética genérica (Zeithaml & Bitner, 2003), o que pode
ser constatado nas fotos analisadas. RAC, v. 11, n. 3, Jul./Set. 2007 Ambiente Externo 73
RAC, v. 11, n. 3, Jul./Set. 2007
Figura 3(4): Fachada do Hospital - http://www.hospitalaleinstein.com.br
Figura 4(4): Fachada do Hotel - http://www.novotel.com.br
Arquitetura Externa: Fachada
O desafio de muitas empresas no momento de ofertar serviços é o de planejar
o espaço e a evidência física, de modo que possam sustentar as necessidades e Figura 3(4): Fachada do Hospital - http://www.hospitalaleinstein.com.br Figura 3(4): Fachada do Hospital - http://www.hospitalaleinstein.com.br
Figura 4(4): Fachada do Hotel - http://www.novotel.com.br
Arquitetura Externa: Fachada Figura 4(4): Fachada do Hotel - http://www.novotel.com.br Figura 4(4): Fachada do Hotel - http://www.novotel.com.br Arquitetura Externa: Fachada Arquitetura Externa: Fachada Arquitetura Externa: Fachada O desafio de muitas empresas no momento de ofertar serviços é o de planejar
o espaço e a evidência física, de modo que possam sustentar as necessidades e 73 RAC, v. 11, n. 3, Jul./Set. 2007 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão preferências do consumidor. Por meio da aparência externa de um prédio, por
exemplo, o cliente pode avaliar o posicionamento da empresa, o tipo de serviço e
o valor a ser pago pelo que é ofertado. O cenário é aquilo que uma organização mostra para o exterior e, por isso,
pode ser fundamental nas impressões iniciais ou para a construção das
expectativas dos clientes – trata-se de uma metáfora visual para um serviço
intangível. Uma instalação bem projetada ou mal projetada do ponto de vista
do cliente pode ser uma experiência prazerosa ou não (Zeithaml & Bitner,
2003). Os dois prédios apresentam similaridades na sua estrutura, por terem
características de modernidade, materiais com novas tecnologias de construção
(vidros arredondados, pinturas especiais). Com formas arquitetônicas
diferenciadas, mas elaboradas, ambos buscam transmitir idéia de organização,
limpeza, beleza e sobriedade. Os prédios são imponentes e com traços elaborados,
o que anteriormente constituía característica própria dos hotéis e está sendo
adotada pelos hospitais para gerenciar impressões. Vale salientar que cenários de serviços podem ser interpretados como forma
de comunicação não-verbal da empresa, transmitindo, por meio sua características
físicas, significados específicos (Zeithaml & Bitner, 2003). Nesse caso, as fotos
(3.4 e 4.4) transmitem imagens de locais sofisticados, podendo ser considerados
como prédios de luxo e, se fosse realizada a análise das fotos das duas fachadas
sem a identificação das empresas, provavelmente não seria possível ao observador
distinguir qual, dentre elas, é efetivamente hospital ou hotel. Sinais, Símbolos e Objetos: Fachada Em determinado ambiente, os símbolos, sinais e objetos podem transmitir
significados de forma direta ou indireta aos clientes, sendo aspectos fundamentais
na formação das impressões sobre o serviço. Esses critérios fornecem
comunicadores explícitos, como a entrada de um local, que tem o propósito de
orientar o cliente no ambiente a que se dirige. Oferecem também indicativos
implícitos relacionados à qualidade do material utilizado na construção das
instalações, as cores, os móveis e o layout que transmitem mensagens que
agregam valor na percepção dos clientes acerca do serviço que estão comprando
(Zeithaml & Bitner, 2003). As características arquitetônicas dos dois prédios propiciam sinais de
organização, manutenção eficiente, higiene, cuidados com a aparência e com
os jardins, verificando-se claramente a relação entre a embalagem para o
produto e o cenário externo para os serviços, pois as características relacionadas
aos símbolos, sinais e objetos permitem ao cliente formar pela imagem da “parte
de fora” o que deve estar dentro da empresa (Zeithaml & Bitner, 2003). Vale
destacar que não se observou a existência de diferenças extremas entre os
cenários analisados, ratificando a utilização de elementos do gerenciamento de
impressões. Paisagismo: Fachada Com base em Edwards e Shackley (1992), pode-se argumentar que os jardins
externos, comunicam significados simbólicos e criam impressão estética genérica
da empresa Pelas fotos verifica-se que o hotel e o hospital realizaram
investimentos semelhantes no aspecto paisagístico, criando harmonia entre a
estrutura dos prédios e espaços disponíveis, oferecendo aspecto mais humanizado
ao ambiente. Cores: Fachada As cores utilizadas nas fachadas dos dois prédios se assemelham pela tonalidade
clara utilizada na pintura. Isto permite considerar que os matizes de cores utilizadas
nas áreas externas do hospital, também observadas no hotel, buscam
descaracterizar o aspecto mais austero, neutro e sem atrativos, que, ao longo dos
tempos, foi sendo associado aos hospitais. De acordo com os psicólogos ambientais, tanto cenários naturais quanto aqueles
criados pelo homem ocasionam reações emocionais (Mintz, 1956; Maslow &
Mintz, 1956 como citado em Turley & Milliman, 2000). Pode-se, portanto,
argumentar que as cores utilizadas exercem função tão importante para a criação
de imagem desses dois tipos de negócios quanto a estrutura e o estilo arquitetônico
dos edifícios. 74 RAC, v. 11, n. 3, Jul./Set. 2007 Fotografias Como um Recurso de Pesquisa em Marketing Tamanho do Prédio: Fachada Os clientes freqüentemente procuram indicadores do ambiente que os auxiliem
a qualificar o local para, então, formar suas expectativas sobre a qualidade do
serviço (Sharma & Stafford, 2000). Um destes indicadores é o tamanho do prédio,
que fornece sinais de imponência, segurança, status, consolidação da empresa no
mercado, dentre outros. Completada a análise, algumas considerações finais e proposições merecem
ser feitas em relação ao tema proposto neste estudo, visando colaborar com
futuros trabalhos voltados para a utilização dos métodos visuais na pesquisa em
administração. Entrada: Fachada As entradas do hospital e do hotel apresentam similaridades quanto à aparência
e forma, pois possibilitam uma visualização e identificação clara de sua utilidade,
o que é fator importante, tanto para o acesso de pacientes como de hóspedes. As
duas entradas sociais principais apresentam traços de arquitetura diferentes, mas
não demonstram divergências aparentes quanto ao seu uso, o que pode levar o
cliente a avaliar a fachada do hospital como a de um hotel, criando expectativas
positivas sobre a empresa. 75 RAC, v. 11, n. 3, Jul./Set. 2007 J. Ricardo C. de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão PROPOSIÇŐES EXTRAÍDAS A PARTIR DA ANÁLISE DAS IMAGENS Como resultado das análises realizadas, pôde-se chegar a algumas
proposições relativas à questão da utilização do gerenciamento de impressões
a partir dos cenários de serviços, como estratégia mercadológica para atrair
e reter clientes. a) P1 Nas áreas comuns aos hospitais e restaurantes
(apartamento e fachada) não se percebe diferenças marcantes na sua
estrutura física e decoração. b) P2 Os hospitais estão utilizando ferramentas
do gerenciamento de impressões para se tornarem mais parecidos com os
hotéis aos olhos do cliente. c) P3 O investimento na estrutura física e
decoração dos hospitais leva o consumidor a criar uma imagem mais positiva
dos serviços de saúde. d) P4 O cenário de serviços é ferramenta importante
para utilização pelos hospitais, como forma de oferecer aos clientes
informações que lhe permitem fazer inferências sobre a qualidade do que é
ofertado. e) P5 O cenário de serviços pode ser utilizado como estratégia de
diferenciação entre as empresas de serviços de saúde. As proposições extraídas desta análise se caracterizam como insights para o
aprofundamento de pesquisas na área de marketing de serviços e para maior
utilização dos métodos visuais em estudos dessa natureza. Feitas essas colocações, parte-se para algumas considerações finais do
trabalho mais voltadas para a utilização dos métodos visuais na pesquisa em
administração. RAC, v. 11, n. 3, Jul./Set. 2007 76 Fotografias Como um Recurso de Pesquisa em Marketing CONSIDERAÇŐES FINAIS CONSIDERAÇŐES FINAIS As imagens analisadas permitiram verificar as similaridades e as divergências
existentes entre dois ambientes de duas empresas de serviços, bem como extrair
sinais e informações em quantidade e qualidade suficientes para chegar-se às
proposições constantes neste trabalho. Essas proposições podem vir a contribuir para o entendimento relativo às
mensagens que os cenários físicos transmitem, como proposição das empresas, e
influenciam as imagens que são formadas pelos consumidores de serviços,
principalmente levando-se em conta que as empresas empregam, nas suas
mensagens visuais, táticas relativas ao gerenciamento de impressões. Isso demonstra que a utilização dos métodos visuais como forma de coleta e
interpretação de dados, na pesquisa de administração, pode contribuir para o
aprofundamento de questões relacionadas ao marketing de serviços, pois as
organizações constituem sistemas de significados simbólicos e os seus artefatos
acabam por influenciar as ações organizacionais e a percepção da realidade
(Gagliardi, 2002). A concretização dos sentidos pode dar-se por meio da análise de imagens,
considerando-se que todos os aspectos da vida humana usam o sentido da visão
(Duffield, 1998) e, neste estudo, essa concretização ocorreu com a avaliação de
fotografias dos cenários de duas empresas de serviços. As informações foram
coletadas a partir de imagens estáticas que constituíram dados empíricos passíveis
de seleção e interpretação, visto que o cenário, enquanto aspecto tangível do
encontro de serviço, solicita profunda análise visual, já que exerce grande influência
na percepção do cliente, pois a fotografia é considerada como registro precioso
da realidade material (Collier, 1973). Nesse sentido, as empresas estão permanentemente tentando “gerenciar imagens”
e produzir “retratos”, com alguns apelos aos seus vários públicos, para posicionar-
se de modo positivo (Alvesson, 1990) e, portanto, comportamentos não verbais,
como a decoração de ambiente, são fundamentais para alcançar esse objetivo. Com esse intuito, o marketing de serviços utiliza-se da metáfora teatral para
entender e explicar os encontros de serviços, procurando verificar como as
empresas criam e gerenciam impressões diante da audiência (Grove & Fisk,
1989). O cenário físico ganha importância nesse contexto, e a observação das
imagens desse ambiente tem papel importante no aprofundamento da teoria
relacionada a esse tema, bem como pode permitir maior desenvolvimento de
metodologias, como a empregada neste trabalho com o uso de imagens. 77 RAC, v. 11, n. 3, Jul./Set. 2007 J. Ricardo C. Artigo recebido em 18.10.2004. Aprovado em 18.10.2005. Artigo recebido em 18.10.2004. Aprovado em 18.10.2005. CONSIDERAÇŐES FINAIS de Mendonça, Maria de Lourdes de Azevedo Barbosa e André Falcão Durão Considerada como uma técnica de observação indireta da realidade social, o
método visual, como qualquer outro método de coleta de dados, possui algumas
limitações. Ao discutir o problema da observação e a natureza da fotografia, Collier
(1973) aponta que uma das limitações da câmera são as próprias limitações de seu
usuário. Ainda de acordo com o autor, enquanto os pesquisadores de campo não
souberem o que fotografar, quando, quantas vezes e por que fotografar, a câmera
não será vista como recurso funcional. Além disso, se os pesquisadores não
dispuserem de recursos seguros para penetrar no conteúdo das fotografias, se não
souberam o que é evidência de confiança e valor e o que é intangível e estritamente
impressionístico, as fotografias não serão utilizadas de forma adequada. Apesar das possíveis dificuldades da transposição dos métodos visuais adotados
na Sociologia e na Antropologia, acredita-se que a discussão metodológica e a
ilustração do uso de métodos visuais de coleta e análise de dados realizadas neste
ensaio contribuíram para o enriquecimento do debate e para a busca do
aprimoramento das ferramentas metodológicas em administração, especialmente
na área de marketing de serviços. NOTAS 1 O termo imagem utilizado no contexto em que foi citado significa um conceito ou conjunto de
opiniões subjetivas de um indivíduo, do público ou de um grupo social, a respeito de pessoa,
produto, empresa etc. 2 Para Collier (1973) as fotografias contêm um número suficiente de “verdades” não verbais, que
permitem ao observador reconstruir a realidade esquemática e formar conceitos. 3 Conforme Harper (2000, p. 724) “para expandir a idéia de dados empíricos de uma única imagem
para uma seqüência de fotos, foi introduzido o conceito de narrativa visual”. 3 Conforme Harper (2000, p. 724) “para expandir a idéia de dados empíricos de uma única imagem
para uma seqüência de fotos, foi introduzido o conceito de narrativa visual”. 4 Da expressão original reflect back. 4 Da expressão original reflect back. 5 Da expressão original photo elicitation. 5 Da expressão original photo elicitation. 6 Características físicas que envolvem um serviço e ajudam os clientes a avaliar a prestação do
serviço (Hoffman & Bateson, 2003). 6 Características físicas que envolvem um serviço e ajudam os clientes a avaliar a prestação do
serviço (Hoffman & Bateson, 2003). 7 A formação de impressões ocorre quando as pessoas descodificam as mensagens e formam
impressões e imagens sobre outras pessoas. 8 Idéia, sentimento ou opinião que o indivíduo tem sobre alguém ou algo, ou que alguém ou algo
transmite ao indivíduo; pode ainda significar o efeito que uma experiência ou uma pessoa tem sobre
alguém ou algo (baseado no Oxford – Advanced learner’s dictionary, 2000). 8 Idéia, sentimento ou opinião que o indivíduo tem sobre alguém ou algo, ou que alguém ou algo
transmite ao indivíduo; pode ainda significar o efeito que uma experiência ou uma pessoa tem sobre
alguém ou algo (baseado no Oxford – Advanced learner’s dictionary, 2000). 9 A formação de impressões ocorre quando as pessoas descodificam as mensagens e formam
impressões e imagens sobre outras pessoas. 9 A formação de impressões ocorre quando as pessoas descodificam as mensagens e formam
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¿Hacia nuevos contratos sociales para la conservación? Revelando las luchas por legitimidad en las áreas protegidas de América Latina
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Debates en Sociología N° 54, 2022, pp. 5-12
e-ISSN 2304-4584
Presentación:
¿Hacia nuevos contratos sociales para la conservación?
Revelando las luchas por legitimidad en las Áreas
Protegidas de América Latina
Deborah Delgado Pugley1
Mattias Borg Rasmussen2
1
Pontificia Universidad Católica del Perú. Correo electrónico: deborah.delgado@pucp.pe https://
orcid.org/0000-0002-5595-0359
2
Universidad de Copenhague, Dinamarca. Correo electrónico: mbr@ifro.ku.dk https://orcid.
org/0000-0002-1869-3525
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Debates en Sociología N° 54, 2022 / e-ISSN 2304-4584
Vivimos en tiempos de emergencias socioambientales múltiples, en los cuales los
umbrales de los sistemas ecológicos planetarios vienen siendo sobrepasados uno a
uno. En este contexto, si bien la historia de la conservación no está libre de patrones
de violencia y despojo, desde un punto de vista sociopolítico conducir esquemas
de preservación de la naturaleza es indispensable para tener un futuro habitable en
un mundo de estructuras moldeadas por las relaciones del capital. Este escenario
nos presenta el reto de reconciliar los legados de la conservación y responder a la
urgencia de encontrar salidas colectivas al aprieto actual. Este número especial nos
llevan a preguntarnos cuáles son las posibilidades para articular nuevos contratos
sociales de este orden, enfocándonos en las luchas por la legitimidad en las áreas
protegidas de América Latina.
La literatura sobre la conservación desde las ciencias sociales ha documentado
los efectos adversos del régimen de Áreas Naturales Protegidas del mundo (West
et al., 2006). Históricamente, la conservación como la conocemos en las Américas
ha estado ligada a la conformación de grandes espacios estatales, manejados con
una lógica vertical, regidos por un espíritu marcado por el legado colonial y capitalista. Sin embargo, la espacialidad de las iniciativas de conservación y los modos
de gestionarla no son algo estático y menos monolítico. El modelo de conservación
como fortaleza, dedicada exclusivamente a la protección de la vida «salvaje» y no
humana, fue acuñado en los parques nacionales de los Estados Unidos hacia fines
del siglo XIX (Taylor, 2016), para luego ser exportado hacia otros continentes. En
el contexto norteamericano, los nativos americanos y los blancos tenían puntos de
vista diferentes sobre el uso de la tierra y esto provocó muchos conflictos, así como
la privación de derechos de los pueblos indígenas.
La historia de la formación del movimiento ambiental demuestra la importancia
de considerar como centrales las diferencias raciales, de género y de clase (Taylor,
2016). Así también lo demuestra la amplitud de su crítica y el surgimiento de paradigmas de conservación más recientes, como el manejo comunitario de los recursos
naturales o los esquemas de pagos por servicios ambientales. Estas corrientes emergieron como intentos de remediar los problemas de ineficiencia en la gestión de la
conservación, y, de forma más trascendente, de violación de derechos fundamentales
sociales, culturales y humanos de los modelos anteriores. Las buenas intenciones y
la incorporación de la crítica a experiencias pasadas, no obstante, presentan ya resultados mixtos. Usando criterios como sensibilización, efectividad en la gobernanza,
legitimidad social y capacidad de movilización, los resultados son dispares.
Si la conservación tiene un historial deficiente en términos de promoción de
derechos fundamentales, en la actualidad enfrenta al menos dos desafíos sustanciales: las áreas protegidas se superponen en su mayoría a las economías de frontera,
donde las instituciones públicas y privadas en competencia, el capital legal y las
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Delgado Pugley y Borg Rasmussen / Presentación
economías ilegales luchan por asegurar el acceso a cualquier recurso que tenga valor
para ellos (Delgado, 2019; Hecht, 2005; Messina et al., 2006; Larsen, 2015; Norris,
2017; Wilson y Coomes, 2019). En segundo lugar, el proyecto de conservación es
inherentemente un dilema espacial: se basa aún en imaginarios coloniales de sedentarismo y de límites estáticos (Adams, 2020; Noe, 2010). Al pretender establecer
límites estables para situar lo que concibe como natural, transforma relaciones
sociales que podrían ser más adecuadas de gestionar el territorio.
Nuevos espacios y respuestas desde las ciencias sociales se abren frente a estos
dilemas. La mirada crítica sobre la historia de la conservación sigue vigente, pero
hay un giro hacia una mayor atención a la necesidad de crear regímenes e instituciones que velen por la preservación de la naturaleza. Vías para dar un lugar central
a las miradas de los pueblos indígenas a problemáticas ambientales globales se van
haciendo paso (Wilson et al., 2022). Se sintetizan y discuten aproximaciones que
surgen de la práctica de la conservación a partir de la valoración de las prioridades y
los activos y no de las carencias de los actores locales (Wali et al., 2017).
En los últimos años, han surgido propuestas para la conservación de convivialidad, en particular desde los trabajos de Bram Buscher y Robert Fletcher (2020) y
otras investigadoras e investigadores que trabajan de cerca en casos situados en Asia
y África (Krauss, 2021). Estos autores proponen una tercera vía entre los intentos
del neoconservacionismo de reservar cada vez más territorios, que tuvo su máxima
expresión en la ambición del trabajo de O.E. Wilson (2016) llamado Half-Earth,
y otras intervenciones conservacionistas basadas en lógicas del mercado capitalista,
como los pagos por servicios ambientales o el REDD+. El problema —sostienen los
autores— es que ambas soluciones reproducen relaciones coloniales y capitalistas.
Para enfrentar esta situación, proponen que busquemos una conservación de convivialidad basada en principios de postcrecimiento y descolonización.
Es una propuesta interesante e importante que también demanda contextualización y enraizamiento territorial. En particular, para el caso de América Latina,
creemos que vale la pena explorar las diferentes formas y experiencias que ya se
están implementando en los territorios de conservación (Paredes y Kaulard, 2021).
Estamos interesados en las posibilidades de construir territorios de conservación
enmarcados por lo que Astrid Ulloa (2011) llama «autonomías relacionales»,
pensando en las formas de gobernanza que surgen en los encuentros entre los
proyectos de conservación y las autonomías enraizados en territorios vivos. Para
Ulloa, debemos comprender los reclamos por autonomía o gobernanza en su
contexto particular, considerando sus articulaciones específicas con instituciones
estatales. Pensemos la conservación desde los territorios y a través de sus prácticas e
interacciones cotidianas y las negociaciones entre diversos actores desde un análisis
de su contexto particular.
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Debates en Sociología N° 54, 2022 / e-ISSN 2304-4584
Los encuentros y desencuentros en torno a la conservación, así como su
influencia en la reconfiguración de los territorios, producen debates, enfrentamientos y diálogos sobre el porqué, el quién y el cómo de las prácticas ambientales
(Rasmussen, 2022). Como han señalado con mucha claridad los estudios empíricos
sobre la conservación, los encuentros de conservación siempre son marcados por
asimetrías de poder. Las autonomías relacionales como proceso implican reconocimiento mutuo, una articulación por un contrato social con lo provisorio que esto
sea. Es ahí donde ubicamos nuestra preocupación por la legitimidad. Una conservación diferente, basada en las autonomías relacionales, requiere un entendimiento
por los procesos de legitimación que autorizan el ejercicio de poder en el control de
recursos y territorios.
Este número especial de Debates en Sociología discute las posibilidades de tener
nuevos acuerdos sociales en torno a la conservación en América Latina. El desafío
que aborda es el de observar aquellas pistas que van más allá de la conservación como
un proyecto dirigido por estados territoriales y definido de forma externa al territorio en que se impone. Examina las iniciativas de conservación en varios países de
América Latina, donde comunidades locales y Pueblos Indígenas han interactuado
con los regímenes de conservación, negociándolos y transformándolos mediante el
establecimiento de reservas comunales, áreas protegidas privadas y áreas naturales
de manejo integrado. En efecto, este número muestra lo difícil —pero posible—
que es diferir de la conservación basada en ordenes impuestos de arriba hacia abajo,
criticada a fondo en la literatura (Brosius et al., 2005). Esto es posible gracias a
oportunidades cuyo potencial legal ha sido apropiado —y, en ocasiones, reformulado— por comunidades de la región amazónica y andina (Orihuela, 2020). Podría
decirse que representa una renegociación de los términos de la conservación en la
que las iniciativas de conservación están cada vez más dirigidas por la comunidad
(Artelle et al., 2019).
En este dossier se agrupan trabajos empíricos y teóricos que siguen diferentes
metodologías y acercamientos conceptuales usando casos en varios países de
América Latina. En esta colección indagamos acerca de diferentes formas de relacionarse con los proyectos territoriales de los sistemas de áreas naturales protegidas.
Desde Colombia, Santiago Martínez-Medina, Hanne Cottyn, Ana María
Garrido Corredor y Joshua Kirshner discuten de una manera innovadora e interdisciplinaria sobre la conservación a partir del caso de Chingaza, en Colombia.
En su artículo, «Osos vaqueros en el páramo incomún: hacia una conservación
cosmopolítica del oso andino en el páramo de Chingaza, Colombia», nos proponen
una vía incomún para la conservación, confrontando a los expertos en el estudio
y la conservación de este animal desde relatos de las comunidades campesinas que
refieren la existencia de dos tipos de oso.
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Graciela María Preda, Mercedes Ejarque, Sofía Lammel y Florencia Pasetto,
de Argentina, en su artículo «Entre la conservación y la producción: las familias
crianceras del Área Natural Protegida El Tromen (Neuquén, Argentina)», traen
un enfoque histórico para entender los usos y sentidos múltiples de territorios de
conservación. Enfocándose en un grupo de crianceros con prácticas de trashumancia, las autoras reconstruyen sus trayectorias y las prácticas que los enraizan en
estos territorios. El Área Natural Protegida El Tromen surge en este contexto como
un actor importante para mediar sus relaciones con el entorno. Si bien la normativa
no considera las actividades productivas, en las prácticas cotidianas se establecen
vínculos y acuerdos para buscar compatibilizar los principios de conservación y de
producción. El artículo muestra así la importancia de tener en cuenta las dinámicas
de las poblaciones y sus trayectorias históricas en la formulación de políticas de
conservación.
Ana Watson y Conny Davidsen nos llevan a la intersección entre la agenda
extractiva y la protección de áreas en territorios ancestrales de la Amazonía peruana.
En su artículo «Conservation Utilitarianism and Indigenous Tactics: Protected Area
and Extractive Agendas in the Peruvian Amazon», narran los efectos contradictorios
del proyecto hidrocarburífero de Camisea. Muestran cómo las comunidades indígenas y sus líderes intentan navegar entre dos proyectos territoriales. Enfatizan las
estrategias indígenas en contextos donde tanto la conservación como la extracción
reproducen jerarquías de saberes y de poder de decisión. El artículo muestra con
sensibilidad etnográfica cómo la conservación puede ser un aliado en luchas sociales
por el territorio en arreglos que son siempre provisorios.
Jean Paul Benavides, de la Universidad Católica de Bolivia, nos trae al caso de la
comunidad del río Ivirgarzama, que forma parte del pueblo Yuracaré, y su interacción con pueblos indígenas colonos altoandinos dedicados a la producción de hoja
de coca en sus territorios ancestrales. En su artículo «Territorios indígenas versus
colonizadores campesinos en Bolivia. Estudio de caso de la Comunidad Indígena
Yuracaré del río Ivirgarzama - CIYRI», busca fundamentar que el desconocimiento
de los derechos reconocidos a los Pueblos Indígenas resulta contraproducente para
la sostenibilidad de los bosques y recursos naturales. Esto sucede en un contexto en
el cual la identidad indígena en general es reivindicada por el Estado. Muestra que
la titulación de territorios no es una medida suficiente para garantizar los medios
de vida indígenas y el bosque asociado de acuerdo con su propio proyecto colectivo
y reflexiona sobre las perspectivas para la conservación en la Amazonía boliviana.
Jorje Zalles discute las reservas de la biosfera desde la experiencia latinoamericana en un ensayo que nos ubica frente a los límites y posibilidades de esta forma de
conservación, que fue una de los pioneras en reconocer la importancia de las sociedades aledañas para las reservas. En su ensayo «Territorios sustentables: legitimando
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Debates en Sociología N° 54, 2022 / e-ISSN 2304-4584
la conservación en reservas de biosfera», demuestra cómo el concepto de reservas de
biosfera ha sido adoptado con entusiasmo en América Latina y el Caribe. El artículo
examina las condiciones bajo las cuales se evalúa el éxito de una reserva de biosfera
y reflexiona sobre la aptitud de este modelo de conservación en la región.
Cerramos el número especial con una entrevista a Fermín Chimatani Tayori,
líder del pueblo Harakbut y presidente de la Asociación Nacional de Ejecutores
de Contrato Administrativo para las Reservas Comunales - ANECAP del Perú.
Podemos escuchar de primera mano la diferencia que existe entre un paradigma
que prefiere dedicarse a la conservación y otro que ofrece alternativas económicas
a los pueblos indígenas que de una u otra manera se ven involucrados en el trabajo
intenso que implica la conservación.
***
Adicional al dossier de este número especial, contamos con dos artículos agrupados en la sección Varia de la revista. Ambos giran en torno a estudios de sexualidad
y género y están centrados específicamente en relaciones homosexuales de España
y Argentina. El artículo «Llegó la ruptura: analizando los divorcios de matrimonios
del mismo sexo en España», de los autores Diego Becerril, José Jiménez-Cabello,
José Luis Paniza e Inmaculada Puertas, ofrece una mirada poco frecuente y muy
necesaria a los matrimonios del mismo sexo. Eso por encontrarse al reverso de
discursos de aceptación y legalización de la diversidad. Finalmente, el artículo de
Maximiliano Marentes, «Seducir, levantar, estabilizar: amor y política entre varones
gays argentinos» analiza la intersección entre trayectorias amorosas y políticas,
encontrando una profunda imbricación entre ambas esferas.
Referencias
Adams, W.M. (2020). Geographies of conservation III: Nature’s spaces. Progress in Human
Geography, 44(4), 789-801. https://doi.org/10.1177%2F0309132519837779
Artelle, K.A., Zurba, M., Bhattacharyya, J., Chan, D. E., Brown, K., Housty, J. y Moola, F.
(2019). Supporting resurgent Indigenous-led governance: A nascent mechanism for
just and effective conservation. Biological Conservation (240), 108284. https://doi.
org/10.1016/j.biocon.2019.108284
Brosius, P.J., Lowenhaupt Tsing, A. y Zerner, C. (eds.) (2005). Communities and conservation: histories and politics of community-based natural resource management. Altamira
Press.
Buscher, B. y Fletcher, R. (2020). The conservation revolution: radical ideas for saving nature
beyond the Anthropocene. Verso Trade.
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Delgado, D. (2018). Rights, justice, and REDD+: Lessons from climate advocacy and
early implementation in the Amazon Basin. En Duyck S., Jodoin S., Johl A. (eds.),
Routledge Handbook of Human Rights and Climate Governance (pp. 183-198).
Routledge.
Krauss, J.E. (2021). Decolonizing, conviviality and convivial conservation: towards
a convivial SDG 15, life on land? Journal of Political Ecology, 28(1). https://doi.
org/10.2458/jpe.3008
Hecht, S.B. (2005). Soybeans, development and conservation on the Amazon
frontier.
Development
and
Change,
36(2),
375-404.
https://doi.
org/10.1111/j.0012-155X.2005.00415.x
Larsen, P. (2015). Post-frontier resource governance: indigenous rights, extraction and conservation in the Peruvian Amazon. Springer.
Messina, J.P., Walsh, S.J., Mena, C.F. y Delamater, P. L. (2006). Land tenure and deforestation patterns in the Ecuadorian Amazon: Conflicts in land conservation in
frontier settings. Applied Geography, 26(2),113-128. https://doi.org/10.1016/j.
apgeog.2005.11.003
Noe, C. (2010). Spatiality and «borderlessness» in transfrontier conservation areas. South
African Geographical Journal, 92(2),144-159. https://hdl.handle.net/10520/
EJC93345
Norris, T.B. (2017). Shared Social License: Mining and Conservation in the Peruvian
Andes. Antipode, 49(3), 721-741. https://doi.org/10.1111/anti.12300.
Orihuela, J. C. (2020). Embedded Countermovements: The Forging of Protected Areas and
Native Communities in the Peruvian Amazon. New Political Economy, 25(1),140155. https://doi.org/10.1080/13563467.2019.1570101.
Paredes, M. y Kaulard, A. (2020). Fighting the climate crisis in persistently unequal land
regimes: Natural Protected Areas in the Peruvian Amazon. Journal of Cleaner
Production (265), 121605. https://doi.org/10.1016/j.jclepro.2020.121605
Rasmussen, M.B. (2022). Convivencia negociada y gobernanza ambiental en áreas naturales protegidas del Perú. Íconos. Revista de Ciencias Sociales (72), 161-183. https://
doi.org/10.17141/iconos.72.2022.4953
Taylor, D.E. (2016). The Rise of the American Conservation Movement. Duke University
Press. https://doi.org/10.1515/9780822373971
Ulloa, A. (2011). The politics of autonomy of indigenous peoples of the Sierra Nevada
de Santa Marta, Colombia: A process of relational indigenous autonomy. Latin
American and Caribbean Ethnic Studies, 6(1), 79-107. https://doi.org/10.1080/17
442222.2011.543874
Wali, A., Alvira, D., Tallman P., Ravikumar, A. y Macedo, M. (2017). A new approach to
conservation: using community empowerment for sustainable well-being. Ecology
and Society, 22(4), 6. https://doi.org/10.5751/ES-09598-220406
West, P., Igoe, J. y Brockington, D. (2006). Parks and peoples: the social impact of protected
areas. Annual Review of Anthropology, 35, 251-277. https://doi.org/10.1146/
annurev.anthro.35.081705.123308
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Wilson, E.O. (2016). Half-earth: our planet’s fight for life. WW Norton & Company.
Wilson, S.J. y Coomes, O. T. (2019). «Crisis restoration» in post-frontier tropical environments: Replanting cloud forests in the Ecuadorian Andes. Journal of Rural Studies,
67, 152-165. https://doi.org/10.1016/j.jrurstud.2019.02.023
Wilson, N.J., Lira, M.G. y O’Hanlon, G. (2022). A systematic scoping review of Indigenous
governance concepts in the climate governance literature. Climatic Change, 171, 32.
https://doi.org/10.1007/s10584-022-03354-7
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Effects of self-reported sensitivity and road-traffic noise levels on the immune system
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PloS one
| 2,017
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cc-by
| 6,122
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RESEARCH ARTICLE Editor: Markus M. Bachschmid, Boston University,
UNITED STATES Received: April 4, 2017
Accepted: October 15, 2017
Published: October 30, 2017 Copyright: © 2017 Kim et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Data Availability Statement: All relevant data are
included within the paper and its Supporting
Information files. Funding: This study was funded by the Korea
Ministry of Environment (MOE) as “the
Environmental Health Action Program” to CSS and
JHS received funding from Korea Ministry of
Environment, grant number: 2014001350001
(URL: http://www.me.go.kr/home/web/main.do). The funder had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript. Ahra Kim1, Joo Hyun Sung2, Jin-Hee Bang1, Seung Woo Cho3, Jiho Lee3, Chang
Sun Sim3* Ahra Kim1, Joo Hyun Sung2, Jin-Hee Bang1, Seung Woo Cho3, Jiho Lee3, Chang
Sun Sim3* 1 Environmental Health Center, University of Ulsan College of Medicine, Ulsan, Republic of Korea,
2 Department of Occupational and Environmental Medicine, Gyeongsang National University Changwon
Hospital, Gyeongsang National University School of Medicine, Changwon, Republic of Korea, 3 Department
of Occupational and Environmental Medicine, Ulsan University Hospital, University of Ulsan College of
Medicine, Ulsan, Republic of Korea a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 * zzz0202@naver.com * zzz0202@naver.com Abstract Sensitivity to noise, particularly road traffic noise, can increase cortisol levels and result in
changes in immune system biomarkers. Therefore, continuous exposure to noise can have
an effect on immune function, hormonal levels, and cardiovascular function, leading to
hypertension and stress. The purpose of this study was to investigate the changes in stress-
and immune system-related biomarkers according to the self-reported sensitivity to noise
and exposure to road traffic noise, to ultimately determine the potential effects of noise on
health. A survey was conducted through questionnaire (ISO/TS 15666) sent to 172 female
subjects in Korea, including 128 from Ulsan and 44 from Seoul. The average noise level
was calculated, and blood samples were collected for measurements of cortisol levels,
Natural killer (NK) / Natural killer T (NKT) cell populations, and NK cell activity (through mea-
surements of interleukin-12 (IL-12) and interferon-gamma (INF-γ) concentrations). Multivari-
ate linear regression analysis of the measured biomarkers according to the road traffic noise
level and self-reported noise sensitivity was conducted adjusting for the effects of age, alco-
hol status, smoking status, regular exercise, and residence period. IL-12 levels increased,
whereas the NKT cell population decreased with increasing noise levels. The results further
suggested that cortisol levels are more influenced by the subject’s sensitivity to noise than
to the level of chronic road traffic noise. Therefore, noise appears to have the largest effect
on IL-12 levels as well as the population and activity of NKT cells. In conclusion, our results
suggest that low-level road traffic noise and sensitivity to noise can affect health by causing
changes in the immune response through mechanisms other than increased cortisol. OPEN ACCESS Citation: Kim A, Sung JH, Bang J-H, Cho SW, Lee
J, Sim CS (2017) Effects of self-reported sensitivity
and road-traffic noise levels on the immune
system. PLoS ONE 12(10): e0187084. https://doi. org/10.1371/journal.pone.0187084 Citation: Kim A, Sung JH, Bang J-H, Cho SW, Lee
J, Sim CS (2017) Effects of self-reported sensitivity
and road-traffic noise levels on the immune
system. PLoS ONE 12(10): e0187084. https://doi. org/10.1371/journal.pone.0187084 Editor: Markus M. Bachschmid, Boston University,
UNITED STATES Editor: Markus M. Bachschmid, Boston University,
UNITED STATES Immune response to road-traffic noise [6] and stress [7, 8]. Therefore, there has been much research interest on the potential health
effects of exposure to environmental noise. Competing interests: The authors have declared
that no competing interests exist. Competing interests: The authors have declared
that no competing interests exist. Through meta- analysis, a small number of studies have shown that noise-induced cardio-
vascular disease is associated with a higher risk for men than for women and a higher risk for
people aged older than 65 years [9]. Recent studies have also reported that sleep quality and noise sensitivity are not related to
vascular function or noise sensitivity, but rather that night noise increases the risk of cardio-
vascular disease due to increased blood pressure in patients and controls [10]. Road traffic
noise is a particular noise source that affects a large portion of urban populations. Indeed,
more than 40% of the populations in European Union countries are exposed to noise of 55 dB
or above; 20% are exposed to daytime noise of 65 dB and above, and more than 30% are
exposed to nighttime noise of 55 dB or above [1]. The World Health Organization stated that
noise has negative effects on health by triggering physiological changes such as impairment of
hearing function and increases in stress hormones and sensitivity [11, 12]. The most well-known mechanism mediating the response to such stress is the hypotha-
lamic-pituitary- adrenal axis (HPA axis). When the HPA axis receives a signal of a stress
response, corticotropin releasing factor is secreted from the hypothalamus, releasing adreno-
corticotropic hormone from the pituitary gland. Adrenocorticotropic hormone then promotes
the secretion of cortisol from the adrenal cortex through the blood, which triggers responses to
various kinds of stress. The secretion of cortisol in response to stress inhibits the function of
the HPA axis to disrupt the secretion of neurohormones and neurotransmitters as well as
influencing the endocrine system, thereby disturbing homeostasis of the body, which can
induce the development of various stress-related diseases[13]. Recently Meyer et al. [14]
reported that mental stress and noise exposure could activate inflammatory cytokines such as
interleukin (IL)-6 and IL-1β. These cytokines also interact with each other. Extracllular IL-6
induces cortisol from the zona fasciculate of the adrenal cortex, and has been reported to affect
the synthesis of cortisol even when the HPA axis is inhibited. Introduction Environmental noise can be defined as the noise emitted from all sources, other than noise
at an industrial workplace [1]. Several studies have shown that environmental noise such as
that due to traffic, aircrafts, and construction can have physiological and psychological conse-
quences [2, 3] such annoyance, sleep disturbance [4], cardiovascular disease [5], hypertension PLOS ONE | https://doi.org/10.1371/journal.pone.0187084
October 30, 2017 1 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0187084
October 30, 2017 Thereby, the immune, endocrine
and nervous systems are related to each other. Thus, environmental noise has direct effects on health as well as indirect effects through the
release of stress hormones such as cortisol [15, 16], dopamine [17, 18] and changes of alpha-
amylase levels [19]. Increased cortisol can further cause changes in the immune system such as
cellular proliferation, cytokine secretion, antibody production and cytotoxicity [20, 21]. Indeed,
one study showed that the activity of natural killer (NK) cells was decreased by increased corti-
sol [22]. NK cells are a leukocyte subset and important components of innate immunity. The
innate immune system provides a rapid, non-specific host response against foreign agents such
as bacteria, viruses, or tumors before triggering the adoptive immune system [23–25]. Innate
immunity includes the antigen-presenting cells, monocytes/macrophages, dendritic cells (DCs),
NK cells, and NKT cells. Moreover, cytokine signaling is essential for intercellular communication in the immune
system to mediate and control immune functions. IL-12 is an important cytokine mediating
immune responses, which is mainly produced by monocytes, macrophages, and DCs in
response to bacterial products, intracellular pathogens, or upon interaction with activated T
cells. IL-12 has been shown to play a critical role in the pathogenesis of a variety of immune-
related diseases. This cytokine can induce interferon-gamma (IFN-γ) production [26], cell
proliferation, and cytotoxicity mediated by NK cells and T cells. Hence, exposure to environmental noise affects immunity by inducing stress and increasing
the secretion of stress hormones. As sensitivity to noise itself will affect the stress response and
such sensitivity varies among individuals, the effects and response of exposure to identical
environmental noise can show high individual variation. 2 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0187084
October 30, 2017 Immune response to road-traffic noise However, studies on the negative effects of environmental noise, including road traffic
noise, on health are still lacking in Korea. Therefore, the aim of our study was to analyze the
immune response against noise generated from road traffic as well as the self-reported sensitiv-
ity to noise in two metropolitan cities in Korea. Specifically, the level of exposure to road traffic
noise was estimated using the noise map generated for Ulsan Nam-gu and Seoul Yangchun-
gu, and the changes of immune response parameters according to road traffic noise and sensi-
tivity were statistically analyzed. Questionnaires The questionnaires included age, residence period, education level, monthly income, alcohol
status, smoking status, and exercise status. Education level was divided into a high school
degree or below and a community college degree or above. Monthly income was divided into
less than 3 million Won and above. Smoking status was divided into current smokers and cur-
rent non-smokers (past smokers and non-smokers). Alcohol status was divided into current
drinker and current non-drinker (past drinker and non-drinker). Exercise status was divided
into participants currently exercising regularly and those who were not. To assess the sensitiv-
ity to noise, an 11-point visual analogue scale was generated and used in this study based on
ISO/TS 15666 [27]. All research procedures were approved by the Ulsan University Hospital Institutional
Review Board (UUh 2014-08-008-012). Blood sampling and NK cell preparation and flow cytometry After the subjects were selected, 7 mL of venous whole blood was taken from the respondents
between 9.00 am to 12.00 pm. Peripheral blood mononuclear cells (PBMCs) were isolated from all 172 subjects. The NK
cell population was analyzed by fluorescence-activated cell sorting (FACs) at Green Cross Lab-
Cells Corporation (Seoul, Korea) to identify the proportion of CD45+CD16+CD56+ cells. Cor-
tisol levels were measured by Quantikine enzyme-linked immunosorbent (ELISA) analysis
using serum from Green Cross LabCells Corporation. The concentrations of IL-12 and INF-γ
in the sera were measured using the Quantikine enzyme-linked immunosorbent (ELISA) kit,
according to the manufacturer’s instructions (Bio Legend San Diego, CA, USA). Immune response to road-traffic noise the survey was used to calculate the mean noise levels of the buildings the participants reside
in as a road traffic noise index. The noise index used in this study was the day-night average sound level (Ldn), which is a
qualitative index of the weighted equivalent day/night noise level that divides a 24h period into
6 am–10 pm and nighttime. Actual noise exposure levels were assessed using a sound level meter (Ulsan: NA-28, Rion,
Japan; Seoul: B&K 2250, Bru¨el & Kjær, Denmark). Statistical analysis The statistical analysis was performed with SPSS v.21 for Window (IBM SPSS Inc., Chicago,
IL, USA). The data regarding immune response parameters such as cortisol, NK cell popula-
tion, and cytokines showed positively skewed distributions; thus, logarithmic transformations
were performed for these variables to facilitate further statistical analyses under normal data
assumptions. Statistical analyses were performed using multiple linear regression and Pearson’s correla-
tion analysis. The results were considered statistically significant when P < 0.05. Participants and noise measurement All subjects participated voluntarily and provided written informed consent. We recruited 1,000 people in Yangcheon-gu (Seoul) and Nam-gu (Ulsan), respectively
from July 2015 to December 2015. Participants were stratified according to noise exposure
level based on noise map data. Although the residents of Yangcheon-gu are exposed to aircraft
and road traffic noise, those of Nam-gu are exposed only to road traffic noise. A total of 336 participants each in Nam-gu, Ulsan and Yangcheon-gu, Seoul finally agreed
to the blood test and included in the study for analysis (Fig 1). The questionnaires and blood test results of 336 participants were reviewed. Questionnaires
were missing for four subjects, 93 subjects were excluded because of underlying chronic dis-
eases, and 67 subjects were men; thus, 172 female subjects were finally selected. The male participants were excluded because they tend to have more activity in environ-
ments outside of the residence target area, which could influence the sensitivity to the envi-
ronmental noise around the residence. Subjects with any chronic illness (hypertension,
hyperlipidemia, stroke, myocardial infarction, angina, arrhythmia, diabetes, and other dis-
eases) were also excluded, because of a potential interference on the influence of noise on
the immune response. To examine the level of environmental noise that the participants were exposed to, a three-
dimensional noise map generated in 2014 based on the participants’ addresses confirmed from Fig 1. Location of residents in Ulsan (A, Nam-gu), Seoul (B, Yangcheon-gu) in noise map. It shows the
noise level of each area and the location of participants in Nam-gu of Ulsan (A) and Yancheon-gu of Seoul (B). https://doi.org/10.1371/journal.pone.0187084.g001 Fig 1. Location of residents in Ulsan (A, Nam-gu), Seoul (B, Yangcheon-gu) in noise map. It shows the
noise level of each area and the location of participants in Nam-gu of Ulsan (A) and Yancheon-gu of Seoul (B). https://doi.org/10.1371/journal.pone.0187084.g001 3 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0187084
October 30, 2017 Immune response to road-traffic noise Table 1. General characteristics of the subjects. Ulsan
Seoul
Total
N
Mean
95% CI
N
Mean
95% CI
N
Mean
95% CI
Age (years)
128
44.9
42.9–46.8
44
45.6
41.8–49.5
172
45.1
43.3–46.8
20–29 years
11 (8.6)
5 (11.4)
16 (9.3)
30–39 years
29 (22.7)
7 (15.9)
36 (20.9)
40–49 years
40 (31.3)
14 (31.8)
54 (31.4)
50–59 years
39 (30.5)
13 (29.5)
52 (30.2)
Over 60 years
9 (7.0)
5 (11.4)
14 (8.1)
Residence period (years)
128
9.6
8.3–10.9
42
7.9
5.5–10.3
170
9.2
8.0–10.3
Noise sensitivity
128
5.4
5.1–5.8
44
6.5
5.9–7.2
172
5.72
5.38–6.05
Education
level
High school and less
62
23
85 (49.4)
College and more
66
21
87 (50.6)
Income
Under 3,000,000 (KWR)
16
19
35 (20.3)
Over 3,000,000 (KWR)
112
25
137 (79.7)
Alcohol
status
No
48
28
76 (44.2)
Yes
80
16
96 (55.8)
Smoking
status
No
125
44
169 (98.3)
Yes
3
0
3 (1.7)
Regular
exercise
No
70
12
82 (47.7)
Yes
58
32
90 (52.3)
Unit: Number (percentage)
https://doi.org/10.1371/journal.pone.0187084.t001 Table 1. General characteristics of the subjects. was more than 60 years in Ulsan and Seoul. In the case of Seoul, the subjects in their 20s also
showed the lowest participation rate. The mean age of the participants was 45.1 years and the mean residence period at the
current residence was 9.2 years. The mean sensitivity to noise was 5.72. With respect to edu-
cation level, 49.4% of the participants had a community college degree or below and 50.6%
had a community college degree or above. For monthly income, 79.7% of the participants
reported earning 3,000,000 Won or more and 20.3% reported earning less than 3,000,000
Won. The frequency of current and non-drinkers was 55.8% and 44.2% respectively, and
the frequency of current and non-smokers was 1.7% and 98.3%, respectively. In addition,
52.3% of the participants reported exercising regularly and 47.7% reported that they did not
exercise regularly. As shown in Table 2, road traffic noise and IL-12 were significantly positively correlated
(r = 0.2333). Road traffic noise and the NKT cell distribution rate showed a significant negative
correlation (r = 0.214; P < 0.01). IL-12 and the NKT distribution were also negatively corre-
lated (r = 0.622), whereas IL-12 and INF-γ showed a significant positive correlation (P < 0.01). Results The sociodemographic variables and sensitivity to noise for 128 participants in Ulsan Nam-gu
and the 44 participants in Seoul Yangchun-gu are summarized in Table 1. In both Ulsan and Seoul, the distribution rate of participants was highest at 31.3% and
31.8%, respectively. The average age of all participants in Ulsan was 42.9 years and the average
age of all participants in Seoul was 45.6 years. The age group with the lowest participation rate PLOS ONE | https://doi.org/10.1371/journal.pone.0187084
October 30, 2017 4 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0187084
October 30, 2017 Immune response to road-traffic noise Table 2. Correlation among noise levels, noise sensitivity and immune response. Variables
Ldn
Noise
sensitivity
Cortisol
NK cells
NKT cells
IL-12
Noise
sensitivity
-0.103
Cortisol
0.065
0.138
NK cells
0.147
0.074
0.057
NKT cells
-0.214**
0.106
-0.138
-0.120
IL-12
0.233**
-0.134
0.149
0.007
-0.620**
INF-γ
0.127
0.017
0.041
-0.167
-0.148
0.251**
**. Correlation is significant at the 0.01 level (2-tailed), Immunologic profiles were transformed by natural log. https://doi.org/10.1371/journal.pone.0187084.t002 Table 2. Correlation among noise levels, noise sensitivity and immune response. mean value of Ldn and the noise sensitivity interval are shown as correlation curves through
the adjusted predicted value of each biomarker. mean value of Ldn and the noise sensitivity interval are shown as correlation curves through
the adjusted predicted value of each biomarker. The change in biomarker for Ldn and noise sensitivity are shown using correlation curves in
Fig 2. Multivariate linear regression analysis was performed to analyze the relationships among
road traffic noise, individual sensitivity, and age-dependent biomarkers, adjusting for the
effects of alcohol status, smoking status, regular exercise, and residence period. As shown in
Table 3, as the sensitivity level increased by 1 step, the cortisol level increased by 0.032 μg/dL. Moreover, as the road traffic noise increased by 1 dB, the percentage of NKT cells decreased
by—0.038%, whereas the IL-12 level increased by 0.006 pg/mL. The percentage of NK cells
and INF-γ levels were not significantly associated with road traffic noise or sensitivity
(P > 0.05), and therefore were meaningless as regression coefficients. As shown in Fig 3, the 5 / 13 PLOS ONE | https://doi.org/10.1371/journal.pone.0187084
October 30, 2017 PLOS ONE | https://doi.org/10.1371/journal.pone.0187084
October 30, 2017 https://doi.org/10.1371/journal.pone.0187084.t002 Discussion In this study, the correlations among road traffic noise, sensitivity to noise, stress hormones,
and immunity-associated factors were investigated. Previous studies have shown that a high level of noise or sensitivity to noise induces sleep
disorders [28, 29], hypertension [30–32], and cardiovascular disease [33, 34]. The result of traffic noise, depression, and anxiety are somewhat limited and somewhat
controversial; however, Beutel et al. [35] found that strong noise discomfort was related to
higher depression and anxiety in the general population. Participants with a mental health
problem may also have higher noise sensitivity and report higher discomfort levels [36]. p
y
g
y
p
g
When exposed to heavy nighttime aircraft, patients with coronary heart disease reported
chest pain caused by a typical heart disease [37]. Moreover, these consequent increases in the levels of the stress hormone cortisol from noise
or sensitivity can have negative effects on health by reducing the activity of NK cells [38, 39]. Duggal et al. [40] suggested that the observed association between NK cell immunesenes-
cence and cortisol was more strongly affected by mental stress rather than by physical stress. Munzel et al. [41] suggested a mechanism of the noise-triggered activation of the immune
system using a mouse model, indicating that noise exposure not only increased levels of nor-
adrenalin (NA), adrenalin (A) and angiotensin II (Ang II), but also increased cortisol thereaf-
ter. Increased Ang II has been reported to activate endothelial NADPH oxidase, which causes
oxidative stress that can lead to direct scavenging of nitric oxide and enhanced nitric oxide
synthatse uncoupling. Since then, the increase in the NADPH oxidase subunit NOX-2 has
been reported to increase immune cell activation and infiltration, such as NK cells, myelomo-
nocytic cells, leukocytes, and macrophages/monocytes. Therefore, noise stress causes blood pressure and vascular dysfunction associated with oxi-
dative stress. In the present study, the IL-12 and INF-γ levels were positively correlated, suggesting that
an increase of cortisol decreases the IL-12 level with a consequent decrease of NK cells and
INF-γ, a marker of NKT cell activation. Hence, it is predicted that an increase in cortisol levels would reduce immune function by
decreasing the activity of NK cells and NKT cells. Although there are few studies on the relationship between NKT cells and noise, these cells
can be activated in both antigen-dependent and independent manners. PLOS ONE | https://doi.org/10.1371/journal.pone.0187084
October 30, 2017 Discussion Furthermore, because PLOS ONE | https://doi.org/10.1371/journal.pone.0187084
October 30, 2017 6 / 13 Immune response to road-traffic noise Fig 2. Each real correlation curve for biomarker, Ldn and noise sensitivity. (A-E) It shows
correlation curve for Ldn and biomarker such as cortisol (A), NK(B) and NKT cells (C), cytokines
and it also shows each the real correlation curve for noise sensitivity and biomarker(F-J). https://doi.org/10.1371/journal.pone.0187084.g002 Fig 2. Each real correlation curve for biomarker, Ldn and noise sensitivity. (A-E) It shows each the real
correlation curve for Ldn and biomarker such as cortisol (A), NK(B) and NKT cells (C), cytokines (D and E))
and it also shows each the real correlation curve for noise sensitivity and biomarker(F-J). Fig 2. Each real correlation curve for biomarker, Ldn and noise sensitivity. (A-E) It shows each the real
correlation curve for Ldn and biomarker such as cortisol (A), NK(B) and NKT cells (C), cytokines (D and E))
and it also shows each the real correlation curve for noise sensitivity and biomarker(F-J). https://doi.org/10.1371/journal.pone.0187084.g002 https://doi.org/10.1371/journal.pone.0187084.g002 PLOS ONE | https://doi.org/10.1371/journal.pone.0187084
October 30, 2017 7 / 13 https://doi.org/10.1371/journal.pone.0187084.t003 Immune response to road-traffic noise Table 3. Immune responses to road traffic noise, noise sensitivity, and stress response. Variables
Multivariate analysis
Dependent
Independent
β±SE
p-value
R2
ln_Cortisol (㎍/dL)
Ldn
0.003±0.003
0.319
0.057
Sensitivity
0.032±0.014
0.020
ln_NK cells (%)
Ldn
0.006±0.004
0.111
0.193
Sensitivity
0.018±0.016
0.263
ln_NKT cells (%)
Ldn
-0.033±0.014
0.021
0.079
Sensitivity
0.046±0.063
0.464
ln_IL-12 (pg/mL)
Ldn
0.006±0.002
0.010
0.097
Sensitivity
-0.010±0.011
0.365
ln_INF-γ(pg/mL)
Ldn
0.007±0.005
0.118
0.105
Sensitivity
0.007±0.020
0.750
SE: standard error, Immunologic profiles were transformed by natural log. Linear regression models adjusted for age, alcohol consumption, smoking status,
regular exercise, residence period. Table 3. Immune responses to road traffic noise, noise sensitivity, and stress response. Variables
Multivariate analysis
Dependent
Independent
β±SE
p-value
R2
ln_Cortisol (㎍/dL)
Ldn
0.003±0.003
0.319
0.057
Sensitivity
0.032±0.014
0.020
ln_NK cells (%)
Ldn
0.006±0.004
0.111
0.193
Sensitivity
0.018±0.016
0.263
ln_NKT cells (%)
Ldn
-0.033±0.014
0.021
0.079
Sensitivity
0.046±0.063
0.464
ln_IL-12 (pg/mL)
Ldn
0.006±0.002
0.010
0.097
Sensitivity
-0.010±0.011
0.365
ln_INF-γ(pg/mL)
Ldn
0.007±0.005
0.118
0.105
Sensitivity
0.007±0.020
0.750
SE: standard error, Immunologic profiles were transformed by natural log. Linear regression models adjusted for age, alcohol consumption, smoking status,
regular exercise, residence period. Table 3. Immune responses to road traffic noise, noise sensitivity, and stress response. SE: standard error, Immunologic profiles were transformed by natural log. Linear regression models adjusted for age, alcohol consumption, smoking status,
regular exercise, residence period. 4.t003
Fig 3. Adjusted predicted value change of biomarker for Ldn and noise sensitivity interval. The values of
cortisol (pg/dL), NK and NKT cells (%), cytokines (pg/ml) were adjusted for age, alcohol consumption, smoking
status, regular exercise, residence period. The mean values of cortisol (A), NK/NKT cells (B), cytokines (C) for
each interval for Ldn are shown, and D-F shows the mean value of each biomarker for the noise sensitivity
interval. 03 Fig 3. Adjusted predicted value change of biomarker for Ldn and noise sensitivity interval. The values of
cortisol (pg/dL), NK and NKT cells (%), cytokines (pg/ml) were adjusted for age, alcohol consumption, smoking
status, regular exercise, residence period. The mean values of cortisol (A), NK/NKT cells (B), cytokines (C) for
each interval for Ldn are shown, and D-F shows the mean value of each biomarker for the noise sensitivity
interval. https://doi.org/10.1371/journal.pone.0187084.g003 https://doi.org/10.1371/journal.pone.0187084.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0187084
October 30, 2017 8 / 13 Immune response to road-traffic noise Fig 4. Schematic representation of the potential immune response by exposure noise levels or noise
sensitivity. Low chronic noise affects DCs and Macrophages and increases IL-12 concentration, but noise
sensitivity increases the concentration of cortisol in the body. Therefore, we expect to decrease INF-γ activity,
NKT and NK cells population by Ldn and noise sensitivity, which are two influencing factors. https://doi.org/10.1371/journal.pone.0187084.g004 Fig 4. Schematic representation of the potential immune response by exposure noise levels or noise
sensitivity. Low chronic noise affects DCs and Macrophages and increases IL-12 concentration, but noise
sensitivity increases the concentration of cortisol in the body. Therefore, we expect to decrease INF-γ activity,
NKT and NK cells population by Ldn and noise sensitivity, which are two influencing factors. https://doi.org/10.1371/journal.pone.0187084.g004 https://doi.org/10.1371/journal.pone.0187084.g004 they have pro-inflammatory and immunoregulatory characteristics, they are known to play an
important role in autoimmune diseases [42, 43], viral infections [44, 45] and cancer [46]. In this study, the level of road traffic noise showed a positive correlation with IL-12 levels
but did not affect the NK cell population. However, the proportion of NKT cells was negatively
correlated with road traffic noise. In addition, multivariate linear regression analysis on the relationships between road traffic
noise level or noise sensitivity to immune and stress parameters, controlling for the potentially
confounding variables of age [47–49], alcohol status, smoking status, regular exercise, resi-
dence period, showed that the increase of noise level increased the IL-12 level but decreased PLOS ONE | https://doi.org/10.1371/journal.pone.0187084
October 30, 2017 9 / 13 Immune response to road-traffic noise the NKT cell distribution rate, similar to results of the correlation analysis. Because cortisol is
considered to be more strongly affected by sensitivity than chronic road traffic noise and road
traffic noise does not largely affect cortisol unlike the biological response to > 80 dB noise
exposure [15], it appears that the IL-12 level and NKT cell frequency and activity are likely
regulated by different mechanisms [15]. That is, cortisol increases through the HPA-axis,
which is stimulated by a sensitivity response to activate DCs and macrophages, and the chronic
noise itself can increase the levels of IL-12, a pro-inflammatory cytokine, as a synergistic effect. However, noise stress will decrease the NKT cell population to induce a pro-inflammatory
response, with negative consequences for health [18]. The limitations of this study are as follows. First, as a cross-sectional study, although correla-
tions could be determined, the cause-and-effect relationship cannot be established from these
data. Acknowledgments The authors would like to thank occupational & environmental medical center, Ulsan Univer-
sity Hospital and Korea Ministry of Environment (MOE). The authors also are grateful to the
participants of survey. Supporting information S1 Dataset. Full dataset of tables. (PDF) Conclusions Low-level environmental noise and sensitivity to noise are likely to have negative effects on
health by triggering changes in the immune response through a mechanism distinct from an
increase in cortisol (Fig 4). Second, the effects of extreme noise levels such as occupational noise were not considered. Finally, not all potentially confounding variables were controlled in the analysis. Nevertheless, the clear associations detected in the present study and previous work suggest
the importance of carrying out longitudinal studies on the effects of chronic noise on health
and to determine the mechanism underlying the immune response to chronic loud noise. Although previous health effect evaluations associated with noise were mostly based on survey
results, heart rate variability, stress hormones (e.g., cortisol, norepinephrine, epinephrine), or
studies of the activation of NK cells in response to loud noise and noise sensitivity through in
vitro experiments, clinical studies on a large number of subjects of similar ages should be per-
formed to more objectively analyze the in vivo effects of exposure to a low level of environmen-
tal noise with respect to the involvement of NK cells, and NKT cells population and activation. Such investigations are expected to provide better indices for future health effect evaluations
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quences for health in older adults. Ageing Res Rev. 2013; 12(4):1069–78. https://doi.org/10.1016/j.arr. 2013.04.003 PMID: 23660515 48. Chidrawar SM, Khan N, Chan YL, Nayak L, Moss PA. Ageing is associated with a decline in peripheral
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PAPER • OPEN ACCESS PAPER • OPEN ACCESS Physiological Measurement Physiological Measurement Monitoring the quality of frozen-thawed venous segments using
bioimpedance spectroscopy To cite this article: M Amini et al 2020 Physiol. Meas. 41 044008 Abstract Objective: Storage at temperatures as low as −80 ◦C and below (cryopreservation) is considered a
method for long-term preservation of cells and tissues, and especially blood vessel segments, which
are to be used for clinical operations such as transplantation. However, the freezing and thawing
processes themselves can induce injuries to the cells and tissue by damaging the structure and
consequently functionality of the cryopreserved tissue. In addition, the level of damage is
dependent on the rate of cooling and warming used during the freezing-thawing process. Current
methods for monitoring the viability and integrity of cells and tissues after going through the
freezing-thawing cycle are usually invasive and destructive to the cells and tissues. Therefore,
employing monitoring methods which are not destructive to the cryopreserved tissues, such as
bioimpedance measurement techniques, is necessary. In this study we aimed to design a
bioimpedance measurement setup to detect changes in venous segments after freezing-thawing
cycles in a noninvasive manner. Approach: A bioimpedance spectroscopy measurement technique
with a two-electrode setup was employed to monitor ovine jugular vein segments after each cycle
during a process of seven freezing-thawing cycles. Main results: The results demonstrated changes
in the impedance spectra of the measured venous segments after each freezing-thawing cycle. Significance: This indicates that bioimpedance spectroscopy has the potential to be developed into a
novel method for non-invasive and non-destructive monitoring of the viability of complex tissue
after cryopreservation. Monitoring the quality of frozen-thawed venous segments using
bioimpedance spectroscopy M Amini1, E Niemi2, J Hisdal3, H Kalvøy4, C Tronstad4, H Scholz2,5, A Rosales6 and Ø G Martinsen1,4 M Amini , E Niemi , J Hisdal , H Kalvøy , C Tronstad , H Scholz , A Rosales and Ø G Mart
1 Department of Physics, University of Oslo, Oslo, Norway
2 Department of Transplant Medicine and Institute for Surgical Research, Oslo University Hospital, Oslo, Norway
3 Vascular Investigations and Circulation Lab, Aker Hospital, Oslo University Hospital, Oslo, Norway
4 Department of Clinical and Biomedical Engineering, Rikshospitalet, Oslo University Hospital, Oslo, Norway
5 Hybrid Technology Hub, Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway
6 Department of Vascular Surgery, Aker Hospital, Oslo University Hospital, Oslo, Norway Keywords cryopreservation, freezing-thawing, non-invasive, venous, bioimpedance spectroscopy © 2020 Institute of Physics and Engineering in Medicine To cite this article: M Amini et al 2020 Physiol. Meas. 41 044008 To cite this article: M Amini et al 2020 Physiol. Meas. 41 044008 View the article online for updates and enhancements. This content was downloaded from IP address 77.18.51.186 on 08/05/2020 at 06:54 This content was downloaded from IP address 77.18.51.186 on 08/05/2020 at 06:54 https://doi.org/10.1088/1361-6579/ab85b7 Physiol. Meas. 41 (2020) 044008 Physiological Measurement Physiological Measurement OPEN ACCESS
RECEIVED
29 November 2019
REVISED
11 March 2020
ACCEPTED FOR PUBLICATION
1 April 2020
PUBLISHED
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citation and DOI. 1.1. Cryopreservation y p
In order to preserve the viability and structural integrity of living cells and tissues for a long period, they are
stored under very low (cryogenic) temperatures where all the chemical, biological and physical reactions in
living cells would be suspended or dramatically reduced. This process is called cryopreservation (Karlsson
and Toner 1996, Pegg 2007, Jang et al 2017). Cryopreservation is usually performed at −80 ◦C and below as
traces of unfrozen solution can still be found in tissue above this temperature, and consequently the optimal
duration of cryopreservation would play an important role in the prevention of any kind of damage to the
cells (Mazur 1984). However, if the cryopreservation is performed at −130 ◦C and below then all biological
activities, including biochemical reactions that can lead to cell death, would stop. Furthermore, at −196 ◦C
(liquid nitrogen temperature) there would not be enough thermal energy to drive any type of biochemical
process (Mazur 1984, Gao and Critser 2000, Woods et al 2016). Under cryogenic temperatures, background
ionizing radiation (which causes photophysical reactions) could be the only cause of damage to tissues, and
this is not considerable (Woods et al 2016). Thus, in temperatures lower than −130 ◦C the duration of Physiol. Meas. 41 (2020) 044008 M Amini et al Figure 1. Physical changes in the cells under different cooling rates: slow and rapid. Figure 1. Physical changes in the cells under different cooling rates: slow and rapid. cryopreservation does not play a very important role in the viability of the cells, so the cryopreservation
period can range from short (Ku et al 1992, Müller-Schweinitzer 1994a) to undefined periods (Elmore et al
1991, Bujan et al 2000). j
The advantage of cryopreserved cells and tissues is their constant availability for both research and
clinical application, where their quality can be tested for suitability for transplantation purposes (Ibars et al
2016, Jang et al 2017). However, experimental findings have shown that the cryopreservation process can be
damaging to the cells (Mazur 1963, Gao and Critser 2000). One of the methods for cryopreservation of cells
and tissues is the freezing-thawing procedure (Karlsson and Toner 1996). Although storage under cryogenic
temperatures is beneficial for long-term preservation of cells and tissues, the freezing and thawing processes
can be detrimental to their viability (Karlsson and Toner 1996, Bia et al 2006, Müller-Schweinitzer et al 2007). 1.1. Cryopreservation However, the challenge is not only to withstand storage at high subzero temperatures but also to withstand
the transition phases and to traverse the temperature range of −15◦C to −60◦C. By the time the tissue
reaches the temperature of −15 ◦C, ice crystals are already formed in the extracellular matrix but the cell
membrane blocks the growth of ice in the intracellular matrix. Consequently, water has started flowing out
of the cells osmotically, due to its higher chemical potential in comparison to the water in the extracellular
matrix. Therefore, the temperature zone in which both ice crystallization in the extracellular matrix and loss
of intracellular water happen is considered a lethal zone for cells, especially when they need to traverse it twice
(once during freezing and once during thawing) (Mazur 1963, Gao and Critser 2000, Woods et al 2016). Both cooling (freezing) and warming (thawing) rates play important roles in determining the viability of
cells and tissue (Pegg 2007) and should optimally result in the recovery and survival of cell and tissue
structure (Karlsson and Toner 1996). The rate of temperature change controls the transport of water out of
the cells during freezing and into the cells during thawing, and thus indirectly influences the concentration of
the solution surrounding the cells (Mazur 1963, Pegg 2007). At low temperatures, permeability of the cell
membrane to water is higher than to the corresponding solute, which makes the cell membrane
semipermeable (Lynch and Diller 1981, Karlsson and Toner 1996). In other words, formation of intracellular
ice is influenced by cooling rate and what happens to that ice is controlled by warming rate (Pegg 2007). Freezing rates that are too rapid or too slow can both cause damage to the cells, however the damage
mechanisms are different (Gao and Critser 2000). On the other hand, if cells are cooled too rapidly (figure 1) the intracellular water volume remains almost
the same, as water does not have time to leave the cells fast enough by osmosis to maintain equilibrium
(Karlsson and Toner 1996). In this way, icy crystals would be formed inside the cell (which is called
‘Intracellular Ice Formation’ or IIF), which can be lethal to the cells (Mazur 1963, Mazur et al 1972, Gao and
Critser 2000). Therefore, damage to the cells during rapid cooling is due to IIF or osmotic rupture (Karlsson
and Toner 1996). 1.1. Cryopreservation Changes in temperature and concentrations of intra- and extracellular solutes damage the lipid-protein
complexes in the cell membrane (Lovelock 1953, Gao and Critser 2000), causing leakage and also changes in
membrane permeability (Pegg 2007, Müller-Schweinitzer 2009). Therefore, an optimal cooling rate (which
would be high enough to minimize the solution effect and low enough to avoid IIF) is necessary to maintain
the viability and integrity of the cells and their membranes (Mazur 1963, Mazur et al 1972, Gao and Critser
2000). 2 Physiol. Meas. 41 (2020) 044008 M Amini et al Warming or thawing can be as challenging for the survival of the cells as the cooling or freezing process
(Mazur 1984, Gao and Critser 2000). Studies have shown that maximum thermal stress to the cells occurs
during the warming or thawing process (Zhang et al 2005, Müller-Schweinitzer 2009) as small intracellular
ice crystals show different behaviors when they are treated by slow or rapid warming (Pegg 2007). Therefore,
the extent to which the warming rate would affect the tissue under cryopreservation actually depends on
whether the prior rate of cooling has caused cellular dehydration or intracellular ice formation (Gao and
Critser 2000, Woods et al 2016). Slow warming gives enough time for the small intracellular ice crystals to recrystallize, join and grow into
harmful ice crystals (Pegg 2007). Therefore, although there is no change in the amount of ice in the cells,
what causes injury is the size and shape of the ice. In addition, during slow warming the cells would be
exposed to concentrated solutes for a longer period of time, which increases the chances of toxicity caused by
the solutes (Woods et al 2016). In contrast, during rapid warming (which is generally applied in a 37 ◦C water bath) (Bellon et al 1999)
there is not enough time for the ice crystals to recrystallize, merge and grow bigger, so the ice crystals just
melt (Pegg 2007). In this way, rapid warming prevents the formation of large ice crystals that can be
damaging to the cells and thus helps rescue them (Mazur 1984, Gao and Critser 2000). 1.2. Cryopreservation of vascular tissue Previous research has shown that blood vessel cells and tissues can be seriously damaged when they are
subjected to cooling and warming (freezing and thawing). This damage can range from loss of contractility
in the smooth muscle layer to loss of function in endothelial cells (Müller-Schweinitzer 2009). According to
some studies, storage of blood vessels at −80 ◦C would provide better preservation than lower temperatures
such as −160 ◦C/−190 ◦C (Ku et al 1994, Müller-Schweinitzer 1994a, 1994b, Bujan et al 2000). Storage of
vascular tissues in temperatures of around −80 ◦C for a short period of time (3–4 weeks) can preserve the
structural integrity of the tissues. For example, studies have shown that the viscoelastic properties of human
veins would not change during the cryopreservation period (Bia et al 2006, Müller-Schweinitzer et al 2007). However, storage under these temperatures for a long period would not guarantee the survival of
mammalian cells (Müller-Schweinitzer et al 1986, Ku et al 1994). According to previous clinical research,
slow warming of arteries and animal vessels (with a rate of 1 ◦C min−1) would cause less damage to the
endothelial cell layer in comparison to rapid warming (which can cause fractures in the vascular wall) (Hunt
et al 1994, Wassenaar et al 1995, Pegg et al 1997, Bujan et al 2000, Pascual et al 2004, Pasquinelli et al 2006,
Müller-Schweinitzer et al 2007). 2. Methods Six ovine jugular veins were subjected to seven cycles of freezing-thawing. Studies have shown that
preservation in −80 ◦C freezers provides long-term storage of tissues and cells from weeks to months, and is
one of the most commonly used storage methods. The combination of slow freezing and fast thawing
methods employed in this study is considered to be an optimal cryopreservation method, which induces less
injury and damage to the cells and tissue (Shu et al 2015). For the freezing part, slow cooling down to −80 ◦C with a freezing rate of 1 ◦C min−1 was performed in
order to minimize the effects of ice injury to the venous tissue. For the thawing part, fast warming was
performed by using a water bath at 37 ◦C in order to reduce recrystallization and ice injury. The venous
segments were preserved in the University of Wisconsin (UW) solution during the whole storage and
freezing-thawing periods. Bioimpedance spectroscopy measurement was performed on all veins after each freezing-thawing cycle,
with at least a 48 h interval between each two consecutive measurements for each vein. A two-electrode
bioimpedance spectroscopic measurement setup (figure 2) was applied and the same pair of electrodes was
used as both current-carrying and voltage pick-up electrodes. The electrodes consisted of a gold wire going
through the glass tubes and the venous segment, and a titanium plate placed at the bottom of the main
container. The venous segments were fixed between two glass tubes in the main container. The container, glass tubes
and venous segment were filled with saline, with a slightly positive pressure inside the vein to keep it
expanded and prevent contact between the electrode and the wall of the venous segment. The measurement
device employed was a Solartron complex impedance measurement system (SI 1260/1294, Solartron Group
PLC., Hampshire, UK), using a controlled electrical excitation voltage of 500 mV rms to record the
impedance modulus and phase of the veins in the frequency range of 1 Hz to 1 MHz (figure 3). The change in impedance over cycles was assessed statistically by selecting the impedance modulus at one
frequency of interest and fitting these values from all veins in a linear mixed effects (LME) model. The
change in impedance was modeled as a linearly dependent function ofthe number of cycles (as a fixed effect)
and with a random intercept for the different veins. 1.3. Monitoring the quality of the frozen tissues tissue are related to their physiological and morphological properties, bioimpedance spectroscopy can b
employed for evaluation of the tissue of interest (Dean et al 2008, Grimnes and Martinsen 2015). The aim of the present study was therefore to employ bioimpedance spectroscopy as a non-invasive and
non-destructive measurement technique, and to evaluate its ability in monitoring changes in venous
segments which have undergone multiple freezing-thawing cycles. 1.3. Monitoring the quality of the frozen tissues It is important to evaluate the viability and structural integrity of the cells and tissues after cryopreservation
(freezing-thawing cycle) and before their clinical application (such as transplantation). Some of the
monitoring methods that are currently employed for this purpose include microcomputed tomography
imaging (Bischof et al 2007); magnetic resonance swift imaging (Zhang et al 2014, Manuchehrabadi et al
2017); FT-IR spectroscopy (Giugliarelli et al 2016); label-free fluorescence imaging with optical fiber
(Alfonso-Garcia et al 2018); light and cryomicroscopy, scanning and transmission electrode microscopy
imaging (Bujan et al 2000); and general histological evaluation methods (Merdassi et al 2011). In addition
there are mechanical tests such as tonometry for evaluating the biomechanical properties and functionality
of tissues, such as their viscoelasticity (Bia et al 2006). However, these methods are mostly invasive and
destructive to the tissues as they require segmenting the tissue into tiny rings or pieces, and also staining. Therefore, there is a need for a non-destructive method which would be able to monitor the tissue quality
after its cryopreservation in a non-destructive manner. Electrical bioimpedance measurements facilitate non-destructive miniaturized methods for real-time
evaluation of the state or properties of biological materials. The bioimpedance measurement technique has
been used in clinical applications to determine fluid content, electrolytic composition, cellular morphology,
organ perfusion, inflammation, body composition, etc. The spatial resolution can range from molecular-size
to whole-body, and can be focused by proper development of measurement setup and electrode design
(Grimnes and Martinsen 2015). Structural tissue properties are usually highly detectable in a bioimpedance
measurement, as are the concentration of cells and their geometry. Destruction of cell membrane and cell
death are examples of detectable vital signs in a tissue (Amini et al 2018). Therefore our hypothesis is that a potential change in the viability of the cells due to freezing/thawing will
be detectable with a properly designed bioimpedance setup. Bioimpedance spectroscopy is one of the
bioimpedance measurement techniques, which records the electrical impedance of a tissue over a frequency
range to detect the frequency-dependent electrical properties of the tissues. As electrical properties of the 3 Physiol. Meas. 41 (2020) 044008 M Amini et al Figure 2. Bioimpedance spectroscopy measurement set-up with two-electrode configuration for monitoring veins after each
freezing-thawing cycle. Figure 2. Bioimpedance spectroscopy measurement set-up with two-electrode configuration for monitoring veins after each
freezing-thawing cycle. 3. Results Comparison of the bioimpedance spectra recorded after each freezing-thawing cycle demonstrated obvious
changes in both the modulus and phase angle of the measured bioimpedance. For four of the six monitored
veins, this change followed a trend where the impedance modulus decreased systematically from the first to
the seventh cycle. For the other two veins, following the measurement protocol and subsequently completing
the measurements was impossible. This was due to the presence of tiny holes in the walls of the venous
segments and consequent leakages from the walls; this made expanding the venous segment and preventing
the venous wall from collapsing and coming into direct contact with the electrode inside the vein impossible. Therefore, measurements from these two veins were not reliable and were excluded from the results. In figure
4 the bioimpedance spectra from all four veins, including the impedance modulus (|Z|) and the phase angle
(Theta), are shown. Figure 5 shows the changes in impedance modulus (at the particular frequency of 10 kHz) over the seven
cycles of freezing-thawing for all four veins. The reason for choosing 10 kHz was that this frequency is above
the frequency range corresponding to contribution from electrode polarization impedance, and below the
frequency range were inductive properties are demonstrated. Based on the spectra shown in figure 4, the impedance modulus at 10 kHz (figure 5) was selected for
statistical analysis of the change in impedance over cycles. Fitting the 10 kHz impedance to the LME model
gave an estimated change of −1.26 Ohms per cycle, with a 95% confidence interval from −1.49 to
−1.04 Ohms per cycle (p < 0.001). 2. Methods The LME was implemented using the fitlme() function
in Matlab 2018b. 4 Physiol. Meas. 41 (2020) 044008 M Amini et al Figure 3. Block diagram of the bioimpedance spectroscopy measurement system. Figure 3. Block diagram of the bioimpedance spectroscopy measurement system. 4. Discussion Each step of the cryopreservation process can cause injury to the cells and influence the viability and
functionality of the cells or tissues, therefore monitoring and evaluating the tissues after cryopreservation
and before their clinical application is important. The majority of current monitoring methods are invasive
and destructive. As an example, methods for histological evaluation of cells and tissues can be mentioned,
which are highly accurate and considered as the gold standard. Thus, the use of an evaluation method that is
not destructive to cryopreserved tissue is warranted. Bioimpedance measurement techniques such as
bioimpedance spectroscopy have shown their suitability as real-time, non-invasive methods for
characterizing tissues and evaluating their viability and integrity in a non-destructive manner (Grimnes and
Martinsen 2015). The main finding of the present study, which employed bioimpedance spectroscopy as the monitoring
method, was observation of a decreasing trend in the impedance modulus and phase angle in four of the six 5 Physiol. Meas. 41 (2020) 044008 M Amini et al (a)
(b)
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Figure 4. Bioimpedance spectra from four veins recorded after each freezing-thawing cycle, including the impedance modulus |Z|
and phase angle Theta of all four veins: (a) vein 1, (b) vein 2, (c) vein 3, and (d) vein 4. 4. Discussion In addition, the results of
statistical analysis of impedance changes at 10 kHz, with the confidence interval of 95% and p-value < 0.001,
indicate that the decrease in impedance modulus per cycle and for all veins is statistically significant. The observed decrease in impedance measurements between each freezing-thawing cycle can be an
indication of a decrease in the structural integrity of the endothelial cell layer. This can be due to
solution-effect injury in addition to ice injury damage to the cells and their membranes during thawing,
which would consequently affect the functionality of the venous segment. Looking closer at the low frequency part of the plots in figure 4 (in the impedance modulus spectra from
the first and second cycles in the frequency range around 1 kHz) a small arc can be detected, which flattens
out for the subsequent cycles. The corresponding circle segments are also evident in the phase plot. Both
of these findings strongly suggest the presence of an α-dispersion for the first and second cycles. The
α-dispersion can usually be seen in the frequency range of 1 Hz to 100 kHz (Grimnes and Martinsen 2010,
2015) and is associated with tissue interfaces, such as cell membranes. Therefore, there is reason to believe
that this α-dispersion can be used to detect a change in the properties of cell membranes or intracellular
structures after consecutive freezing-thawing cycles (Ivorra et al 2005). g
g y
However, the arc representing α-dispersion (which is mostly evident from a few hundred Hz) should not
be confused with the impedance modulus curve below 100 Hz, which has a large increase in impedance
towards 1 Hz due to electrode polarization impedance (EPI). The presence of EPI or electrode impedance at
the electrode interface with the tissue/solution in the frequency range below 1 kHz, and its contribution to
changes in the impedance modulus and phase angle, is often inevitable — especially when a two-electrode
configuration is employed (Grimnes and Martinsen 2015). Generally, electrode material and size, sample
impedance, measurement frequency and some other factors affect the influence of EPI in the measurements. Therefore, by being aware of this dependency it is possible to design the measurement setup such that the
contribution of EPI would be reduced or minimized (Kalvøy et al 2011, Amini et al 2018). 4. Discussion (a)
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Frequency [Hz] (d) (b) Figure 4. Bioimpedance spectra from four veins recorded after each freezing-thawing cycle, including the impedance modulus |Z|
and phase angle Theta of all four veins: (a) vein 1, (b) vein 2, (c) vein 3, and (d) vein 4. Cycle 1
Cycle 2
Cycle 3
Cycle 4
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Vein 1
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Figure 5. Changes in impedance modulus |Z| at the particular frequency of 10 kHz over seven freezing-thawing cycles for all four
veins. Figure 5. Changes in impedance modulus |Z| at the particular frequency of 10 kHz over seven freezing-thawing cycles for all four
veins. 6 Physiol. Meas. 41 (2020) 044008 Physiol. Meas. 41 (2020) 044008 M Amini et al measured veins (in the frequency range of 1 Hz to 1 MHz) after each freezing-thawing cycle. In the other two
veins it was not possible to follow the measurement protocol and complete the measurements, due to leakage
caused by the formation of tiny holes in the blood vessel walls. The plots in figure 5 show that all four veins demonstrate linear changes in the same direction along with
the cycles, and strongly suggest that these results are not random changes. 4. Discussion In our
measurement setup, which used gold as the electrode material (which demonstrated one of the lowest EPIs
among the other electrode materials tested in our pilot measurements) and specific dimensions for the gold
electrode (diameter of 1 mm and length covering the whole length of the venous segment), it is expected that
the contribution of EPI would be low. In addition, changes in impedance modulus after each
freezing-thawing cycle are most evident in higher frequency ranges (above 100 Hz), since the contribution
from EPI is inversely proportional to frequency. The positive phase angle seen in the MHz range can be interpreted as if the veins demonstrate inductive
properties. However, studies have shown that the existence of inductive properties in tissue samples and cell
membranes under stable conditions in passive bioimpedance measurements is not very likely (Cole 1972, Riu
2004). Nevertheless, the positive phase angle does not need to be a sign of measurement error and can be
caused by the contribution of self-inductance when measuring low impedance values (<50Ω) at high
frequencies, such as in our case. Especially when a two-electrode setup is used, the self-inductance of the
measuring lead and wire would be in series with the tissue impedance and in this way contribute to the
measurements. Hence, the lower the impedance value is, the larger the inductive influence would be
(Grimnes and Martinsen 2007). This study has shown that a bioimpedance spectroscopy technique is capable of detecting changes in
venous grafts after freezing-thawing cycles. However, the mechanisms and types of morphological changes in
the vascular grafts that have led to these distinct changes in the impedance spectra are unknown and require
further investigations. In addition, optimization of the measurement setup and electrode configuration, in
order to minimize the contribution of electrode impedance polarization, is recommended. In this way, the
arcs representative of α-dispersion, which are the result of structural changes in the cells and the venous
segment, would become even more visible. Conflict of Interest The authors do not have any potential conflicts of interest to declare. 5. Conclusion From the results of this study, it can be concluded that bioimpedance spectroscopy has the potential to be
developed into a novel method for non-invasive and non-destructive monitoring of the viability of complex
tissue after cryopreservation. 7 M Amini et al Physiol. Meas. 41 (2020) 044008 M Amini https://orcid.org/0000-0001-8835-9998 M Amini https://orcid.org/0000-0001-8835-9998 Ethical approval The research related to animal use has complied with all the relevant national regulations and institutional
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English
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Correction: The population genetics of human disease: The case of recessive, lethal mutations
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PLOS genetics
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cc-by
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1.
Amorim CEG, Gao Z, Baker Z, Diesel JF, Simons YB, Haque IS, et al. (2017) The population genetics
of human disease: The case of recessive, lethal mutations. PLoS Genet 13(9): e1006915. https://doi.
org/10.1371/journal.pgen.1006915 PMID: 28957316 Correction: The population genetics of human
disease: The case of recessive, lethal
mutations Carlos Eduardo G. Amorim, Ziyue Gao, Zachary Baker, Jose´ Francisco Diesel, Yuval
B. Simons, Imran S. Haque, Joseph Pickrell, Molly Przeworski Carlos Eduardo G. Amorim, Ziyue Gao, Zachary Baker, Jose´ Francisco Diesel, Yuval
B. Simons, Imran S. Haque, Joseph Pickrell, Molly Przeworski The authors note that the citation of Wright (1937) and Nei (1968) is incorrect in the “Models
of mutation-selection balance” section in Results. Wright (1937) not only showed the equilib-
rium frequency of a deleterious allele for a population of infinite size (Eq 1) but also derived
the full equilibrium frequency distribution of a deleterious allele under a finite effective popu-
lation size. He also derived the mean (Eq 2) allele frequency of a fully recessive deleterious
allele and gave approximations for large and small effective population sizes. Nei (1968) fur-
ther derived the approximate variance (Eq 3) in allele frequency of a fully recessive mutation
by using Wright’s general formula. The authors confirm that this error does not affect the con-
clusions of the article. OPEN ACCESS Citation: Amorim CEG, Gao Z, Baker Z, Diesel JF,
Simons YB, Haque IS, et al. (2018) Correction: The
population genetics of human disease: The case of
recessive, lethal mutations. PLoS Genet 14(7):
e1007499. https://doi.org/10.1371/journal. Reference a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 Published: July 2, 2018 Copyright: © 2018 Amorim 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. 1 / 1 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1007499
July 2, 2018
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Fane: A Firewall Appliance For The Smart Home
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Annals of Computer Science and Information Systems
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Proceedings of the Federated Conference on
Computer Science and Information Systems pp. 449–458
DOI: 10.15439/2019F177
ISSN 2300-5963 ACSIS, Vol. 18 g
Computer Science and Information Systems pp. 449–458 ISSN 2300-5963 ACSIS, Vol. I. INTRODUCTION I
N THE the last years, the proliferation of Smart Home
installations has gained momentum. Today, the consumer
market offers a huge number of different Internet-of-Things
(IoT) devices. I We show that it is possible to develop a generic IT-
Security concept for IoT devices in a Smart Home installation
by making few realistic assumptions, e.g., the IoT network
segment is only used by single-purpose IoT devices, which
do not fundamentally change their communication profiles. We have implemented this security concept in FANE. Our
evaluation indicates that FANE can secure the IoT network
segment without requiring the user to possess network-security
expertise. Smart thermostats, cameras, speakers and even toothbrushes
contain information technology that connects the IoT device
over the Internet with cloud services or other IoT devices. For
example, IoT devices from the Google Nest family [1] provide
a straightforward, user-friendly way to control heating, cooling
and home security. Smart speakers like Amazon Alexa [2]
allow to control many daily activities via voice control. From
the perspective of the manufacturers, the Smart Home concept
allows new business models, e.g., to sell new product features
as digital upgrades for IoT devices. Paper structure: In Section II, we review related work. In Section III we provide a problem statement. We describe
FANE in Section IV, followed by a proof-of-concept imple-
mentation in Section V and an experimental evaluation in
Section VI. Section VII concludes. On the other hand, consumers might be tempted to overlook
that the IoT devices pose an IT-Security risk. For example, the
lifetime of a traditional security camera ends when the device
is broken. In contrast, the lifetime of an IoT security camera
that connects over the Internet should come to an end when
its manufacturer discontinues security updates, even if the IoT
security camera is still working. Otherwise, the IoT security
camera might end up as, say, part of the Mirai bot network,
which consisted of approx. 500,000 devices in 2016 [3]. FANE: A Firewall Appliance for the Smart Home Erik Buchmann
Hochschule für Telekommunikation
in Leipzig
Gustav-Freytag-Straße 43-45, 04277 Leipzig, Germany
Email: buchmann@hft-leipzig.de Christoph Haar
Hochschule für Telekommunikation
in Leipzig
Gustav-Freytag-Straße 43-45, 04277 Leipzig, Germany
Email: haar@hft-leipzig.de Abstract—With the advent of the Internet of Things (IoT),
many domestic devices have been equipped with information
technology. By connecting IoT devices with each other and
with the Internet, Smart Home installations exist that allow the
automation of complex household tasks. A popular example is
Google Nest that controls cooling, heating and home security. However, Smart Home users are tempted to neglect that such IoT
devices pose IT-Security risks. Examples like the Mirai malware
have already shown that insecure IoT devices can be used for
large-scale network attacks. Thus, it is important to adapt secu-
rity approaches to Smart Home installations. In this paper, we
introduce FANE, our concept for a Firewall AppliaNcE for Smart
Home installations. FANE makes a few realistic assumptions on
the network segmentation and the communication profile of IoT
devices. This allows FANE to learn firewall rules automatically. Our prototypical implementation indicates that FANE can secure
a wide range of IoT devices without requiring network-security
expertise from the Smart Home user. explore options to integrate a firewall into typical Smart Home
installation that can detect and deter such attacks. This is
challenging, since the firewall must be compliant with the
typical modes of use of a Smart Home installation, and a
consumer cannot be expected to evaluate firewall rules or
identify false alarms. On the other hand, the IoT devices used
differ from general-purpose devices such as smartphones and
desktop computers. This might allow for pre-configuration to
some extent. In particular, we make the following contributions: 1) We systematically compare the lifecycle of a classical
firewall with the lifecycle of IoT devices in a typical
Smart Home installation. 2) We propose FANE, a Firewall AppliaNcE on a Wi-Fi
bridge in Smart Home installations. 3) We describe a proof-of-concept implementation of FANE
based on a Raspberry Pi, and we evaluate it with three
different IoT devices. II. RELATED WORK In this section we provide a brief definition of Internet
of Things and Smart Home, and we introduce related work
on firewalls, firewall management and approaches to generate
firewall rules automatically. PROCEEDINGS OF THE FEDCSIS. LEIPZIG, 2019 PROCEEDINGS OF THE FEDCSIS. LEIPZIG, 2019 450 B. Security Challenges To assess the security properties of Smart Home installa-
tions, it is important consider the basic security challenges
that occur in installations of IoT devices. One study [6] lists
six major security issues: Identity and Authentication: In IoT environments, numer-
ous devices need to authenticate each other in order to provide
trustable services. Thus, reliable techniques for identification
and authentication are needed. Access Control: To create new services it is necessary to
aggregate data from different providers. This is challenging,
because in typical IoT scenarios each provider has its own
access control policy. Design and
Selection
Policy Definition
Implementation and
Configuration
Plan Firewall
Rules
Implement Firewall
Rules
Review Firewall
Operations
Determine
appropriate Actions
IT-Security Operations
Configuration Management
Information Security
Management
Policy
System
Architecture
Firewall
Configuration
Firewall
Ruleset
Activity
Document
Control Flow
Information Flow
Fig. 1. Traditional Firewall-Lifecycle Protocol and Network Security: If IoT devices commu-
nicate with each other in a distributed network architecture,
distributed schemes for key management are needed. Protocol and Network Security: If IoT devices commu-
nicate with each other in a distributed network architecture,
distributed schemes for key management are needed. distributed schemes for key management are needed. Privacy: The Smart Home concept means that numerous
IoT devices monitor the actions of its users in order to devise
meaningful responses. Thus, privacy very important from a
user perspective. Policy Trust and Governance: In IoT architectures there are two
dimensions of trust. The first dimension is between users and
their IoT devices. The other dimension is between the IoT
devices. Device A needs to trust the accuracy and integrity of
the data produced by device B. Data governance goes in the
same direction, in a sense of data and access governance. Fault Tolerance: Mechanisms for fault tolerance need to
be established to counteract faulty or tampered devices. Other studies [7] list similar challenges. D. Firewall Lifecycle connected cars over smart buildings to connected machinery in
an Industry 4.0 setting. The concept "‘Smart Home"‘ narrows
down this range to devices that let end users to control, monitor
or access everyday objects of the daily routine [5]. Traditionally, a firewall must be part of the IT-Security
process, as described by the ISO 270xx standards family [10],
the German BSI Grundschutz Standard 200-2 [11] or the ITIL
process for security Management [12]. The IT-Security process
starts with a IT-Security policy that has been passed by the
management. Based on this policy, business objectives, the
assets to be protected and a risk classification can be identified. Subsequently, measures can be defined and implemented that
restrict the IT-Security risks to acceptable levels. In the follow-
ing, the effectiveness of these measures needs to be monitored. Based on this information, corrective actions can be planned
and executed [13]. Note that all process steps require a person
with IT-Security expertise, which cooperates with various IT
experts from the operations department. A. Internet of Things and Smart Homes From the perspective of a consumer without in-depth ex-
pertise of network security, it is next to impossible to find
out if the IoT devices present in a Smart Home installation
are subject to attacks over the Internet. In this paper, we The "‘Internet of Things"’ (IoT) refers to physical appli-
ances, which have been equipped with information technology
in order to connect them with other devices directly or over
the Internet [4]. IoT includes a wide range of appliances, from IEEE Catalog Number: CFP1985N-ART c⃝2019, PTI 449 E. Firewall Rules It is a labor-intensive task for a domain expert to create a
rule set for firewalls for manually. One option to obtain firewall
rules (semi-)automatically is to use data mining or machine
learning on a training set consisting of network packets. This
option is based on the assumption all user applications operate
as intended while the training set is recorded. Respective
approaches [15]–[17] have been proposed for Intrusion De-
tection systems, but might be adaptable to firewalls as well. By using k-Means, C4.5 decision tree algorithms, Naive Bayes
classifier, Neural Networks or Support Vector Machines, it is
possible to derive common characteristics of allowed network
connections. Those characteristics could be translated into
firewall rules. It is also possible to generate firewall rules by
mining the firewall log [18] instead of a dump of network
packets. However, all approaches require an IT-Security expert
to decide which generated rules are relevant to meet the
security requirements, and the quality of the generated rules
still needs further research. b) Network Architecture: Firewalls depend on the net-
work segmentation. With traditional use cases, a network
installation might contain multiple segments protected by
multiple firewalls. A prominent example is a perimeter net-
work [20], which contains assets such as Web servers that
must be accessible from an external network. Two sets of
firewall rules protect the perimeter network against the external
network and the internal network against the perimeter and
the external network. However, the number and architecture
of the network segments might be individually different for
each network installation. In contrast, a typical Smart Home installation with IoT
devices produces three network segments with different se-
curity properties: (a) the untrusted Internet, (b) the home
network with trusted devices such as the user’s laptop and
printer, and (c) an IoT network segment that contains all IoT
devices. Since the IoT device and its software comes as an
integrated package, the user has little options to influence the
security of the IoT device, e.g., by disabling unused network
protocols or by removing unused software functionality. Thus,
the IoT network segment should be separated from the home
network [23], which is used for sensible tasks such as online
banking or online shopping. E. Firewall Rules All devices in the IoT network
segment can be expected to require an Internet connection, to A different option to generate firewall rules is to deduce
them from a formal specification of security requirements by
using argumentation logic [19]. This approach allows to auto-
matically obtain a detailed, comprehensive set of rules from a
high-level specification. However, creating a specification of
the security requirements for a certain system architecture still
requires expert knowledge in IT-Security. A. Does a Firewall Fit into the Smart Home Concept? To find out in which ways traditional firewall use cases
differ from Smart Home use cases, we consider the modes of
use, network architecture, application scenario, user roles and
information technology used. a) Modes of Use: A firewall is an access control mech-
anism that allows or blocks network traffic between two
network segments that have different security properties [20],
e.g., an internal network and the Internet that is open for
anybody. The firewall enforces a set of firewall rules that allow
or prohibit network packets to travel from one segment into the
other one. The firewall rules depend on the use cases that are
executed over both network segments. For example, a business
workflow "‘Answer customer requests"’ might require that a
set of machines in the internal network is allowed to send
and receive email to/from the Internet. Thus, firewall rules
must be defined by a network-security expert with domain
knowledge. If the workflows, the applications or the segment
boundaries are changed, the expert must adapt the firewall
rules as well. Traditionally, firewalls are tailored for complex
multi-purpose scenarios where the hosts execute numerous
different applications that change over time. Smart Home use cases are fundamentally different [21]: A
typical IoT device is a physical object that has been extended
with information technology to improve its usefulness. For
example, a smart toothbrush [22] can tell its user if a tooth has
gone unbrushed. Thus, IoT devices are constructed for a single
purpose that does not change over time. It only makes sense to
install a toothbrush control software on a smart toothbrush. As
a result, IoT devices are single-purpose objects. If the device
is not needed any more, it will be disposed. CHRISTOPH HAAR, ERIK BUCHMANN: FANE: A FIREWALL APPLIANCE FOR THE SMART HOME 451 A firewall fits into the IT-Security process [14] as shown in
Figure 1. In the Information-Security Management phase, the
management defines a security policy based on company-wide
security objectives. This policy is independent from technical
realities. Based on the security policy, an IT-Security expert
designs the architecture of the firewall system and selects
the firewall system components. In the implementation and
configuration phase, the IT-Security expert adapts the firewall
system to the system architecture with its network segments,
hosts and applications. This includes a preliminary set of
firewall rules that define which network packets are allowed
to pass the firewall. In the next step, a plan-do-check-act cycle
takes place where the firewall rules are designed, implemented,
reviewed and improved in a repetitive way. Typically, this
cycle is part of IT-Security operations. It allows to adjust the
firewall rules to changes such as new business applications,
hosts moving from one network segment to another one,
or in case of detecting new attacks. Note that not only the
management of firewall rules is a cyclic process, but also
the IT-Security process. If the management observes that the
security policy is ineffective, this policy can be changed as
well, and it has an impact on all design decisions further down
the IT-Security process chain. C. Firewalls State of the Art Firewalls are able to control and log the network traffic
based on rules set by an administrator or security expert. In
literature different firewall generations are distinguished [8]. 1st generation firewalls are known as packet filters which
operates on the transport layer. The filtering is based on
source and destination IP addresses, ports and protocols. 2nd
generation firewalls are also operating on the transport layer
and they are known as stateful packet inspection. State tables
are used to keep track of the network traffic and filtering is
based on state and context of packets. 3rd generation firewalls
are operating on the application level and require different
proxies for each service. The proxy acts as a middleman
between source and destination to reestablish a new session. Current firewall technologies are called next generation fire-
walls. These next generation firewalls are looking deep into
packets and combine traditional firewall technologies with
network filtering capabilities on the application level [9]. However, all these generations have in common that an expert
is needed to define rules or check them for correctness which
motivates our new approach. mplement Firewall
Rules Fig. 1. Traditional Firewall-Lifecycle A. Network Architecture A firewall separates network segments with different secu-
rity properties. Typical IoT devices do not allow its user to
observe security properties, and to configure security-related
aspects, such as disabling unused functions. Furthermore, an
IoT device is designed to be used like a classical, non-smart
device, i.e., its users are tempted to forget that the device might
pose IT-Security risks. For this reason, IoT devices should be
placed in network segments that are isolated from all other
network segments of the Smart Home. In contrast, an IoT device for a Smart Home usually is pre-
configured by the manufacturer for typical use cases. The end
user can deploy and configure the IoT device with minimal
efforts, does not need to monitor it later on and does not need
expert knowledge. e) Information Technology: IoT devices make use of
network protocols which have been well established. They
use Linux-based operating systems, Cloud resources and Open
Source programming libraries. The network security of IoT
devices is based on mechanisms for encryption, certification
and signatures that have been in use for years. Thus, from a
technical point of view, off-the-shelf firewalls can be directly
used to control the network traffic of IoT devices. Thus, FANE operates as a Wi-Fi bridge that connects the
IoT network segment to the Internet and includes a firewall,
as shown in Figure 2. The IoT network segment only contains
single-purpose IoT devices, and the Wi-Fi bridge is the only
connection of the IoT segment to other network segments and
the Internet. We observe that this allows us to specify the
security concept in advance. IV. FANE: A FIREWALL APPLIANCE In this section, we introduce FANE, a concept for a Firewall
AppliaNcE that is compatible with the Smart Home paradigm. Homes must differ in the following properties from traditional
firewalls: On the opposite side, one of the fundamental principles of
the Smart Home concept is to let IoT devices use sensors
to observe its environment, in order learn appropriate actions
with a minimum of user interaction and without requiring
the user to scrutinize the operations of the IoT device on a
regular basis. For example, the nest thermostat observes the
temperature preferences of its user and if he or she is at home,
and controls the heating system accordingly. Furthermore, the
duration of use of IoT devices is an one-dimensional process
that starts with the deployment of a device and ends with
it’s disposal, just like non-smart devices [24], i.e., it does not
follow a periodic lifecycle where it is constantly monitored and
improved. For example, a smart light switch never changes its
function, and it cannot be adapted to different needs. We have ruled out a cloud-based approach [25], [26] that
externalizes the firewall to a trusted third party on the In-
ternet. Although such an approach might fulfil the properties
described, it requires a permanent Internet connection. In ad-
dition, a cloud-based firewall would transfer security-relevant
information into the cloud. Thus, both the Internet connection
of the firewall and the trusted third party would be a valuable
target for an attacker. d) User Roles: Setting up a traditional firewall typically
requires three distinct roles: The role "‘Information Security
Management"’ defines a security policy by considering the
assets and (business) objectives that are relevant for a certain
part of the IT infrastructure. Based on the policy, the role
"‘Configuration Management"’ designs a firewall system, se-
lects appropriate firewall components, and provides an initial
installation and configuration of the system. Finally, a role
"‘IT-Security Operation"’ constantly monitors and improves
the firewall system, both on the level of the firewall rules and
of the firewall hard- and software. PROCEEDINGS OF THE FEDCSIS. LEIPZIG, 2019 PROCEEDINGS OF THE FEDCSIS. LEIPZIG, 2019 452 Homes must differ in the following properties from traditional
firewalls: Homes must differ in the following properties from traditional
firewalls: provide a service, to obtain updates and upgrades, to allow a
remote control via smartphone app, etc. provide a service, to obtain updates and upgrades, to allow a
remote control via smartphone app, etc. c) Application Scenario: Firewalls follow the IT-Security
lifecycle, as explained in Section II. Based on a general
security policy that has been defined from a management
perspective, a network-security expert defines the position
of the firewall(s) in the network architecture and a set of
firewall rules. By using a plan-do-check-act-cycle, the firewall
rules as well as the firewall hard- and software must be
constantly monitored, evaluated and adapted to changes in the
IT infrastructure. c) Application Scenario: Firewalls follow the IT-Security
lifecycle, as explained in Section II. Based on a general
security policy that has been defined from a management
perspective, a network-security expert defines the position
of the firewall(s) in the network architecture and a set of
firewall rules. By using a plan-do-check-act-cycle, the firewall
rules as well as the firewall hard- and software must be
constantly monitored, evaluated and adapted to changes in the
IT infrastructure. P1 The firewall must be usable without expert knowledge. P2 The firewall must fit to the durations of use of Smart
Home components. P3 The firewall must operate in a way that is typical for IoT
devices in the Smart Home. P3 The firewall must operate in a way that is typical for IoT
devices in the Smart Home. P1 implies not only that the configuration and installation of
a firewall in a Smart Home must not require network security
expertise. It also means that a user cannot be expected to tell
false alarms from real alarms, or to decide if a certain firewall
rule is applicable to the home network. From P2 it follows that
such a firewall must deal with IoT devices that are bought once
for a certain purpose and never change its basic properties until
disposal, and it must operate in the same way. Furthermore, the
firewall must operate in the same way. P3 means that a firewall
in a Smart Home needs to operate without permanent care
from the user, i.e., it must monitor the network traffic, deduce
meaningful firewall rules and provide appropriate reactions to
forbidden network packets. III. PROBLEM STATEMENT In this section, we explore the differences between tradi-
tional firewalls and firewalls needed for IoT devices in a Smart
Home installation. In addition, we derive requirements for a
Smart Home firewall. B. Security Concept From a technical perspective, it would be a simple exercise
for a network security expert to set up a firewall that controls
the network traffic of an IoT device. However, this procedure
conflicts with the general understanding how IoT devices
should operate in a Smart Home. Thus, a firewall for Smart From Section III it follows that a traditional firewall ap-
proach is complex, because the underlying network segmen-
tation and the processes executed over the boundaries of
these segments are complex, too, and might change from 453 CHRISTOPH HAAR, ERIK BUCHMANN: FANE: A FIREWALL APPLIANCE FOR THE SMART HOME 453 IP Forwarding
Internet Router
NAT, DNS, DHCP
Firewall
Wi-Fi Access Point
Internet
(untrusted)
Home Network
(trusted)
FANE
IoT Network
(less trusted)
Smartphone
Cloud Server
IoT Device
Fig. 2. System Architecture IoT Network
(less trusted) Internet
(untrusted) Firewall Cloud Server Home Network
(trusted) Smartphone Fig. 2. System Architecture 3) An IoT device is allowed to open a connection to devices
in other (trusted) network segments of the Smart Home,
if this is part of its normal operation. time to time if a new software is installed on a device in
the network. With our network architecture, we have reduced
this complexity. We only need to consider three kinds of
communication activities: 4) A device from a trusted segment is allowed to open
connections to the IoT network segment. • An IoT device wants to communicate with a server
on the Internet. For example, a smart thermostat wants
to communicate with the user’s smartphone, which is
mediated over a cloud service. 5) IoT devices are allowed to open connections to other
devices in the IoT network segment. 5) IoT devices are allowed to open connections to other
devices in the IoT network segment. A. Our FANE prototype We have realized FANE on the basis of a Raspberry Pi,
which executes several linux shell scripts to configure and
operate an iptables packet filter (see Subsection II-C). Figure 4
illustrates our hardware configuration and the main software
packages used. Implement Firewall
Rules Smart Home Operations Review Smart
Home Operations IP Forwarding
Dnsmasq
Hostapd
IPtables
USB Power
Raspberry Pi Model B
Ethernet
Port
SD Card Slot
HDMI Port
Ubuntu Mate OS
USB
Port
USB
WiFi
Adapter
Fig. 4. Our FANE prototype USB
WiFi
Adapter Ethernet
Port Alert
Security Expert Activity Control Flow Document Information Flow Fig. 4. Our FANE prototype a) Hardware: From Section IV it follows that FANE
must provide a Wi-Fi access point that creates a network
segment for IoT devices. The IoT devices might want to
communicate with other devices in the same segment, the
home network segment and the Internet. Thus, FANE must
be connected to the Internet, and its firewall must control all
incoming and outgoing network packets of the IoT network
segment. V. PROOF-OF-CONCEPT IMPLEMENTATION In this section, we describe the software and hardware
components of our FANE prototype, how FANE learns firewall
rules and in which way it interacts with the user. PROCEEDINGS OF THE FEDCSIS. LEIPZIG, 2019 PROCEEDINGS OF THE FEDCSIS. LEIPZIG, 2019 454 Implementation
and Configuration
Learn
Firewall Rules
Implement Firewall
Rules
Review Smart
Home Operations
Re-evaluate
Firewall Rules
Smart Home Operations
Pre-Configuration Management
System
Architecture
Firewall
Configuration
Firewall
Ruleset
Activity
Document
Control Flow
Information Flow
Alert
Security Expert
Policy
Fig. 3. FANE Operations that cannot be handled automatically by FANE. For example,
the IoT network segment might face a denial-of-service attack
from the Internet, or an IoT device has been taken over and
tries to connect to the attacker’s command and control server
on the Internet. In such cases, the user might decide to call
the customer support of the IoT device, or ask an IT-Security
expert for further investigations. Firewall
Configuration C. Smart Home Firewall Operations • An IoT device wants to communicate with a device in
another network segment. For example, the user installs
a control application on a laptop to configure the smart
thermostat. FANE has to meet conflicting requirements: It must meet
the expectations provided by Smart Home components (P2). In
particular, this means that FANE must operate without constant
supervision (P3). At the same time, as a security component
it must not neglect the IT-Security process, including a plan-
do-check-act cycle to refine firewall rules. However, this
must be possible without requiring the user to possess expert
knowledge (P1). • An IoT device wants to communicate with another IoT
device in the same network segment. For example, our
smart thermostat wants to directly communicate with the
smart air condition. Since FANE operates as a bridge to the Internet, only the
first two kinds of communication have to be monitored, and the
security properties of the endpoints of the communication can
be specified at production-time of FANE: The open Internet
is insecure by default, the IoT devices are less secure, and
the devices in other network segments of the Smart Home are
trustworthy. This allows to pre-configure the security concept
of FANE in advance, i.e., it does not need a user with network-
security expertise (Property P1): We circumvent these conflicts, as shown in Figure 3):
We distinguish between pre-configuration management and
Smart Home operations. Because we restrict FANE to the
network architecture described in Subsection IV-A, the policy
definition, the firewall design and a baseline configuration of
firewall rules can be done at pre-configuration time. Thus,
we shift the initial parts of the IT-Security process into the
responsibility of the Smart Home firewall manufacturer who
possess IT-Security expertise. Furthermore, we propose to
automate the configuration and the plan-do-check-act cycle in
a way that it’s phases can be started without expert knowledge
at operation time. Finally, we define a process step in a way
that the user is informed when an IT-Security expert is needed. 1) No device on the Internet is allowed to open a network
connection to the IoT network segment. 2) An IoT device is allowed to open a connection to the
Internet, if this is part of its normal operation. VI. EXPERIMENTAL EVALUATION In this section, we explore the applicability of FANE with
three different Smart Home appliances. Fig. 5. Firewall rules learned from an adjusted packet log Note that this procedure can be extended easily to extended
firewall features, e.g., to include the iptables options for
stateful inspection. At the end of the rule generation phase,
FANE installs the rules and is ready for operation. When FANE is connected to power and Internet for the first
time, or if the user wants FANE to learn new rules, it enters the
monitoring phase for a certain period of time. In this phase,
FANE waits for new IoT devices connecting to the access
point, and logs the network packets. We have implemented
this phase as follows: If an IoT device is not working properly, if a new IoT device
is added to the IoT network segment or if an existing device is
used in a way it has never been used before, the user can order
FANE to re-evaluate the rule set. In this case, the user has the
option to discard rules from preceding learning procedures,
and to re-start the monitoring- and rule-generation phase. At boot time, a cron task with the time prefix @reboot
starts a script that finds out if the set of firewall rules is the
one that has been pre-configured from the security concept
(Subsection IV-B). Alternatively, a user command starts the
monitoring phase manually. In the monitoring phase, FANE
uses the monitoring tool inotify to find out if the dhcp.leases
file changes. This indicates new devices using the access point. In this case, inotify executes a script that obtains the IP address
of the device from dhcp.leases. At the same time, FANE uses
tcpdump to create a log file containing all network packets
sent or received during the monitoring phase. CHRISTOPH HAAR, ERIK BUCHMANN: FANE: A FIREWALL APPLIANCE FOR THE SMART HOME Firewall rules learned from an adjusted packet log D. User Interaction After having defined the operations of FANE, we can
define the user interactions needed. Observe that no interaction
requires expert knowledge (Property P1). FANE comes as an
IoT device that runs out-of-the-box after being connected to a
power outlet and the Internet. When FANE is connected to the Smart Home installation
for the first time or if new IoT devices are added, the user
can tell FANE to learn new firewall rules by observing the
network packets of the IoT devices. Assume an IoT device uses
a functionality that has not been used during the learning stage,
or the device has been updated and a new network connection
is now blocked by FANE. In this case, the user has the option
to let FANE re-evaluate the rule set. That is, FANE executes
a learning stage on a certain device with the option to discard
rules that have been learned before. The rules from the security
concept (Subsection IV-B) cannot be discarded. We have implemented this approach on a third-generation
Raspberry Pi model B. This is a credit-card sized single board
computer containing a quad-core processor with 1.2GHz, 1 GB
main memory and various network and connection interfaces. Because the on-board Wi-Fi chip cannot be configured as
a Wi-Fi access point, we have connected an external Wi-
Fi module via USB. We have used a 32 GB SD Card for
permanent storage. We would need only two switches to initiate the learning-
and re-evaluation stage of the user interface, and one LED
indicating an alert. The IT-Security expert, which might be
needed to handle serious attacks on the IoT nework segment,
would be able to obtain firewall logs and other information
by using an SSH connection. This way, our FANE prototype If FANE blocks a large number of network packets per time-
interval, it generates an alert. The alert shows the user that
immediate action needs to be taken, i.e., something happens CHRISTOPH HAAR, ERIK BUCHMANN: FANE: A FIREWALL APPLIANCE FOR THE SMART HOME B. Learning Firewall Rules 7
15:25:07 IP 10.200.65.101.8554 > 35.157.255.122.80:
8
iptables -A FORWARD -s 10.200.65.101 -sport
1024:65535 -d 35.157.255.122 -dport 80
-p tcp -j ACCEPT 7
15:25:07 IP 10.200.65.101.8554 > 35.157.255.122.80:
8
iptables -A FORWARD -s 10.200.65.101 -sport
1024:65535 -d 35.157.255.122 -dport 80
-p tcp -j ACCEPT For our FANE prototype, we have used a straightforward ap-
proach to learn firewall rules. For more elaborate approaches,
see Section II. The learning stage consists of two phases, a
monitoring phase and a rule generation phase. We assume
that all network traffic recorded during the monitoring phase
is allowed, i.e., we assume that no IoT device has been
manipulated or attacked before the monitoring phase ends. CHRISTOPH HAAR, ERIK BUCHMANN: FANE: A FIREWALL APPLIANCE FOR THE SMART HOME 455 costs less than 60 EUR. However, to ease development we
have used an external USB keyboard and a LCD monitor. costs less than 60 EUR. However, to ease development we
have used an external USB keyboard and a LCD monitor. reduced to unique entries in a second step. The odd lines in
Figure 5 show, how the set of addresses and ports looks like
after FANE has removed surplus information and duplicats
from the log file. In a third step, a shell scripts parses the
remaining addresses and ports into firewall rules that allow
such packets for the iptables chain "‘FORWARD"’. The odd
lines in Figure 5 illustrate this step. We have used the iptables
policy "‘DROP"’, i.e., FANE drops all packets that are not
allowed by the rules generated. b) Software: We have used the Ubuntu Mate Linux
operating system as a basis of our software configuration. On top of a minimal OS installation, we need the following
software packages and services: • awk (script language to edit text files) • cron (timed execution of processes) • dnsmasq (DHCP client and DNS cache) • hostapd (Wi-Fi access point) 1
15:23:18 IP 10.200.65.101.1080 > 35.158.162.95.80:
2
iptables -A FORWARD -s 10.200.65.101 -sport
1024:65535 -d 35.158.162.95 -dport 80
-p tcp -j ACCEPT • inotify-tools (monitor changes in files) • iptables (network address translation and firewall) • iptables (network address translation • tcpdump (record network packets) By configuring the Ethernet interface eth0 as a DHCP client,
our Raspberry Pi can be connected to any Internet router
without further configuration. We have configured the wlan0
interface with a static IP address and subnet mask, and we
have configured it as a Wi-Fi access point by using hostapd. Our Smart Home firewall must act as a bridge between eth0
(Internet) and wlan0 (Wi-Fi segment for IoT devices). Thus,
we have used iptables and sysctl to activate IP forwarding,
including network-address translation and masquerading. With
dnsmasq, we have realized a DHCP service. 3
15:23:22 IP 10.200.65.101.8553 > 35.157.158.75.1883:
4
iptables -A FORWARD -s 10.200.65.101 -sport
1024:65535 -d 35.157.158.75 -dport 1024:65535
-p tcp -j ACCEPT 5
15:24:36 IP 10.200.65.101.8653 > 35.156.40.103.1883:
6
iptables -A FORWARD -s 10.200.65.101 -sport
1024:65535 -d 35.156.40.103 -dport 1024:65535
-p tcp -j ACCEPT 7
15:25:07 IP 10.200.65.101.8554 > 35.157.255.122.80:
8
iptables -A FORWARD -s 10.200.65.101 -sport
1024:65535 -d 35.157.255.122 -dport 80
-p tcp -j ACCEPT
Fig. 5. Fig. 7. Fragment of the packet log of the IoT relay 1) An electrical IoT relay. C. IoT Power Outlet 2) An IoT power outlet. 2) An IoT power outlet. Our second use case is an IoT power outlet "‘Smart Wi-
Fi Socket Model SWA1"’, sold for 18 EUR, produced by
Shenzhen Lingan Intelligent Technology [28]. Similarly to
the IoT relay, the IoT power outlet can be turned on or off
via smartphone app. In addition, it can be controlled with
Amazon Alexa or Google Home, which allows to integrate
non-smart electrical devices into an elaborate Smart Home
concept without requiring a technician. Any command to the
IoT power outlet is handled by a cloud service over the
Internet. 3) An IoT security camera. 3) An IoT security camera. The IoT devices do not communicate directly with each
other, but with the user’s smartphone and the Internet. Thus,
for our experiments we do not need to preconfigure rule 5
from our security concept (see Subsection IV-B). We have
configured each device for FANE’s IoT network segment. We have used a monitoring phase of 40 minutes, and we
have operated each device periodically during this phase. In
the following, we briefly introduce each IoT device, and we
describe what we have learned by using FANE as described. In our monitoring phase, we have used the IoT power outlet
via smartphone in the same way as the relay for 40 minutes. At the end of the monitoring phase, FANE has collected a
packet log of approx. 2,600 lines, all of them TCP packets. The rule generation phase has generated rules that allow five 5
13:41:31.669239 IP 52.71.154.91.443 >
10.200.65.109.55161: Flags [P.], ... Fig. 6. Our experimental setup A. Setup Figure 6 shows our experimental setup. FANE is directly
connected to the Internet router, and its integrated access
point spans a Wi-Fi network segment for IoT devices. The
Internet router creates a Wi-Fi home network that connects a
smartphone to the Internet. Different cloud services connect
the smartphone to the IoT devices. A cloud service might use
a load balancer, i.e., the IP addresses the IoT devices connect
to might change from time to time. At the end of the monitoring phase, FANE stops tcpdump
and enters the rule generation phase. In this phase, FANE
parses the log file from tcpdump into firewall rules according
to the IP addresses of the IoT devices that have used the
access point in the monitoring phase. In particular, FANE uses
a sed command to filter the log for incoming and outgoing
IP addresses and ports. This set of addresses and ports is We have tested three different devices, which communicate
differently with a control app on the user’s smartphone: PROCEEDINGS OF THE FEDCSIS. LEIPZIG, 2019 PROCEEDINGS OF THE FEDCSIS. LEIPZIG, 2019 456 smartphone app, which allows a technician to integrate non-
smart electrical devices into a straightforward Smart Home
installation. Sending commands from the app to the relay
requires an Internet connection, i.e., there is no option to
directly connect the smartphone app to the IoT device. After
the relay is connected to the access point provided by FANE,
and the user has installed the smartphone app, the relay is
ready to use. Internet
FANE
Cloud
Server
IoT Devices
Smartphone of
the User
eth0
wlan0
IoT Network
Internet Router
Home
Network
Fig. 6. Our experimental setup Cloud
Server Cloud
Server In our monitoring phase of 40 minutes, we have switched
the relay on and off frequently via smartphone app for 10
minutes. After that, we have waited for a period of 20 minutes. Finally, we have operated the relay for further 10 minutes. After completing the monitoring phase, FANE has written
1,800 lines in the packet log. All packets followed the TCP
protocol and were sent/received to/from one singular IP ad-
dress located at a dedicated server leased from Amazon. Thus,
the rule generation phase has generated only one rule for in-
and outgoing packets. The IoT relay was working properly
after FANE has activated the firewall rule set generated. Figure 7 shows an example from the traffic log FANE has
recorded from the IoT relay. Internet wlan0 1
13:41:31.551813 IP 10.200.65.109.55147 >
52.71.154.91.443: Flags [F.], ... Home
Network 2
13:41:31.551870 IP 10.200.65.109.55145 >
52.71.154.91.443: Flags [F.], ... 3
13:41:31.551914 IP 10.200.65.109.55161 >
52.71.154.91.443: Flags [.], ... 3
13:41:31.551914 IP 10.200.65.109.55161 >
52.71.154.91.443: Flags [.], ... 4
13:41:31.668878 IP 52.71.154.91.443 >
10.200.65.109.55161: Flags [.], ... 5
13:41:31.669239 IP 52.71.154.91.443 >
10.200.65.109.55161: Flags [P.], ... 1
13:41:31.551813 IP 10.200.65.109.55147 >
52.71.154.91.443: Flags [F.], ... VII. CONCLUSION The last years have brought a plethora of Internet-of-Things
(IoT) devices dedicated to Smart Home installations. While
such IoT devices have numerous practical use cases, observa-
tions have shown that many of them come with IT-Security
risks. For example, the Mirai botnet consisted of approax. 500,000 baby-phones, security cameras and other insecure
IoT devices that were able to execute distributed denial-of-
service attacks with 1 Tbit/s network bandwidth. However,
typical Smart Home users do not possess the network-security
knowledge needed to identify and deter attacks on IoT devices. Furthermore, the Smart Home concept encourages the users to
leave IoT devices unattended for long periods of time. We have observed that the IoT security camera was not
working properly, after FANE started to filter network packets. Our investigations have shown that this due to a specific load
balancer. The IP address of the load balancer was allowed by
the firewall rule set generated. But the load balancer referred
the IoT security camera frequently to IP addresses unknown
to FANE. However, it would be possible to adapt the learning
approach to cope with such a load balancer. For example,
FANE could detect and accept IP addresses that are close by
addresses that are already allowed by the rule set. The packet log has also shown that the IoT security camera
first tries to reach the smartphone app in the same network
segment directly, via multicast. Thus, even if the IoT security
camera makes use of the Internet connection, it might be able
to provide its basic functionality without the Internet. From
this observation we conclude that there might be options for
FANE to distinguish between communication needed for the
normal operation of an IoT device, and other communication
needed for advertising purposes or usage analytics that can be
blocked without undesired side-effects. In this paper, we have introduced FANE, our concept for
a Firewall AppliaNcE for Smart Home installations. FANE
makes a few realistic assumptions on the network segmen-
tation and the communication profile of IoT devices. This
allows to pre-configure FANE with a generic security concept. It also enables FANE to learn firewall rules automatically by
observing the network traffic of IoT devices. Experiments with a prototypical implementation have pro-
vided evidence that FANE can secure ordinary IoT devices
without requiring network-security expertise from the Smart
Home user. CHRISTOPH HAAR, ERIK BUCHMANN: FANE: A FIREWALL APPLIANCE FOR THE SMART HOME 457 different IP addresses, all of them in the address range of the
Amazon AWS cloud. E. Discussion Our three use cases have provided evidence that a straight-
forward learning approach is applicable to many IoT devices
used in Smart Home scenarios. Two of our three IoT devices
remained fully operative after FANE has monitored the net-
work activities of our devices for 40 minutes, and has sub-
sequently generated and activated firewall rules. Furthermore,
our observations have shown that it would be easily possible
to extend our learning approach to consider load balancers. As there is no communication standard for IoT devices, it is
problematic to generalize our findings to all IoT devices used
in the Smart Home. However, using a cloud service seems
to be typical for many use cases. Only network packets can
pass FANE that are allowed by a specific rule. Thus, FANE
increases the security of the Smart Home installation. The IoT power outlet was fully operational after FANE
has started to filter network connections. We have observed
that only one of the five addresses in the firewall rule set
was actually used to operate the outlet via smartphone app. We assume that some network connections are used only for
analyzing customer behavior or similar purposes, i.e., blocking
them would not reduce the functionality of the device. D. IoT Security Camera The most complex IoT device tested was a "‘720P HD IP
Wireless security camera"’, sold for 37 EUR and manufactured
by XinweiYa [29]. The IoT security camera sends a live
video stream to the smartphone of the user. Furthermore, the
smartphone app allows to restart the IoT security camera,
and to rotate it around two axes. After connecting the IoT
security camera to a power outlet, it can be configured with a
smartphone app to use FANE’s access point. FANE operates without requiring the user to possess expert
knowledge, by making three assumptions: First, the network
segment created by FANE’s access point contains IoT devices
only. This allows to specify a security policy in advance,
before FANE is delivered to the user. Second, the IoT devices
operate as single-purpose appliances that do not fundamentally
change their communication profiles. Due to this assumption,
FANE can learn a rule set that remains stable over a long
period of time, which makes it compatible with the Smart
Home concept. Third, we assume that the IoT devices are
working properly during the monitoring phase. This allows
FANE to learn firewall rules unattended. During our monitoring phase of 40 minutes, we have
restarted the IoT security camera, we have let the IoT security
camera sent a live video stream of 10 minutes to the smart-
phone, we have waited for 20 minutes, and we have restarted it
again for another live stream of 10 minutes. After 40 minutes,
FANE has collected 8 MB packet log of approx. 27,000 lines,
most of them UDP packets. The rule generation phase produces a rule set of 20 rules
for this device. Those rules allow services like Network Time
Protocol (NTP) or Domain Name System (DNS) as well as
cloud services hosted on Amazon AWS, the Microsoft cloud
and the Alibaba cloud. B. IoT Relay Our first use case is an electrical relay "‘10A Wi-Fi
smart switch"’, sold for less than 9 EUR, manufactured by
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Injection and Combustion Analysis of Pure Rapeseed Oil Methyl Ester (RME) in a Pump-Line-Nozzle Fuel Injection System
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Energies
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Article
Injection and Combustion Analysis of Pure Rapeseed Oil
Methyl Ester (RME) in a Pump-Line-Nozzle Fuel
Injection System Flavio Caresana 1
, Marco Bietresato 2
and Massimiliano Renzi 2,* 1
Dipartimento di Ingegneria Industriale e Scienze Matematiche, Università Politecnica delle Marche,
I-60131 Ancona, Italy; f.caresana@univpm.it
2
Facoltà di Scienze e Tecnologie, Libera Università di Bolzano, I-39100 Bolzano, Italy;
marco.bietresato@unibz.it y
p
2
Facoltà di Scienze e Tecnologie, Libera Università di Bolzano, I-39100 Bolzano, Italy;
marco.bietresato@unibz.it *
Correspondence: massimiliano.renzi@unibz.it; Tel.: +39-0471-0171816; Fax: +39-0471-017009 *
Correspondence: massimiliano.renzi@unibz.it; Tel.: +39-0471-0171816; Fax: +39-0471-017009 Abstract: This work suggests an interpretation to the quantitatively higher formation of NOx in a
compression ignition (CI) engine when fueled with pure biodiesel (B100). A comparative study about
the use of rapeseed oil methyl ester (RME) and diesel fuel mixtures on injection timing, in-chamber
pressure, heat release rate, and NOx emissions were carried out using a diesel engine equipped with
a pump-line-nozzle injection system. Such engines are still widely adopted mainly in agriculture, as
the fleet of agricultural machinery is particularly old (often over 20 years) and the use of biofuels can
reduce the environmental footprint of the sector. This work aims to supply some general explanations
and figures useful to interpret the phenomena occurring within the fuel line and in the combustion
process when using biodiesel, as well as in engines with different construction characteristics and
fueling systems. Given the contradictory results available in the literature, the so-called “biodiesel
NOx effect” cannot be explained solely by the different physical properties of biodiesel (in particular,
a higher bulk modulus). Experimental results show that, with the same pump settings, the start
of injection with the RME is slightly advanced while the injection pressure values remain almost
the same. With the RME, the pressure in the injection line increases faster due to its greater bulk
modulus but the pressure rise starts from a lower residual pressure. The start of combustion takes
place earlier, the heat release during the premixed phase is steeper, and a higher peak is reached. The NOx emissions with the RME are at least 9% higher when compared to mineral diesel fuel. The
greater amount of the RME injected per cycle compensates for its minor lower heating value, and the
brake torque at full load is similar to the two analyzed fuels. Finally, a variation of the pump line
timing is evaluated in order to assess the effect of the delay and the advance of the injection on the
performance of the engine and on the emissions.
Citation: Caresana, F.; Bietresato, M.;
Renzi, M. Injection and Combustion
Analysis of Pure Rapeseed Oil Methyl
Ester (RME) in a Pump-Line-Nozzle
Fuel Injection System. Energies 2021,
14, 7535. https://doi.org/10.3390/
en14227535 Academic Editor: Rocio Maceiras Academic Editor: Rocio Maceiras
Received: 27 September 2021
Accepted: 8 November 2021
Published: 11 November 2021 Keywords: compression ignition engine; biodiesel; B100; heat release rate; injection timing advance;
injection pressure; biodiesel NOx effect Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Article
Injection and Combustion Analysis of Pure Rapeseed Oil
Methyl Ester (RME) in a Pump-Line-Nozzle Fuel
Injection System A viable and simple solution in the variation of the
injection strategy is suggested to counterbalance the biodiesel NOx effect. Keywords: compression ignition engine; biodiesel; B100; heat release rate; injection timing advance;
injection pressure; biodiesel NOx effect Citation: Caresana, F.; Bietresato, M.;
Renzi, M. Injection and Combustion
Analysis of Pure Rapeseed Oil Methyl
Ester (RME) in a Pump-Line-Nozzle
Fuel Injection System. Energies 2021,
14, 7535. https://doi.org/10.3390/
en14227535 energies energies 1. Introduction Other properties, compared
to mineral diesel, include a higher cetane number, lubricity, and flash point, while the
toxicity is reduced. Among the detrimental characteristics, it is possible to list its lower
volatility, the calorific value, the oxidation stability [10,11], and the higher viscosity [12]
and pour point [13] that, however, can be adjusted due to new processes that improve
the low temperature performance of biodiesels [5–7]. When the biodiesel is blended
with diesel fuel at high percentages, it can also cause reliability problems for the engine
components, as it can cause corrosion and chemically attack some plastic components of
seals and coatings [14]. g
Different oils are used to produce biodiesel fuels depending on the local availability. The most common in the United States is soybean oil (from Glycine max (L.) Merr.); coconut
oil (from Cocos nucifera L.) and palm oils (mainly from Elaeis guineensis Jacq.) are used in
Malaysia and Indonesia; the Jatropha (Jatropha curcas L.), Karanja (Pongamia pinnata L.),
and Mahua trees (Mahua Indica J. F. Gmel) are used as a significant fuel sources in India
and southeast Asia; while rapeseed oil-based esters (from Brassica napus L.) are commonly
used in many European countries [15,16]. In Austria and Germany, neat biodiesel is widely
used, whereas in France, Italy, Spain, Sweden, the Czech Republic, and other European
countries, fuel blends of diesel fuel with up to 25–30% rapeseed oil methyl ester (RME)
are popular [17]. Some reviews on the effects of biodiesel fuels on compression-ignited
(CI) engine performance and emissions report the following main outcomes [18–22]: (i) the
fuel consumption increases proportionally to the loss of the heating value of the fuel;
(ii) the particulate matter (PM) emissions show a consistent reduction; (iii) a minimal or
absent loss of power is reported, except when maximum power is required. As regards
the brake thermal efficiency (BTE), its value is not significantly affected with biodiesel,
but a slight increase or a reduction in the BTE can be noted depending on the engine
load, the type of biodiesel, and the blend with mineral diesel fuel [23]. Improvements in
the efficiency are mainly due to the synergic blending effects that could be caused by the
reductions in friction losses [24]; instead, lower efficiencies are typically reported at high
engine loads and rotational speeds because of the poor spray characteristics, poor mixing,
high viscosity, and the lower calorific value of biodiesel [25]. 1. Introduction The role of “alternative” fuels to meet the reduction targets in the impact of the energy
production and transport sectors has increased in the last few years. Currently, “bioenergy”
is the largest contributor to the global renewable energy supply, especially for thermal
energy production, but has not yet reached a large-scale production for liquid biofuels,
as this must overcome some complex research uncertainties on the management of those
resources, their lifecycle emissions, their effects on water, the soil management, the process
efficiencies, as well as facing their potential negative effects [1]. In particular, among the
biomass-derived fuels, and despite the existence of some serious problems associated with
their application with respect to feedstock requirements and land availability—the “food 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/energies Energies 2021, 14, 7535. https://doi.org/10.3390/en14227535 Energies 2021, 14, 7535 2 of 25 vs. fuel” debate—the so-called “first generation biofuels” have grown mainly thanks to
government subsidies and legislative pressures [2]. vs. fuel” debate—the so-called “first generation biofuels” have grown mainly thanks to
government subsidies and legislative pressures [2]. Meanwhile, novel biofuels have been introduced that use nonfood biomass, dedicated
energy crops, and biomass coproducts and waste from many different sectors, such as
agriculture, horticulture, forestry, paper and pulp processing, and wastes (e.g., municipal
solid waste (MSW)) [3]. Novel improved technologies are being developed to increase the
production yield of qualitatively acceptable biodiesel [4], and new processes have been
proposed to improve the low-temperature performance of biodiesels [5–7]. Among the available biofuels, “biodiesel” is one of the candidates in this class and its
use is expanding throughout the world [8]. As an example, total biofuel consumption in the
EU was around 17.0 Mtoe in 2018, and in terms of energy content, while biodiesel’s share
was 82.0%, bioethanol was 17.1%, and biomethane fuel was 0.9%, respectively [9]. The main
advantage of the use of biodiesel resides in its renewability, which allows for the reduction
of the burdens of greenhouse gases (GHG). From the environmental point of view, it is
possible to enumerate other important benefits deriving from the chemical composition of
biodiesel, which has a minimal sulfur and aromatic content. 1. Introduction As regards the emissions,
all of the authors agree in the reduction of the carbon monoxide (CO), hydrocarbon (HC),
and PM emissions due the use of biodiesel [26]. With regard to the nitrogen oxides
(NOx) concentration, most of the authors [11,14] report a slight increase with biodiesel;
nevertheless, some studies have actually found very different evidence, spanning from
“NOx increment only in some operating conditions” up to “there is lack of difference
between diesel fuel and biodiesel” or even “NOx emissions decrease with biodiesel”. As it
appears from the literature reviews [27–30], despite the great number of research articles
available, various topics regarding biodiesel use in diesel engines remain unsolved and
need further investigation. In 2007, Lapuerta et al. [27] showed that, although a dominant Energies 2021, 14, 7535 3 of 25 3 of 25 trend could be found in most cases, there are always opposing trends proposed elsewhere
by contrast. As possible reasons for this, they indicate: the large number of different
engine technologies tested, the various operating conditions or driving cycles followed,
the different biodiesel fuels used (i.e., obtained from different feedstocks and with different
qualities), and the various measurement techniques and procedures applied. In 2018,
Chen et al. [31] titled their article “NOx emission of biodiesel compared to diesel: Higher
or lower?”, thus evidencing that the question has remained open. On a deeper analysis, the
need for further research appears less surprising, as the literature on the subject consists
only of results obtained from different engines, with different injection systems, and in
different operating conditions. Moreover, the effect of biodiesel is sometimes addressed by
taking the engine as a “black box”; thus, not all the data necessary to analyze the problem
are reported (or known by the experimenters). Moreover, in several experiments, it is very
complex to isolate the different thermo-chemical phenomena and to control the operating
conditions and devices of the engine that can contribute to the variation of the performance
and the emissions of a motor fueled with blends of biodiesel. 1. Introduction In general, the main causes of the increase in the NOx formation are [29]: (i) the
advancement of the injection (due to the higher bulk modulus of biodiesel) and, therefore,
the higher in-cylinder pressure and temperature; (ii) the lower formation of soot and the
consequent lower radiative heat transfer, which implies a higher combustion temperature;
(iii) the higher oxygen content in the biodiesel composition, which facilitates the combustion
and the formation of NOx; (iv) other physical properties of the fuel, like the different
viscosity and density, which increase the amount of fuel injected with a consequent higher
combustion temperature, lower spray cone angle, and fuel atomization; (v) the higher and
advanced heat release rate (HRR) in the premixed phase of the combustion process. The
various combinations of the factors cited above interfere with each other to determine NOx
emissions: the increasing or decreasing trend depends on the delicate balance due to every
change in the fuel and combustion characteristics or engine operating conditions [22]. g
g
p
g
However, it is worthwhile to note that there is no shared agreement on the reasons for
the higher NOx formation with biodiesel. At first this could seem quite strange, given the
number of researchers that have analyzed the effects of these parameters, starting from the
first organized studies by the National Renewable Energy Laboratory (NREL) to measure
the sound speed of biodiesel and to establish its impact on the injection timing in diesel
engines [32]. In their review article, Jinlin Xue et al. [28], in trying to individuate the factors
of effect on NOx emissions for biodiesel, indicate the higher oxygen content as the main
cause of the NOx emissions increase, but they say that the injection characteristics also
have an impact and that a further study is needed to inquire into the effect of the injection
timing and pressure. A key role of fuel-based oxygen was also found by Graboski and
McCormick [33] after analyzing a large number of test results of various vegetable oil based
fuels. They concluded that the main advantage of feeding biodiesels in diesel engines
seemed related to their high oxygen content. On the other hand, Lapuerta [34] suggests
that the oxygen/fuel mass ratio of biodiesel (equal to 2.92) is lower than the mineral diesel
fuel ratio (3.58) and, therefore, the oxygen content cannot be the reason for the higher
concentration of NOx. 1. Introduction Some authors have reported that full load torque in
unchanged diesel engines does not vary substantially by switching from diesel fuel to
the ME biofuels because the actual energy content delivered per each active stroke of the
plunger is not substantially lower than with the diesel fuel [44,45]. Indeed, the higher
viscosity of the RME reduces the internal leakages of the injection pump, and this increases
the volumetric fuel delivery per stroke. For example, Labeckas et al. [39] found that,
within an unmodified diesel engine, the volumetric fuel delivery per stroke with the RME
increased by about 2.6% with respect to the diesel fuel. y
p
As far as the injection pressure and an advance in the start of ignition (SOI) is consid-
ered, it is necessary to assess in a different way the engines that operate with a common rail
system and with a mechanical injection system. In particular, in the mechanical injection
systems, the injection timing can be modified only by regulating the fuel compression
stroke with some shims. An advanced injection timing leads to enhancements in the turbu-
lence of the mixture, to an earlier and rapid combustion, to a longer ignition delay time,
and to higher combustion chamber pressures and temperatures [30,46]. On the other hand,
a retarded injection timing results in lower chamber pressures and temperatures during
the combustion, as well as in a shorter ignition delay time [30]. Several experimental inves-
tigations [22,30] have revealed that an advance of the injection timing results in a reduction
of CO, unburned HC, and PM emissions. The same studies showed that retarding the fuel
injection can reduce the NOx concentration at the exhaust thanks to the reduction of the
peak temperature in the engine [47]. As a matter of fact, both the injection timing and the
injection pressure are easily controllable only in common rail injection systems, where the
SOI event is commanded by the electronic control unit (ECU) and the injection pressure
in the rail can be easily regulated. For common rails, one could reasonably conclude that
the injection timing is fluid-independent, whilst the pressure in the rail may vary with
biodiesel in case the same torque has to be reached [48–50]. Nevertheless, an advance in
the SOI and a higher in-cylinder pressure was also reported in common rail engines [51]. 1. Introduction Among the different types of biodiesels and fueling systems, this study focuses on
a methyl ester biodiesel used in a pump-line-nozzle (PLN) injection system. The PNL
CI engines are still a widespread technology in small-scale engines for cogeneration,
for industrial applications, in the agricultural sector, and in off-highway diesel engine
applications where more advanced technologies are too expensive and, often, advanced
gas-cleaning solutions cannot be adopted. Indeed, in all of these machines, a simple and
straightforward solution for the reduction of pollutants and the limitation of greenhouse
gas emissions is surely the adoption of alternative (bio)fuels [35]. The engine used here is
also particularly representative of the majority of the engines used in agricultural vehicles,
and also due to a mere numerical issue: in the most developed countries, the agricultural
sector is characterized by a very low turnover of mechanical units (e.g., even less than Energies 2021, 14, 7535 4 of 25 1% in Italy in 2012 [36]) and a consequent high average age (around 25 years in Italy [36]),
while developing countries prefer engines/machines with a simple and robust construction
due to many economic and environmental conditions. In both scenarios, newer engine
technologies (e.g., the common rail) are still struggling to gain market shares. The simplicity
of these engines also has a further appreciable effect in an experimental context: due to their
intrinsic simplicity, it is possible to avoid any masking of the effects of fuel substitution as a
result of the engine’s automatic adjustment mechanisms used in more recent, electronically
controlled engines (first and foremost, the automatic injection advance variation system). g
j
y
Diesel engines operating on neat methyl esters (ME) or their blends with diesel fuel
have already been tested by various researchers evidencing, in line with the majority of
the biodiesels, reductions in CO, HC, and PM emissions and smoke, along with a higher
concentration of NOx in the exhausts. Çelikten [37] reported that in all performance modes,
RME leads to lower CO and to higher NOx emissions than the fossil fuels. Krahl et al. [38]
found similar results on these pollutants, but they could not find a general tendency
regarding the HC emissions. Most of the authors agreed that the reduction of the calorific
value due to the ME mixing with diesel fuel causes an increase in the brake-specific
fuel consumption (BSFC) [39–43]. 1. Introduction However, mechanical injection systems have been shown to be influenced by some fluid
properties, mainly the bulk modulus and the viscosity, and it is almost generally accepted
that, when substituting biodiesel to diesel fuel: (i) the SOI is advanced and (ii) the injection
pressure becomes higher. These conclusions are somehow taken for granted and often used
to explain the biodiesel NOx effect, and their combined effect advances the start of combus-
tion (SOC) and contributes to a higher combustion rate; both enhance the NOx formation
according to the well-documented Zeldovich mechanism [52]. It is worth remembering
that only data from experiments at the fixed position of the injection pump should be
considered. If the engine was set at the same torque level (for a given rotational speed), a
greater amount of biodiesel should be injected to compensate for its lower heating value. In such cases, the injection pump settings should not be the same for the diesel fuel and the Energies 2021, 14, 7535 5 of 25 biodiesel. Moreover, some injection pumps have a fixed end of injection timing and, when
additional quantities of fuel are requested to be injected, the start of injection is advanced. biodiesel. Moreover, some injection pumps have a fixed end of injection timing and, when
additional quantities of fuel are requested to be injected, the start of injection is advanced. In a previous article [53], the differences in the injection timing and the pressure of
the RME as an alternative to diesel fuel in a PLN injection system were discussed. The
final considerations suggested that the injection timing due to the higher bulk modulus of
biodiesel is less advanced than is commonly believed. The aim of the present work is to study in greater detail the injection characteristics of
a CI engine fueled with pure RME (B100) in pump-line-nozzle systems. In such engines, the
adoption of modern injection solutions and gas cleaning solutions is necessarily limited due
to cost issues, and a lower environmental impact can only be obtained by using alternative
(bio)fuels and tuning their management so to optimize the performance and the emissions. This study tries to supply some general explanations and figures that can be helpful to
interpret the phenomena occurring in the fuel line and in the combustion process when
using biodiesel, as well as in engines with different construction characteristics and fueling
systems. 1. Introduction Specifically, the performance variations, the thermal efficiency, and the NOx
concentration in a mechanical PLN engine are reported in this work. Further analyses and
the consequences on combustion behavior are evidenced, in particular the SOI and the
SOC advances are shown, and the trend of the HRR is evaluated with the aim of finding a
correlation with the biodiesel, etc. Moreover, a quick and simple modification of the fuel
injection timing on the pump line was tested in order to highlight the chance of reducing
the NOx increase in this typology of engines without causing any performance decay. Some
authors also delved into the RME combustion characteristics [54–61], and their results are
addressed in the concluding Section 3.4 and compared with those of the present study. 2. Materials and Methods Quantity
Unit
Diesel Fuel
Biodiesel
Bulk modulus
MPa
1490
1650
Cetane number
-
48
54
C/H/O
molar ratio
16:30:0
19:34:2
Density at 15 °C
kg·dm−3
0.835
0.885
Lower heating value on a mass basis (LHVm)
kJ·kg−1
42,739
37,400
Kinematic viscosity at 40 °C
mm2·s−1
2.60
4.50
Carbon content
wt %
86.5
77.4
Hydrogen content
wt %
13.4
12.0
Oxygen content
wt %
0.0
10.5
Sulfur content
wt %
0.05
<0.01
Stoichiometric air-fuel ratio
-
14.5
12.6
Iodine value
g(I2)·(100 g)−1
-
118.2
Pour point
°C
−26
−12
Flash point
°C
72
>150
Biodegradability (after 28 days)
%
18
85
Quantity
Unit
Diesel Fuel
Biodiesel
Bulk modulus
MPa
1490
1650
Cetane number
-
48
54
C/H/O
molar ratio
16:30:0
19:34:2
Density at 15 ◦C
kg·dm−3
0.835
0.885
Lower heating value on a mass basis (LHVm)
kJ·kg−1
42,739
37,400
Kinematic viscosity at 40 ◦C
mm2·s−1
2.60
4.50
Carbon content
wt %
86.5
77.4
Hydrogen content
wt %
13.4
12.0
Oxygen content
wt %
0.0
10.5
Sulfur content
wt %
0.05
<0.01
Stoichiometric air-fuel ratio
-
14.5
12.6
Iodine value
g(I2)·(100 g)−1
-
118.2
Pour point
◦C
−26
−12
Flash point
◦C
72
>150
Biodegradability (after 28 days)
%
18
85
EVIEW
7 of 26 Quantity
Unit
Diesel Fuel
Biodiesel
Bulk modulus
MPa
1490
1650
Cetane number
-
48
54
C/H/O
molar ratio
16:30:0
19:34:2
Density at 15 °C
kg·dm−3
0.835
0.885
Lower heating value on a mass basis (LHVm)
kJ·kg−1
42,739
37,400
Kinematic viscosity at 40 °C
mm2·s−1
2.60
4.50
Carbon content
wt %
86.5
77.4
Hydrogen content
wt %
13.4
12.0
Oxygen content
wt %
0.0
10.5
Sulfur content
wt %
0.05
<0.01
Stoichiometric air-fuel ratio
-
14.5
12.6
Iodine value
g(I2)·(100 g)−1
-
118.2
Pour point
°C
−26
−12
Flash point
°C
72
>150
Biodegradability (after 28 days)
%
18
85
Quantity
Unit
Diesel Fuel
Biodiesel
Bulk modulus
MPa
1490
1650
Cetane number
-
48
54
C/H/O
molar ratio
16:30:0
19:34:2
Density at 15 ◦C
kg·dm−3
0.835
0.885
Lower heating value on a mass basis (LHVm)
kJ·kg−1
42,739
37,400
Kinematic viscosity at 40 ◦C
mm2·s−1
2.60
4.50
Carbon content
wt %
86.5
77.4
Hydrogen content
wt %
13.4
12.0
Oxygen content
wt %
0.0
10.5
Sulfur content
wt %
0.05
<0.01
Stoichiometric air-fuel ratio
-
14.5
12.6
Iodine value
g(I2)·(100 g)−1
-
118.2
Pour point
◦C
−26
−12
Flash point
◦C
72
>150
Biodegradability (after 28 days)
%
18
85
REVIEW
7 of 26
Figure 2. 2. Materials and Methods The experiments were carried out on a single-cylinder Ruggerini RF91 diesel engine
(by Kohler Lombardini, Reggio nell’Emilia, Italy [62]), whose main characteristics are
described in Table 1. The engine was tested on an APIcom RE100 test stand (Cento, Ferrara,
Italy) with commercial diesel fuel and rapeseed biodiesel (Figure 1). At the end of each
trial, the fuel was switched back to diesel fuel and the engine was kept running for a while
before shut-down to flush out the biodiesel from the fuel line and the injection system. The
properties of the two fuels are reported in Table 2. In particular, according to the EASAC
report 2012 classification, the biodiesel used during the experiments is a first generation
one, produced starting from the oil extracted from rapeseed and passed through refining,
transesterification, and subsequent distillation processes [16]. Table 1. Technical characteristics of the tested diesel engine (Ruggerini RF51). Quantity
Specification
Type
Single-cylinder four-stroke direct injection
Nr. of valves
2–OHV
Bore × stroke
90 × 75 mm
Displacement
477 cm3
Compression ratio
18.5
Maximum power
8.1 kW (11 CV) at 3600 rpm
Maximum torque
25 N·m (2.5 kgf·m) at 2500 rpm
Injection advance
16◦CA before top dead center
Injection nozzle opening pressure
210 bar Table 1. Technical characteristics of the tested diesel engine (Ruggerini RF51). Table 1. Technical characteristics of the tested diesel engine (Ruggerini RF51). Quantity
Specification
Type
Single-cylinder four-stroke direct injection
Nr. of valves
2–OHV
Bore × stroke
90 × 75 mm
Displacement
477 cm3
Compression ratio
18.5
Maximum power
8.1 kW (11 CV) at 3600 rpm
Maximum torque
25 N·m (2.5 kgf·m) at 2500 rpm
Injection advance
16◦CA before top dead center
Injection nozzle opening pressure
210 bar Specification
Single-cylinder four-stroke direct injection
2–OHV
90 × 75 mm
477 cm3
18.5
8.1 kW (11 CV) at 3600 rpm
25 N·m (2.5 kgf·m) at 2500 rpm
16◦CA before top dead center
210 bar In-cylinder and injection pressures were measured by using piezoelectric transducers,
specifically an AVL GU12P (Graz, Austria [63]) fitted on the cylinder head, and a PCB
Piezotronics 112A21 (Depew, NY, USA [64]) fitted on the injection pipe about 10 mm
apart (Figure 2). An AVL 3066A0” charge amplifier received the outputs of the pressure
transducers and fed them to an AVL 619 INDIMETER system. 2. Materials and Methods p
Quantity
Unit
Diesel Fuel
Biodiesel
Bulk modulus
MPa
1490
1650
Cetane number
-
48
54
C/H/O
molar ratio
16:30:0
19:34:2
Density at 15 °C
kg·dm−3
0.835
0.885
Lower heating value on a mass basis (LHVm)
kJ·kg−1
42,739
37,400
Kinematic viscosity at 40 °C
mm2·s−1
2.60
4.50
Carbon content
wt %
86.5
77.4
Hydrogen content
wt %
13.4
12.0
Oxygen content
wt %
0.0
10.5
Sulfur content
wt %
0.05
<0.01
Stoichiometric air-fuel ratio
-
14.5
12.6
Iodine value
g(I2)·(100 g)−1
-
118.2
Pour point
°C
−26
−12
Flash point
°C
72
>150
Biodegradability (after 28 days)
%
18
85
Table 2. Main specifications about the used fuels. Quantity
Unit
Diesel Fuel
Biodiesel
Bulk modulus
MPa
1490
1650
Cetane number
-
48
54
C/H/O
molar ratio
16:30:0
19:34:2
Density at 15 ◦C
kg·dm−3
0.835
0.885
Lower heating value on a mass basis (LHVm)
kJ·kg−1
42,739
37,400
Kinematic viscosity at 40 ◦C
mm2·s−1
2.60
4.50
Carbon content
wt %
86.5
77.4
Hydrogen content
wt %
13.4
12.0
Oxygen content
wt %
0.0
10.5
Sulfur content
wt %
0.05
<0.01
Stoichiometric air-fuel ratio
-
14.5
12.6
Iodine value
g(I2)·(100 g)−1
-
118.2
Pour point
◦C
−26
−12
Flash point
◦C
72
>150
Biodegradability (after 28 days)
%
18
85
EVIEW
7 of 26
Figure 2. Detailed view showing the positions on the engine head of the piezoresistive sensors by
AVL used to measure the pressure in the combustion chamber and in the fuel injection subsystem. Table 3 Main specifications about the measuring instruments used in the test campaign
Hole to house the in-
chamber pressure
sensor tip
Fuel injection
pressure sensor
Figure 2. Detailed view showing the positions on the engine head of the piezoresistive sensors by
AVL used to measure the pressure in the combustion chamber and in the fuel injection subsystem. 2. Materials and Methods An AVL 36401 encoder,
connected to the engine output shaft, passed its signal to the AVL 619 INDIMETER through Energies 2021, 14, 7535 6 of 25 an AVL 3064Z03 light pulse converter. For each working condition, pressure signals were
measured over 50 consecutive cycles with 0.5◦resolution, and then averaged. The main
characteristics of the measuring instruments are reported in Table 3. Maximum power
8.1 kW (11 CV) at 3600 rpm
Maximum torque
25 N·m (2.5 kgf·m) at 2500 rpm
Injection advance
16 °CA before top dead center
Injection nozzle opening pressure
210 bar an AVL 3064Z03 light pulse converter. For each working condition, pressure signals were
measured over 50 consecutive cycles with 0.5◦resolution, and then averaged. The main
characteristics of the measuring instruments are reported in Table 3. Maximum power
8.1 kW (11 CV) at 3600 rpm
Maximum torque
25 N·m (2.5 kgf·m) at 2500 rpm
Injection advance
16 °CA before top dead center
Injection nozzle opening pressure
210 bar an AVL 3064Z03 light pulse converter. For each working condition, pressure signals were
measured over 50 consecutive cycles with 0.5◦resolution, and then averaged. The main
characteristics of the measuring instruments are reported in Table 3. Maximum power
8.1 kW (11 CV) at 3600 rpm
Maximum torque
25 N·m (2.5 kgf·m) at 2500 rpm
Injection advance
16 °CA before top dead center
Injection nozzle opening pressure
210 bar (b) (a) (a)
(b)
Figure 1. Frontal (a) and lateral (b) views of the Ruggerini RF51 engine installed at the APIcom RE100 test stand for the
execution of the tests. Figure 1. Frontal (a) and lateral (b) views of the Ruggerini RF51 engine installed at the APIcom RE100 test stand for the
execution of the tests. (a) (b) Figure 1. Frontal (a) and lateral (b) views of the Ruggerini RF51 engine installed at the APIcom RE100 test stand for the
execution of the tests. Figure 1. Frontal (a) and lateral (b) views of the Ruggerini RF51 engine installed at the APIcom RE100 test stand for the
execution of the tests. Table 2. Main specifications about the used fuels. Table 2. Main specifications about the used fuels. Table 2. Main specifications about the used fuels. Table 2. Main specifications about the used fuels. Table 2. Main specifications about the used fuels. Table 2. Main specifications about the used fuels. 2. Materials and Methods Instrument
Specifications
Dyno brake model (APIcom, Cento (FE), Italy)
APIcom Eddy current RE100—X TWIN—4943/02
Torque measurement (APIcom, Cento (FE), Italy)
Range 0–500 N·m; Uncertainty ± 0.25%
Rotational speed measurement (APIcom, Cento (FE), Italy)
Range 0–10,000 rpm; Uncertainty ± 10 rpm
Load indicator (APIcom, Cento (FE), Italy)
Range 250–6000 W; Uncertainty ± 10 W
Temperature indicator
Range 0–900 ◦C; Uncertainty ± 1 ◦C
Fuel Volume
Burette range 1–30 cm3; Uncertainty ± 0.2 cm3
Exhaust gas analyzer
Range NO 0–5000 ppm; Uncertainty ± 50 ppm
Range HC 0–20,000 ppm; Uncertainty ± 10 ppm
Range CO 0–10%; Uncertainty ± 0.03%
Pressure transducer (AVL, Graz, Austria)
Range 0–110 bar; Uncertainty ± 0.1 bar
Crank angle encoder (AVL, Graz, Austria)
Range 0–360◦; Uncertainty ± 0.1◦ Specifications
APIcom Eddy current RE100—X TWIN—4943/02
Range 0–500 N·m; Uncertainty ± 0.25%
Range 0–10,000 rpm; Uncertainty ± 10 rpm
Range 250–6000 W; Uncertainty ± 10 W
Range 0–900 ◦C; Uncertainty ± 1 ◦C
Burette range 1–30 cm3; Uncertainty ± 0.2 cm3
Range NO 0–5000 ppm; Uncertainty ± 50 ppm
Range HC 0–20,000 ppm; Uncertainty ± 10 ppm
Range CO 0–10%; Uncertainty ± 0.03%
Range 0–110 bar; Uncertainty ± 0.1 bar
Range 0–360◦; Uncertainty ± 0.1◦ Specifications
APIcom Eddy current RE100—X TWIN—4943/02
Range 0–500 N·m; Uncertainty ± 0.25%
Range 0–10,000 rpm; Uncertainty ± 10 rpm
Range 250–6000 W; Uncertainty ± 10 W
Range 0–900 ◦C; Uncertainty ± 1 ◦C
Burette range 1–30 cm3; Uncertainty ± 0.2 cm3
Range NO 0–5000 ppm; Uncertainty ± 50 ppm
Range HC 0–20,000 ppm; Uncertainty ± 10 ppm
Range CO 0–10%; Uncertainty ± 0.03%
Range 0–110 bar; Uncertainty ± 0.1 bar
Range 0–360◦; Uncertainty ± 0.1◦ Pressure transducer (AVL, Graz, Austria)
Crank angle encoder (AVL, Graz, Austria) By using appropriate correlations for the heat losses, the gross HRR would have been
derived from the net HRR (NHRR) curve but considering that the aim was comparing the
combustion behavior of diesel fuel and B100 in the same engine, the differences in heat
losses were considered to be the same in all the fueling conditions, thus not influencing
the outcomes of the study. Besides, most of the literature regarding the analysis of RME
combustion, in engines similar to that of the present study, report data on either the net or
the apparent heat release rate. 2. Materials and Methods The SOC was located in correspondence with the crank angle (CA) at which the first
derivative of the in-cylinder pressure with respect to crank angle (dp/dθ) suddenly changes
its slope, going from a negative to a positive value, and so presenting a local minimum [65]. p
g
g
g
p
p
g
During the experimental campaign, the engine was tested at full load and at rotational
speeds spanning from 2000 rpm to 3000 rpm, with steps of 250 rpm. The pump control
rack was fixed at its maximum so that the pump operated in the same way independently
of the delivered fuel. By doing so, only differences in the fuel properties were responsible
for changes in the injection and combustion parameters. Besides, as shown in Section 3.1,
the brake mean effective pressures (BMEPs) at full load were almost the same with the
two fuels. For this reason, the full-load comparisons are also comparisons at nearly the
same BMEP value. 2. Materials and Methods Detailed view showing the positions on the engine head of the piezoresistive sensors by
AVL used to measure the pressure in the combustion chamber and in the fuel injection subsystem. Table 3. Main specifications about the measuring instruments used in the test campaign. Hole to house the in-
chamber pressure
sensor tip
Fuel injection
pressure sensor
Figure 2. Detailed view showing the positions on the engine head of the piezoresistive sensors by
AVL used to measure the pressure in the combustion chamber and in the fuel injection subsystem. Hole to house the in-
chamber pressure
sensor tip
Fuel injection
pressure sensor Figure 2. Detailed view showing the positions on the engine head of the piezoresistive sensors by
AVL used to measure the pressure in the combustion chamber and in the fuel injection subsystem. Figure 2. Detailed view showing the positions on the engine head of the piezoresistive sensors by
AVL used to measure the pressure in the combustion chamber and in the fuel injection subsystem. 7 of 25 Energies 2021, 14, 7535 Table 3. Main specifications about the measuring instruments used in the test campaign. Table 3. Main specifications about the measuring instruments used in the test campaign. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions 3.1. Brake Torque, Fuel Consumption, and NOx Emissions Figure 3 shows the fuel mass flow rate and the corresponding injection quantities
per cycle as a function of the rotational speed. The mass of B100 injected per cycle is
8–10% greater than that of the diesel fuel to compensate for the lower heating value. Consequently, the engine brake torque, as shown in Figure 4, is almost the same with the
two fuels, while the BSFC of the RME is slightly higher, in accordance with the higher fuel
flow rate injected. These results confirm the outcomes of other authors that studied similar
injection systems [44,45]. Figure 5 reports the comparison of NOx emissions with the diesel fuel and the RME. The NOx concentrations are higher with the RME than with the diesel fuel, thus confirming
the well-known biodiesel NOx effect, except for the higher rotational speeds, in correspon-
dence to which the differences between the two fuels become lower. Figure 6 reports the
percentage increase of the NOx concentration in the exhaust with the RME compared to the
diesel fuel as a function of the rotational speed. On the same chart, the variations of the heat
release peak between the diesel fuel and the RME during the combustion process are also
reported. Even though a detailed description of the combustion process is reported in the
next section, this interesting figure is anticipated, as it helps to highlight how the increase
of the NOx emissions with biodiesel is strongly correlated to the increase of the heat release
peak; indeed, the two above-mentioned trends present a similar morphology. This can be
explained by considering the main mechanism of NOx formation in a combustion process: Energies 2021, 14, 7535 8 of 25
with the
higher the thermal formation mechanism. A higher peak in the heat release is obviously correlated
to a higher local temperature and pressure in the combustion chamber, thus to a higher
risk of formation of this polluting compound. injection systems [44,45]. the thermal formation mechanism. A higher peak in the heat release is obviously correlated
to a higher local temperature and pressure in the combustion chamber, thus to a higher
risk of formation of this polluting compound. injection systems [44,45]. g
p
p
,
g
risk of formation of this polluting compound. gure 3. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions Fuel mass flow rate and injected mass per cycle as a function of the engine rotational speed for the two test
els. 20
22
24
26
28
30
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
1750
2000
2250
2500
2750
3000
3250
injected quantity (mg cycle-1)
fuel consumption (g h-1)
engine rotational speed (rpm)
fuel consumption Diesel
fuel consumption RME
injected volume Diesel
injected volume RME
Figure 3. Fuel mass flow rate and injected mass per cycle as a function of the engine rotational speed for the two tested fuels. 4, x FOR PEER REVIEW
Figure 4 Engine torque and BSFC as a function of the rotational speed for the two tested fuels
0
50
100
150
200
250
300
350
400
450
500
15.0
17.5
20.0
22.5
25.0
27.5
30.0
1750
2000
2250
2500
2750
3000
3250
brake specific fuel consumption (g/kWh)
torque (Nm)
rotational speed (rpm)
torque Diesel fuel
torque RME
BSFC Diesel fuel
BSFC RME
Figure 4. Engine torque and BSFC as a function of the rotational speed for the two tested fuels. risk of formation of this polluting compound. 20
22
24
26
28
30
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
1750
2000
2250
2500
2750
3000
3250
injected quantity (mg cycle-1)
fuel consumption (g h-1)
engine rotational speed (rpm)
fuel consumption Diesel
fuel consumption RME
injected volume Diesel
injected volume RME
, x FOR PEER REVIEW ass flow rate and injected mass per cycle as a function of the engine rotational speed for the tw
el mass flow rate and injected mass per cycle as a function of the engine rotational speed for the two tested fue ure 3 Fuel mass flow rate and injected mass per cycle as a function of the engine rotation
Figure 3. Fuel mass flow rate and injected mass per cycle as a function of the engine rotational speed re 3. Fuel mass flow rate and injected mass per cycle as a function of the engine rotational speed for the two
. Figure 3. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions NOx concentration in the exhaust as a function of the rotational speed for the two tested
fuels
0
20
40
60
80
100
120
140
1750
2000
2250
2500
2750
3000
3250
NOx emissions (ppm)
rotational speed (rpm)
Diesel fuel
RME
Figure 5. NOx concentration in the exhaust as a function of the rotational speed for the two tested fuels. Figure 5. NOx concentration in the exhaust as a function of the rotational speed for the two tested
fuels. 0
20
40
60
80
100
120
140
1750
2000
2250
2500
2750
3000
3250
NOx emissions (ppm)
rotational speed (rpm)
Diesel fuel
RME rotational speed (rpm)
unction of the rotational sp
otat o a speed ( p
) Figure 5. NOx concentration in the exhaust as a function of the rotational speed for the two tested
fuels
Figure 5. NOx concentration in the exhaust as a function of the rotational speed for the two tested fuels. g
p
fuels. fuels. Figure 6. NOx emissions and heat release peak variations for the biodiesel at full load as a function
of the rotational speed (ref diesel fueling)
0%
10%
20%
30%
40%
50%
60%
1750
2000
2250
2500
2750
3000
3250
NOx and max heat release variations (%)
rotational speed (rpm)
NOx % variations
max heat release % variations
Figure 6. NOx emissions and heat release peak variations for the biodiesel at full load as a function
of the rotational speed (ref. diesel fueling). 0%
10%
20%
30%
40%
50%
60%
1750
2000
2250
2500
2750
3000
3250
NOx and max heat release variations (%)
rotational speed (rpm)
NOx % variations
max heat release % variations
Figure 6. NOx emissions and heat release peak variations for the biodiesel at full load as a function
of the rotational speed (ref. diesel fueling). Figure 6. NOx emissions and heat release peak variations for the biodiesel at full load as a function
of the rotational speed (ref diesel fueling)
rotational speed (rpm)
Figure 6. NOx emissions and heat release peak variations for the biodiesel at full load as a function
of the rotational speed (ref. diesel fueling). Figure 6. NOx emissions and heat release peak variations for the biodiesel at full load as a function
of the rotational speed (ref. diesel fueling). of the rotational speed (ref. diesel fueling). 3.2. Injection Pressure Characteristics and Combustion Analysis
3.2. Injection Pressure Characteristics and Combustion Analysis 3.2. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions Injection Pressure Characteristics and Combustion Analysis
The fuel pressure in the injection pipe was measured by means of a piezoelectric
transducer, and the recorded signals were pressure referenced according to the method
proposed in [53]. In fact, the piezoelectric transducers can detect rapid pressure variations
but, conversely, give no information on the absolute pressure values; therefore, pressure
referencing is needed if absolute pressure values are required or different situations must
be compared, as in this case. Indeed, an incorrect pressure referencing may lead to errors
in the injection advance and the injection pressure estimation [19]. Thus, in the following
figures (Figures 7–11) the pressure values are reported as relative to the fuel’s vapor pres-
sure (the differences between vapor pressure of B100 and diesel fuel are neglected as
The fuel pressure in the injection pipe was measured by means of a piezoelectric
transducer, and the recorded signals were pressure referenced according to the method
proposed in [53]. In fact, the piezoelectric transducers can detect rapid pressure variations
but, conversely, give no information on the absolute pressure values; therefore, pressure
referencing is needed if absolute pressure values are required or different situations must
be compared, as in this case. Indeed, an incorrect pressure referencing may lead to errors
in the injection advance and the injection pressure estimation [19]. Thus, in the following
figures (Figures 7–11) the pressure values are reported as relative to the fuel’s vapor pres-
sure (the differences between vapor pressure of B100 and diesel fuel are neglected as
The fuel pressure in the injection pipe was measured by means of a piezoelectric
transducer, and the recorded signals were pressure referenced according to the method
proposed in [53]. In fact, the piezoelectric transducers can detect rapid pressure variations
but, conversely, give no information on the absolute pressure values; therefore, pressure
referencing is needed if absolute pressure values are required or different situations must be
compared, as in this case. Indeed, an incorrect pressure referencing may lead to errors in the
injection advance and the injection pressure estimation [19]. Thus, in the following figures
(Figures 7–11) the pressure values are reported as relative to the fuel’s vapor pressure
(the differences between vapor pressure of B100 and diesel fuel are neglected as compared
to the injection pressure). 3.1. Brake Torque, Fuel Consumption, and NOx Emissions Fuel mass flow rate and injected mass per cycle as a function of the engine rotational speed for the two tested fuels
0
50
100
150
200
250
300
350
400
450
500
15.0
17.5
20.0
22.5
25.0
27.5
30.0
1750
2000
2250
2500
2750
3000
3250
brake specific fuel consumption (g/kWh)
torque (Nm)
rotational speed (rpm)
torque Diesel fuel
torque RME
BSFC Diesel fuel
BSFC RME
Figure 4. Engine torque and BSFC as a function of the rotational speed for the two tested fuels. 3. Fuel mass flow rate and injected mass per cycle as a function of the engine rotational speed for the tw
ure 3. Fuel mass flow rate and injected mass per cycle as a function of the engine rotational speed for the two tested fue
Figure 4 Engine torque and BSFC as a function of the rotational speed for the two tested fuels
0
50
100
150
200
250
300
350
400
450
500
15.0
17.5
20.0
22.5
25.0
27.5
30.0
1750
2000
2250
2500
2750
3000
3250
brake specific fuel consumption (g/kWh)
torque (Nm)
rotational speed (rpm)
torque Diesel fuel
torque RME
BSFC Diesel fuel
BSFC RME
Figure 4. Engine torque and BSFC as a function of the rotational speed for the two tested fuels. Figure 4. Engine torque and BSFC as a function of the rotational speed for the two tested fuels. 9 of 25
10 of 26
10 of 26 Energies 2021, 14, 7535
Energies 2021, 14, x FOR
Energies 2021, 14, x FOR Figure 5. NOx concentration in the exhaust as a function of the rotational speed for the two tested
fuels. 0
20
40
60
80
100
120
140
1750
2000
2250
2500
2750
3000
3250
NOx emissions (ppm)
rotational speed (rpm)
Diesel fuel
RME
Figure 5. NOx concentration in the exhaust as a function of the rotational speed for the two tested fuels. Figure 5. NOx concentration in the exhaust as a function of the rotational speed for the two tested
fuels. 0
20
40
60
80
100
120
140
1750
2000
2250
2500
2750
3000
3250
NOx emissions (ppm)
rotational speed (rpm)
Diesel fuel
RME Figure 5. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions While not true, this is a reasonable approximation since with
injection pressures around 200 bar the error in absolute values is of the order of 1/200. 10 of 25
ng to a
etected Energies 2021, 14, 7535 with the RME than with diesel. ure 7. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2000 rpm and at fu
-10
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10
20
30
40
50
60
70
80
0
50
100
150
200
250
300
-30
-20
-10
0
10
20
30
40
50
60
NHRR (J/ °) - in-cylinder pressure (bar)
injection pressure (bar)
crank angle (°)
RME
Diesel fuel
injection pressures
NHRRs
in-cylinder pressures
2000 rpm
Figure 7. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2000 rpm and at full load. 4, x FOR PEER REVIEW
re 8. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2250 rpm and at fu
-10
0
10
20
30
40
50
60
70
80
0
50
100
150
200
250
300
-30
-20
-10
0
10
20
30
40
50
60
NHRR (J/ °) - in-cylinder pressure (bar)
injection pressure (bar)
crank angle (°)
RME
Diesel fuel
injection pressures
NHRRs
in-cylinder pressures
2250 rpm
80
300
Figure 8. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2250 rpm and at full load. ure 7 RME and diesel fuel injection pressures in-cylinder pressures and NHRR at 2000 rpm and at ful
-10
0
10
20
30
40
50
60
70
80
0
50
100
150
200
250
300
-30
-20
-10
0
10
20
30
40
50
60
NHRR (J/ °) - in-cylinder pressure (bar)
injection pressure (bar)
crank angle (°)
RME
Diesel fuel
injection pressures
NHRRs
in-cylinder pressures
2000 rpm
Figure 7. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2000 rpm and at full load. 4, x FOR PEER REVIEW re 7. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2000 rpm and at ful
-10
0
10
20
30
40
50
60
70
80
0
50
100
150
200
250
300
-30
-20
-10
0
10
20
30
40
50
60
NHRR (J/ °) - in-cylinder pressure (bar)
injection pressure (bar)
crank angle (°)
RME
Diesel fuel
injection pressures
NHRRs
in-cylinder pressures
2000 rpm
Figure 7. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2750 rpm and at fu
-10
0
10
20
30
40
50
60
70
80
0
50
100
150
200
250
300
-30
-20
-10
0
10
20
30
40
50
60
NHRR (J/ °) - in-cylinder pressure (bar)
injection pressure (bar)
crank angle (°)
RME
Diesel fuel
injection pressures
NHRRs
in-cylinder pressures
2750 rpm
80
300
3000 rpm
Figure 10. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2750 rpm and at full load. ure 9. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2500 rpm and at ful
-10
0
10
20
30
40
50
60
70
80
0
50
100
150
200
250
300
-30
-20
-10
0
10
20
30
40
50
60
NHRR (J/ °) - in-cylinder pressure (bar)
injection pressure (bar)
crank angle (°)
RME
Diesel fuel
injection pressures
NHRRs
in-cylinder pressures
2500 rpm
Figure 9. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2500 rpm and at full load. 4, x FOR PEER REVIEW re 9. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2500 rpm and at fu
-10
0
10
20
30
40
50
60
70
80
0
50
100
150
200
250
300
-30
-20
-10
0
10
20
30
40
50
60
NHRR (J/ °) - in-cylinder pressure (bar)
injection pressure (bar)
crank angle (°)
RME
Diesel fuel
injection pressures
NHRRs
in-cylinder pressures
2500 rpm
Figure 9. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2500 rpm and at full load. x FOR PEER REVIEW ME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2500 rpm and a
re 9. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2500 rpm and at full load e 10. RME and diesel fuel injection pressures in-cylinder pressures and NHRR at 2750 rpm and at fu
-10
0
10
20
30
40
50
60
70
80
0
50
100
150
200
250
300
-30
-20
-10
0
10
20
30
40
50
60
NHRR (J/ °) - in-cylinder pressure (bar)
injection pressure (bar)
crank angle (°)
RME
Diesel fuel
injection pressures
NHRRs
in-cylinder pressures
2750 rpm
Figure 10. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2750 rpm and at full load. re 10. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2000 rpm and at full load. x FOR PEER REVIEW in-cylinder pressures ME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2000 rpm and a
re 7. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2000 rpm and at full load j
p
y
p
p
re 8. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2250 rpm and at fu
-10
0
10
20
30
40
50
60
70
80
0
50
100
150
200
250
300
-30
-20
-10
0
10
20
30
40
50
60
NHRR (J/ °) - in-cylinder pressure (bar)
injection pressure (bar)
crank angle (°)
RME
Diesel fuel
injection pressures
NHRRs
in-cylinder pressures
2250 rpm
Figure 8. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2250 rpm and at full load. re 8. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2250 rpm and at fu
-10
0
10
20
30
40
50
60
70
80
0
50
100
150
200
250
300
-30
-20
-10
0
10
20
30
40
50
60
NHRR (J/ °) - in-cylinder pressure (bar)
injection pressure (bar)
crank angle (°)
RME
Diesel fuel
injection pressures
NHRRs
in-cylinder pressures
2250 rpm
Figure 8. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2250 rpm and at full load. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2250 rpm an
igure 8. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2250 rpm and at full l 11 of 25 Energies 2021, 14, 7535 e 8. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2250 rpm and at fu
e 9. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2500 rpm and at fu
-10
0
10
20
30
40
50
60
70
80
0
50
100
150
200
250
300
-30
-20
-10
0
10
20
30
40
50
60
NHRR (J/ °) - in-cylinder pressure (bar)
injection pressure (bar)
crank angle (°)
RME
Diesel fuel
injection pressures
NHRRs
in-cylinder pressures
2500 rpm
Figure 9. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2500 rpm and at full load. x FOR PEER REVIEW
10. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2750 rpm and at fu
-10
0
10
20
30
40
50
60
70
80
0
50
100
150
200
250
300
-30
-20
-10
0
10
20
30
40
50
60
NHRR (J/ °) - in-cylinder pressure (bar)
injection pressure (bar)
crank angle (°)
RME
Diesel fuel
injection pressures
NHRRs
in-cylinder pressures
2750 rpm
Figure 10. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2750 rpm and at full load. ME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2750 rpm and a
re 10. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 2750 rpm and at full load 12 of 25 Energies 2021, 14, 7535 re 11. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 3000 rpm and at full lo
-10
0
10
20
30
40
50
60
70
80
0
50
100
150
200
250
300
-30
-20
-10
0
10
20
30
40
50
60
NHRR (J/ °) - in-cylinder pressure (bar)
injection pressure (bar)
crank angle (°)
RME
Diesel fuel
injection pressures
NHRRs
in-cylinder pressures
3000 rpm
Figure 11. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 3000 rpm and at full load. E and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 3000 rpm and at f
1. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 3000 rpm and at full load. By analyzing the injection pressure signals in a higher detail, some interes
derations can be drawn regarding the injection advance and the injection durat
emise, since the needle lift was not measured, the SOI was assumed to coincide
ank angle position at which the fuel pressure reaches the value of the injecto
ening pressure (i.e., 210 bar). Similarly, the end of injection (EOI) was assumed
de with the crank angle position at which the fuel pressure reaches the value
With the biodiesel, the fuel pressure recorded in the pipe just before injection was
lower at all rotational speeds; nevertheless, the injection pressure was reached in advance
(the black circles on the injection pressure curves of Figures 7–11 evidence the crank angular
position at which the injector nozzle opening pressure is reached). 3.1. Brake Torque, Fuel Consumption, and NOx Emissions In fact, due to the lower
compressibility of the biodiesel, the pressure rise produced by the pump was faster and also
propagated faster toward the injectors (as a consequence of its higher sound speed) [32]. In all the investigated cases, the RME injection pressure curves seem smoother, probably
because of the higher viscosity of the RME with respect to the viscosity of diesel fuel. tor nozzle closing pressure (i.e., ~150 bar). As an example, the SOIs, EOIs, and
quent injection durations are reported in Figure 12 in the case of 2500 rpm. g
y
p
y
At the end of injection, cavitation occurred in the pipe. This is identifiable in the
zones where the pressure curves, which had previously fallen steeply (see the pressure
signals at 17◦, 19◦, 21◦, 23◦, and 26◦CA, for engine rotational speeds of 2000 rpm, 2250 rpm,
2500 rpm, 2750 rpm, and 3000 rpm, respectively), rapidly flatten and the pressure remains
almost constant at the vapor pressure despite the transient nature of the phenomenon. At
the same time, it is possible to observe an intense pressure wave travelling to-and-fro in
the pipe. As the speed increases, the angular interval where cavitation is detected is longer
with the RME than with diesel. By analyzing the injection pressure signals in a higher detail, some interesting con-
siderations can be drawn regarding the injection advance and the injection duration. As
a premise, since the needle lift was not measured, the SOI was assumed to coincide with
the crank angle position at which the fuel pressure reaches the value of the injector noz-
zle opening pressure (i.e., 210 bar). Similarly, the end of injection (EOI) was assumed to
coincide with the crank angle position at which the fuel pressure reaches the value of the
injector nozzle closing pressure (i.e., ~150 bar). As an example, the SOIs, EOIs, and the
consequent injection durations are reported in Figure 12 in the case of 2500 rpm. q
j
p
g
p
The effect of the rotational speed on the differences in the injection advance and
injection duration will be commented on by analyzing Figures 13 and 14, respectively. 13 of 25 13 of 25 Energies 2021, 14, 7535
es 2021, 14, x FOR PE
300
2 Figure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at full load. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions The effect of the rotational speed on the differences in the injection
jection duration will be commented on by analyzing Figures 13 and 14, re
0
50
100
150
200
250
-20
-15
-10
-5
0
5
10
15
20
injection pressure (bar)
crank angle (°)
Diesel fuel
Injection duration Diesel fuel
Injection duration RME
SOIRME
SOIdiesel
EOIdiesel
EOIRME Figure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at full load. The effect of the rotational speed on the differences in the injection a
jection duration will be commented on by analyzing Figures 13 and 14, res
-18
-16
-14
-12
-10
-8
-6
-4
-2
0
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
2000
2250
2500
2750
3000
residual pressure difference RME −diesel fuel (bar)
injection advance difference RME −diesel fuel ( )
t ti
l
d (
)
injection advance
residual pressure difference
Figure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at full load. p
j
jection duration will be commented on by analyzing Figures 13 and 14, respe
13. Injection advances with the RME and the diesel fuel and residual pressure difference between the sa
s a function of the engine speed and at full load. -18
-16
-14
-12
-10
-8
-6
-4
-2
0
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
2000
2250
2500
2750
3000
residual pressure difference RME −diesel fuel (bar)
injection advance difference RME −diesel fuel ( )
rotational speed (rpm)
injection advance
residual pressure difference
Figure 13. Injection advances with the RME and the diesel fuel and residual pressure difference between the same two fuels
as a function of the engine speed and at full load. gure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at
Figure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at full load. jection duration will be commented on by analyzing Figures 13 a Figure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at full load. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions The effect of the rotational speed on the differences in the injection
jection duration will be commented on by analyzing Figures 13 and 14,
0
50
100
150
200
250
300
-20
-15
-10
-5
0
5
10
15
20
injection pressure (bar)
crank angle (°)
RME
Diesel fuel
2500 rpm
Injection duration Diesel fuel
Injection duration RME
SOIRME
SOIdiesel
EOIdiesel
EOIRME
-18
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-8
-6
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-2
0
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
residual pressure difference RME −diesel fuel (bar)
injection advance difference RME −diesel fuel ( )
injection advance
residual pressure difference
Figure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at full load. Figure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at full load. The effect of the rotational speed on the differences in the injection ad
jection duration will be commented on by analyzing Figures 13 and 14, res
Injection advances with the RME and the diesel fuel and residual pressure difference between the
0
50
100
150
200
250
-20
-15
-10
-5
0
5
10
15
20
injection pressure (bar)
crank angle (°)
Diesel fuel
Injection duration Diesel fuel
Injection duration RME
SOIRME
SOIdiesel
EOIdiesel
EOIRME
-18
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-8
-6
-4
-2
0
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
2000
2250
2500
2750
3000
residual pressure difference RME −diesel fuel (bar)
injection advance difference RME −diesel fuel ( )
rotational speed (rpm)
injection advance
residual pressure difference
gure 13. Injection advances with the RME and the diesel fuel and residual pressure difference between the same two fuels Figure 12 Injection durations with the RME and the diesel fuel at 2500 rpm and at full load
0
50
100
150
200
250
300
-20
-15
-10
-5
0
5
10
15
20
injection pressure (bar)
crank angle (°)
RME
Diesel fuel
2500 rpm
Injection duration Diesel fuel
Injection duration RME
SOIRME
SOIdiesel
EOIdiesel
EOIRME
Figure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at full load. Figure 12. Injection durations with the RME and the diesel fuel at 2500 rpm and at full load. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions The effect of the rotational speed on the differences in the injection
jection duration will be commented on by analyzing Figures 13 and 14,
-18
-16
-14
-12
-10
-8
-6
-4
-2
0
0 00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
residual pressure difference RME −diesel fuel (bar)
injection advance difference RME −diesel fuel ( )
injection advance
residual pressure difference
I j
i
d
i h h RME
d h
di
l f
l
d
id
l
diff
b
h
-18
-16
-14
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-10
-8
-6
-4
-2
0
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
2000
2250
2500
2750
3000
residual pressure difference RME −diesel fuel (bar)
injection advance difference RME −diesel fuel ( )
rotational speed (rpm)
injection advance
residual pressure difference
gure 13. Injection advances with the RME and the diesel fuel and residual pressure difference between the same two fuel e in
0 -18
0.00
2000
2250
2500
2750
3000
r
13. Injection advances with the RME and the diesel fuel and residual pressure difference between the s
s a function of the engine speed and at full load. Figure 13. Injection advances with the RME and the diesel fuel and residual pressure difference between the same two fuels
as a function of the engine speed and at full load. 14 of 25 14 of 25 Energies 2021, 14, 7535
PEER REVIEW i
E
d d
l f
l
l
d
d
Figure 14. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 3000 rpm and at full load. fuel injection pressures, in-cylinder pressures, and NHRR at 3000 rpm and at full load. Figure 14. RME and diesel fuel injection pressures, in-cylinder pressures, and NHRR at 3000 rpm and at full load. nd diesel fuel injection pressures, in-cylinder pressures, and NHRR at 3000 rpm
d, due to the higher bulk modulus, the injection with the RME is advanced
of 0.27° CA at 2500 rpm and a maximum of around 1° CA at 3000 rpm. confirm the so-called biodiesel inadvertent injection advance effect, but
As expected, due to the higher bulk modulus, the injection with the RME is advanced
by a minimum of 0.27◦CA at 2500 rpm and a maximum of around 1◦CA at 3000 rpm. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions These findings confirm the so-called biodiesel inadvertent injection advance effect, but also
evidence a nonmonotone trend with the rotational speed. The reason for this nonmonotone
behavior can be explained by having a look at the variation in the rotational speed of the
residual pressure differences between the two fuels. j
nonmonotone trend with the rotational speed. The reason for this non-
vior can be explained by having a look at the variation in the rotational
dual pressure differences between the two fuels. hown in the graphs in Figure 13 the decrease in the residual pressure
In fact, as shown in the graphs in Figure 13, the decrease in the residual pressure,
caused by the use of the RME, which partially compensates for the advancing effect of its
higher bulk modulus, has a very similar trend to that of the advance—only the data at
3000 rpm seem against the above-evidenced behavior. hown in the graphs in Figure 13, the decrease in the residual pressure,
e of the RME, which partially compensates for the advancing effect of its
dulus, has a very similar trend to that of the advance—only the data at
against the above-evidenced behavior. reconsider the results in Figure 6. Surprisingly, it appears that the bio-
t is more evident at 2500 rpm where the injection advance differences
Now, let us reconsider the results in Figure 6. Surprisingly, it appears that the biodiesel
NOx effect is more evident at 2500 rpm, where the injection advance differences between
the two fuels are the lowest. However, as already evidenced by Figure 6, this is explainable
by the higher peak of the heat release, which occurs despite the reduced advance. This is
another example of the not always straightforward behavior of biodiesel as concerns the
combustion analysis and NOx formation mechanisms. t is more evident at 2500 rpm, where the injection advance differences
fuels are the lowest. However, as already evidenced by Figure 6, this is
h hi h
k
f th h
t
l
hi h
d
it th
d
d d
As is visible in Figure 14, the total injection angle increases with the rotational speed
for both fuels, but the injection lasts almost 3–4% longer when using RME. he higher peak of the heat release, which occurs despite the reduced ad-
other example of the not always straightforward behavior of biodiesel as
mbustion analysis and NOx formation mechanisms. 3.1. Brake Torque, Fuel Consumption, and NOx Emissions in Figure 14, the total injection angle increases with the rotational speed
These results contribute to explain the increase of the injection rate of the RME with re-
spect to diesel fuel. Overall, the higher injection pressures and the longer injection duration
cause an increase of the injected mass per cycle of RME, which in the end compensates for
the reduction of the fuel LHVm, thus yielding almost the same torque values for both fuels. ut the injection lasts almost 3
ts contribute to explain the in
3.3. Combustion Characteristics As confirmed by the positio
e local minima of the in-cylinder pressure 1st derivative in the Figures 15–19, the
r the biodiesel was almost the same as for the diesel fuel at 2000 rpm; then, as the
onal speed increased, the SOC of the biodiesel was slightly shifted to an earlier
ith the advance reaching about 0.75° CA at 3000 rpm. g
p
gure 15. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2000 rpm and at full loa
gure 16. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2250 rpm and at full loa
0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
Fi
0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
Figure 15. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2000 rpm and at full load. gure 15. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2000 rpm and at full loa
gure 16. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2250 rpm and at full loa
As the advances in the SOC were almost identical to the SOI advance
0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
Fi
0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
Figure 16. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2250 rpm and at full load. ure 15. ut the injection lasts almost 3
ts contribute to explain the in
3.3. Combustion Characteristics s contribute to explain the increase of the injection rate of the RME with
fuel. Overall, the higher injection pressures and the longer injection du-
ncrease of the injected mass per cycle of RME, which in the end compen-
uction of the fuel LHVm, thus yielding almost the same torque values for
As previously shown in Figures 7–11, the NHRR graphs and the general combustion
characteristics are those of a classic direct injection diesel engine for both biodiesel and
diesel fuel. Starting from the SOI, the HRR is slightly negative during the ignition delay
period due to the cooling effect of fuel vaporization; then, in the initial phase of combustion,
a rapid HRR occurs because of the combustion of the fuel that has mixed with air during
the ignition delay. After this phase, the combustion continues slowly until most of fuel is Energies 2021, 14, 7535 15 of 25 15 of 25 burned; the final combustion phase is the late combustion, which continues until the end
of the expansion stroke. Instead, some differences between the biodiesel and the diesel
fuel occurred regarding the SOC and HRR values. As confirmed by the positions of the
local minima of the in-cylinder pressure 1st derivative in the Figures 15–19, the SOC for
the biodiesel was almost the same as for the diesel fuel at 2000 rpm; then, as the rotational
speed increased, the SOC of the biodiesel was slightly shifted to an earlier stage, with the
advance reaching about 0.75◦CA at 3000 rpm. e end of the expansion stroke. Instead, some differences between the biodiesel an
esel fuel occurred regarding the SOC and HRR values. As confirmed by the positio
e local minima of the in-cylinder pressure 1st derivative in the Figures 15–19, the
r the biodiesel was almost the same as for the diesel fuel at 2000 rpm; then, as the
onal speed increased, the SOC of the biodiesel was slightly shifted to an earlier s
ith the advance reaching about 0.75° CA at 3000 rpm. esel fuel occurred regarding the SOC and HRR values. ut the injection lasts almost 3
ts contribute to explain the in
3.3. Combustion Characteristics RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2000 rpm and at full lo
0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
Fi
Figure 15. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2000 rpm and at full load. ure 15. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2000 rpm and at full lo
0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
Fi 15. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2000 rpm and at full l
igure 15. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2000 rpm and at full load. 15. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2000 rpm and at full l ure 16. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2250 rpm and at full lo
0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
ure 16. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2250 rpm and at full lo
0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
Figure 16. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2250 rpm and at full load. ut the injection lasts almost 3
ts contribute to explain the in
3.3. Combustion Characteristics 0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel 16 RME
d di
l f
l i
li d
d
t
f
i
t 2250
d t f
g
( )
re 16. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2250 rpm and at fu
Figure 16. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2250 rpm and at full load Energies 2021, 14, 7535 16 of 25
ure peak
p 16 of 25
ure peak Figure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load
0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
Figure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load. Figure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load
0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel ure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full loa
0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
Figure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load. ure 17. ut the injection lasts almost 3
ts contribute to explain the in
3.3. Combustion Characteristics This behavior can be interprete
considering that the reduction of the chemical ignition delay due to the higher ce
number of the RME is probably compensated by an increased physical ignition delay
t
t
i
ti
d
i t
f
ti
A
h
i
h 3 2 th i j ure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full lo
crank angle (°)
Figure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load. ure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full lo
crank angle ( )
90
2 7. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full
igure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load. 7. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full ure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load. ure 18. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load. 0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
g
y
p
p
p
ure 18. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load. 0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
Figure 18. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load. As the advances in the SOC were almost identical to the SOI advances, the angular
ignition delays were comparable for the two fuels. ut the injection lasts almost 3
ts contribute to explain the in
3.3. Combustion Characteristics RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full loa
0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel ure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load. ure 18. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load. 0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
Figure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load. ure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load. ure 18. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load. 0
15
30
45
60
75
-1
-0.5
0
0.5
1
1.5
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
Figure 18. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load. As the advances in the SOC were almost identical to the SOI advances, the ang
ignition delays were comparable for the two fuels. ut the injection lasts almost 3
ts contribute to explain the in
3.3. Combustion Characteristics i
20 i i
ibl
h
h
f f
li
h
li
i
d ff
p
g
y
g
p
g
g
In order to assess the effect of the variation of the injection timing on the performance
and emissions, and to try to offset the biodiesel NOx effect, the standard injection timing
has been modified starting from the reference value of 16◦CA before top dead center
(BTDC). Starting from this reference injection condition, and based on the manufacturer’s
technical indications, two other configurations were set, properly adjusting the number of
shims in the injection pump: a late injection equal to 14◦CA BTDC, and an early injection
equal to 18◦CA BTDC. igure 20, it is possible to note that a change of fueling has a limited effect
rque. Moreover, consider that, for example, the differences in torque at
he same magnitude of the measurement error, and therefore no precise
e depicted. Nevertheless, in the analysis of the trends one can observe
gher value is obtained with the RME by retarding the injection timing to
ds with the RME with the standard SOI, and by anticipating the injection,
l
i
i h h di
l f
li
Observing Figure 20, it is possible to note that a change of fueling has a limited effect
on the engine torque. Moreover, consider that, for example, the differences in torque at
2500 rpm are of the same magnitude of the measurement error, and therefore no precise
indications can be depicted. Nevertheless, in the analysis of the trends one can observe
that a slightly higher value is obtained with the RME by retarding the injection timing to
+2◦CA. The trends with the RME with the standard SOI, and by anticipating the injection,
are practically overlapping with the diesel fueling. erlapping with the diesel fueling. the specific fuel consumption (Figure 21), a higher influence of the SOI
g can be appreciated. ut the injection lasts almost 3
ts contribute to explain the in
3.3. Combustion Characteristics This behavior can be interpreted by
considering that the reduction of the chemical ignition delay due to the higher cetane
number of the RME is probably compensated by an increased physical ignition delay due ure 17. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2500 rpm and at full load
0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
ure 18 RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load
0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
Figure 18. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load. crank angle (°)
e 18 RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at f
Figure 18. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load esel fuel in-cylinder pressures and rate of pressure rise at 2750 rpm and at full load. esel fuel in cylinder pressures and rate of pressure rise at 2750 rpm and at full load. As the advances in the SOC were almost identical to the SOI advances, the angular
ignition delays were comparable for the two fuels. This behavior can be interpreted by
considering that the reduction of the chemical ignition delay due to the higher cetane
number of the RME is probably compensated by an increased physical ignition delay due
to a worse atomization and mixture formation. As shown in paragraph 3.2, the injection
pressure of the RME and diesel fuel were similar, and the worse mixture formation is
attributable to the RME’s higher surface tension, higher flash point, and poorer volatility. 17 of 25 Energies 2021, 14, 7535
EER REVIEW Figure 19. ut the injection lasts almost 3
ts contribute to explain the in
3.3. Combustion Characteristics RME and diesel fuel in-cylinder pressures and rate of pressure rise at 3000 rpm and at
0
15
30
45
60
75
90
-1
-0.5
0
0.5
1
1.5
2
-40
-20
0
20
40
pressure (bar)
1st pressure derivative (bar/°)
crank angle (°)
1st derivative of in−cylinder pressure
RME
Diesel fuel
in−cylinder pressure
RME
Diesel fuel
Figure 19. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 3000 rpm and at full load. gure 19. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 3000 rpm a
Figure 19. RME and diesel fuel in-cylinder pressures and rate of pressure rise at 3000 rpm and at full load values of NHRR are likely to be a consequence of the biodiesel molecular
deed, as the oxygenated fuel evaporates, the oxygen bound in the fuel is
After the SOC, the NHRR curves of biodiesel became steeper than those of diesel fuel,
and a higher peak was reached due to an increased amount of fuel undergoing premixed
combustion. This also caused higher rates of pressure rise and higher pressure peaks
(Figures 15–19). eed, as the oxygenated fuel evaporates, the oxygen bound in the fuel is
eased, thus enhancing the flame speed and resulting in a higher HRR. ssess the effect of the variation of the injection timing on the performance
d t
t
t
ff
t th bi di
l NO
ff
t th
t
d
d i j
ti
ti
i
g
The higher values of NHRR are likely to be a consequence of the biodiesel molecular
composition; indeed, as the oxygenated fuel evaporates, the oxygen bound in the fuel is
progressively released, thus enhancing the flame speed and resulting in a higher HRR. nd to try to offset the biodiesel NOx effect, the standard injection timing
ed starting from the reference value of 16° CA before top dead center
from this reference injection condition, and based on the manufacturer’s
ions, two other configurations were set, properly adjusting the number
njection pump: a late injection equal to 14° CA BTDC, and an early injec-
CA BTDC. ut the injection lasts almost 3
ts contribute to explain the in
3.3. Combustion Characteristics Specifically, by anticipating the SOI (−2° CA), the
ME becomes comparable with that of the diesel fuel at all the rotational
he lower LHVm of biodiesel, which entails a higher global conversion ef-
oth a standard SOI and a retarding of the injection (+2° CA), the BSFC
l
h h l
f b
d
l
d
d l
h
h
p
y
pp
g
g
As concerns the specific fuel consumption (Figure 21), a higher influence of the SOI
and of the fueling can be appreciated. Specifically, by anticipating the SOI (−2◦CA), the
BSFC with the RME becomes comparable with that of the diesel fuel at all the rotational
speeds despite the lower LHVm of biodiesel, which entails a higher global conversion
efficiency. With both a standard SOI and a retarding of the injection (+2◦CA), the BSFC
increases consistently with the lower LHVm of biodiesel, and a delay in the injection shows
a slightly higher performance at high rotational speeds than at lower rpms. ently with the lower LHVm of biodiesel, and a delay in the injection shows
performance at high rotational speeds than at lower rpms. As concerns the emissions, the use of RME consistently reduced the concentration of
CO in the exhaust at all the operative conditions, as shown in Figure 22. The highest abate-
ment is obtained by anticipating the SOI by two degrees. This outcome is a consequence of Energies 2021, 14, 7535
OR PEER REVIEW 18 of 25
of 26 the higher combustion temperature and pressure, which facilitate the shift of the oxidation
reactions toward a complete conversion to CO2. However, the higher oxygen content and
the higher HRR with the RME grants lower CO emissions for all the injection timings. RME +2CA the higher combustion temperature and pressure, which facilitate the shift of the oxidati
reactions toward a complete conversion to CO2. However, the higher oxygen content a
the higher HRR with the RME grants lower CO emissions for all the injection timings. 20 Torque as a function of the rotational speed at full load for different values of the SOI and for the tw
15
17.5
20
22.5
25
27.5
30
1750
2000
2250
2500
2750
3000
3250
torque (Nm)
rotational speed (rpm)
RME +2CA
Diesel fuel
RME
RME -2CA
Figure 20. ut the injection lasts almost 3
ts contribute to explain the in
3.3. Combustion Characteristics Torque as a function of the rotational speed at full load, for different values of the SOI and for the two tested fuels. e as a function of the rotational speed at full load, for different values of the SOI and for the two tested
15
17.5
20
22.5
25
27.5
30
1750
2000
2250
2500
2750
3000
3250
torque (Nm)
rotational speed (rpm)
RME +2CA
Diesel fuel
RME
RME -2CA rotational speed (rpm)
ent values of the SOI and fo 20 Torque as a function of the rotational speed at full load for different values of the SOI and for the tw
Figure 20. Torque as a function of the rotational speed at full load, for different values of the SOI and for the two tested fuels. nction of the rotational speed at full load for different values of the SOI and for the tw
a function of the rotational speed at full load, for different values of the SOI and for the two tested fuels. 20. Torque as a function of the rotational speed at full load, for different values of the SOI and for the tw
250
300
350
400
450
500
1750
2000
2250
2500
2750
3000
3
brake specific fuel consumption (g/kWh)
rotational speed (rpm)
RME +2CA
Diesel fuel
RME
RME -2CA
Figure 21. Brake specific fuel consumption as a function of the rotational speed at full load, for dif-
ferent values of the SOI and for the two tested fuels. 250
300
350
400
450
500
1750
2000
2250
2500
2750
3000
3250
brake specific fuel consumption (g/kWh)
rotational speed (rpm)
RME +2CA
Diesel fuel
RME
RME -2CA
Figure 21. Brake specific fuel consumption as a function of the rotational speed at full load, for different values of the SOI
and for the two tested fuels. 20. ut the injection lasts almost 3
ts contribute to explain the in
3.3. Combustion Characteristics diesel emissions with 16° CA of injection advance). As is visible in Figure 22, whatever the injection advance, the use of biodiesel allows
reduction of the CO emissions. A further decrease can be obtained when shifting to
RME by reducing the injection advance. The higher percentage decreases can be ob-
ed in correspondence with the low rotational speeds. Moving toward the higher rpms,
i i
ff
d
h
il
i
i
b
2750
As is visible in Figure 22, whatever the injection advance, the use of biodiesel allows
for a reduction of the CO emissions. A further decrease can be obtained when shifting
to the RME by reducing the injection advance. The higher percentage decreases can be
observed in correspondence with the low rotational speeds. Moving toward the higher
rpms, the positive effects decrease somewhat until an inversion occurs at about 2750 rpm. As is visible in Figure 22, whatever the injection advance, the use of biodiesel allows
for a reduction of the CO emissions. A further decrease can be obtained when shifting to
the RME by reducing the injection advance. The higher percentage decreases can be ob-
served in correspondence with the low rotational speeds. Moving toward the higher rpms,
the positive effects decrease somewhat until an inversion occurs at about 2750 rpm. positive effects decrease somewhat until an inversion occurs at about 2750 rpm. Figure 23 shows the comparison of the concentrations of NOx between the diesel and
RME, where the NOx effect can be clearly identified; however, the negative NOx effect
ms to disappear, shifting the SOI to +2° CA (i.e., in correspondence with an angle caus-
h
d
l
i h h
f h di
l f
l)
p
p
p
Figure 23 shows the comparison of the concentrations of NOx between the diesel
and the RME, where the NOx effect can be clearly identified; however, the negative NOx
effect seems to disappear, shifting the SOI to +2◦CA (i.e., in correspondence with an angle
causing the trend to overlap with that of the diesel fuel). Figure 23 shows the comparison of the concentrations of NOx between the diesel and
the RME, where the NOx effect can be clearly identified; however, the negative NOx effect
seems to disappear, shifting the SOI to +2° CA (i.e., in correspondence with an angle caus-
ing the trend to overlap with that of the diesel fuel). ut the injection lasts almost 3
ts contribute to explain the in
3.3. Combustion Characteristics Torque as a function of the rotational speed at full load, for different values of the SOI and for the tw
250
300
350
400
450
500
1750
2000
2250
2500
2750
3000
brake specific fuel consumption (g/kWh)
RME +2CA
Diesel fuel
RME
RME -2CA
Figure 21 Brake specific fuel consumption as a function of the rotational speed at full load for dif-
250
300
350
400
450
500
1750
2000
2250
2500
2750
3000
3250
brake specific fuel consumption (g/kWh)
rotational speed (rpm)
RME +2CA
Diesel fuel
RME
RME -2CA
Figure 21 Brake specific fuel consumption as a function of the rotational speed at full load for different values of the SOI rotational speed (rpm) 1750
2000
2250
2500
2750
3000
rotational speed (rpm)
Figure 21. Brake specific fuel consumption as a function of the rotational speed at full load, for dif
ferent values of the SOI and for the two tested fuels. Figure 21. Brake specific fuel consumption as a function of the rotational speed at full load, for different values of the SOI
and for the two tested fuels. 19 of 25 19 of 25 Energies 2021, 14, 7535 re 22. CO emissions variations for the biodiesel at different values of the SOI and at full load
diesel emissions with 16° CA of injection advance). 0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
1750
2000
2250
2500
2750
3000
3250
CO emissions (%)
rotational speed [rpm]
RME +2CA
Diesel
RME
RME -2CA
Figure 22. CO emissions variations for the biodiesel at different values of the SOI and at full load (ref. diesel emissions with 16◦CA of injection advance). Figure 22. CO emissions variations for the biodiesel at different values of the SOI and at full load
(ref. diesel emissions with 16° CA of injection advance). 0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
1750
2000
2250
2500
2750
3000
3250
CO emissions (%)
rotational speed [rpm]
RME +2CA
Diesel
RME
RME -2CA e 22. CO emissions variations for the biodiesel at different values of the SOI and at full load
diesel emissions with 16° CA of injection advance). Figure 22. CO emissions variations for the biodiesel at different values of the SOI and at full load (ref. diesel emissions with 16◦CA of injection advance). Figure 22. CO emissions variations for the biodiesel at different values of the SOI and at full load
(ref. 3.4. Comments on the Results and Comparison with Literature Studies 3.4. Comments on the Results and Comparison with Literature Studies In this section, the presented performance results are commented on and compared to
other similar studies available in the literature. The analysis focuses on both the combustion
and the emissions. When the RME is used to fuel the studied engine, and having an in-line
fuel pumping system: •
the brake torque at full load is minimally affected, and the variation of the torque
with the diesel oil and the RME fueling is within the accuracy of the measuring
instrumentation; •
the NOx emissions increase between 9% (at higher rotational speeds) and 40% (at
lower rotational speeds); •
the SOI is slightly advanced (of a maximum of about 0.75◦CA) and the SOC takes
place earlier, accordingly; •
when the fuel delivery ends, the pressure in the injection system drops rapidly, causing
severe cavitation in the fuel, and the cavitation period lasts more with the RME as,
due to its higher bulk modulus, the pressure drop is the steeper; •
the pressure in the fuel line just before injection (i.e., the residual pressure) is lower
with the RME, probably due to the combined effects of a more intense cavitation at
the end of the injection and the higher fluid viscosity; •
the maximum fuel pressure during the injection is only slightly higher with the RME
if a correct pressure reference is taken into account, and thus the effect of the residual
pressure decrease with RME is considered; •
the net heat release during the premixed phase is steeper, and a higher peak of heat
release is reached; •
with the same pump settings, the mass of injected fuel is higher with the RME than
with the mineral diesel fuel due to the increased injection duration and the slightly
higher injection pressure; •
the control of the fuel injection timing of the line pump can help to counterbalance the
NOx effect, and with the RME, a retarding of 2◦CA on the standard timing can offset
the increase of the NOx emissions so that the other performance parameters remain
comparable to a standard fueling. The results show that, apart from a slight advance in the SOC caused by the advance
in the SOI, the RME differs from diesel fuel for the higher HRR during the first part of
the combustion. ut the injection lasts almost 3
ts contribute to explain the in
3.3. Combustion Characteristics the trend to overlap with that of the diesel fuel). ure 23. NOx emissions variations for the biodiesel at different values of the SOI and at full load
diesel emissions with 16° CA of injection advance). 0
20
40
60
80
100
120
140
1750
2000
2250
2500
2750
3000
3250
rotational speed (rpm)
RME +2CA
Diesel
RME
RME -2CA
Figure 23. NOx emissions variations for the biodiesel at different values of the SOI and at full load
(ref. diesel emissions with 16° CA of injection advance). 0
20
40
60
80
100
120
140
1750
2000
2250
2500
2750
3000
3250
NOx emissions (ppm)
rotational speed (rpm)
RME +2CA
Diesel
RME
RME -2CA
Figure 23. NOx emissions variations for the biodiesel at different values of the SOI and at full load
(ref. diesel emissions with 16◦CA of injection advance). e 23. NOx emissions variations for the biodiesel at different values of the SOI and at full load
diesel emissions with 16° CA of injection advance). rotational speed (rpm)
Figure 23. NOx emissions variations for the biodiesel at different values of the SOI and at full load
(ref. diesel emissions with 16° CA of injection advance). Figure 23. NOx emissions variations for the biodiesel at different values of the SOI and at full load
(ref. diesel emissions with 16◦CA of injection advance). Energies 2021, 14, 7535 20 of 25 20 of 25 What seems a little surprising is the reduction of NOx in the 2500–2750 rpm range
when the injection of RME is anticipated. This is another example of the not-so-straightforward behavior of biodiesel as concerns
NOx emissions. The factors influencing this behavior are, in effect, not independent but
comprehensive and interactive, and include, besides the biodiesel properties, engine loads
and also, as in this case, engine speeds to influence the final determinant for the NOx
emission of biodiesel combustion temperature [31]. Unfortunately, in this case, we are not able to correlate this behavior to the heat release,
as no in-cylinder pressure recordings were available for these tests. Nevertheless, we can conclude that retarding the SOI by two degrees of CA with
respect to the factory settings seems to be the best compromise for the performance and
emissions of the engine fueled with the RME. ut the injection lasts almost 3
ts contribute to explain the in
3.3. Combustion Characteristics Indeed, (i) the NOx effect is eliminated, (ii) the
CO emissions do not increase compared to diesel combustion, (iii) the torque improves,
and the global efficiency is acceptable. 3.4. Comments on the Results and Comparison with Literature Studies This causes a higher peak of heat release, a higher rise in pressure, and
in-cylinder peak pressure, and thus a higher combustion temperature. Considering that
the atomization of biodiesel is poorer than that of diesel fuel, this behavior is likely to
be due to the oxygen bound in the fuel molecules. This is consistent with the literature
results, in particular with Labeckas G. and Slavinskas S. [39] who, having tested RME on
a four-cylinder, water-cooled, direct injection, naturally aspirated diesel engine, stated
that “in contrast to air-borne oxygen, the fuel-based oxygen accelerates the combustion Energies 2021, 14, 7535 21 of 25 21 of 25 process from within the fuel-rich spray patterns themselves”, and added: “this advantage
is especially beneficial at heavy loads”. As far as the biodiesel NOx effect is concerned, the bound oxygen has a twofold
effect: boosting the thermal production of NOx and supplying the oxygen other than that
which is air borne. This last mechanism of NOx formation is also consistent with the NOx
emissions increase observable in common rail injection systems. Data are in agreement with
researchers that have tested RME on similar engines, but different behaviors can be found in
the literature when biodiesels from different feedstock are used in similar engines or when
the RME is used in different engine types. In fact, in the premixed combustion phase, an
increased HRR with respect to diesel fuel was also found for RME by Tsolakis A. et al. [54];
similarly, Purushothaman K. and Nagarajan G. [55] also evidenced higher HRRs for the
orange oil, and Pradeep V. and Sharma R.P. [56] for the Jatropha oil-based biodiesel. On the contrary, a lower NHRR during the premixed combustion period was found
by Sahoo and Das [57] for Jatropha-, Karanja-, and Polanga-based biodiesels, while Sinha
and Agarwal found it for a rice bran oil-based biodiesel [58] and by Nwafor and Rice [59]
found it for straight rapeseed oil. Qi et al. [60] tested a diesel engine with a neat biodiesel
produced from straight soybean oil and found that, at lower engine loads, the net heat
release for diesel was slightly lower than for biodiesel, but at higher engine loads, the
HRR for diesel fuel was higher. Apart from the biodiesel typology and origin, the engine
type may also influence the fuel injection and combustion characteristics of biodiesel. 3.4. Comments on the Results and Comparison with Literature Studies For
example, Breda [61] tested RME on a bus diesel engine with an M injection system and
found a lower maximum HRR and in-cylinder gas pressure when compared to diesel fuel. y
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Table 4 summarizes the main results available in the literature, reported here for
proper comparisons with the results obtained in this study. All the data refer to engines
adopting a PNL injection system at their own rated full load, unless differently indicated. Table 4. Main characteristics, emissions, and performance of biodiesel-fueled engines in the prese haracteristics, emissions, and performance of biodiesel-fueled engines in the presented literature. Table 4. Main characteristics, emissions, and performance of biodiesel-fueled engines in the presented literature. Author and
Reference Nr. Type of Biodiesel
NOx
Concentration
Peak NHRR
Fuel Injection
System
BSFC
Tsolakis [54]
B100—rapeseed oil
+50%
+20%
PLN unmodified
+15.0%
Purushothaman [55]
B100—orange oil
+12.5%
+33%
PLN unmodified
+10.5%
Pradeep [56]
B100—Jatropha
+8%
+2%
PLN optimized
for B100
+12.3%
Sahoo [57]
B100—Jatropha
N/A
−23%
PLN unmodified
N/A
B100—Karanja
N/A
−22%
N/A
B100—Polanga
N/A
−25%
N/A
Nwafor [59]
B100—neat
rapeseed oil
~ +5%
PLN unmodified—
indirect
injection
+7%
Qi [60]
B100—soybean oil
−1% (full load)
~ +2% (min load)
~ −5% (max load)
PLN unmodified
+10% (full load)
Breda [61]
B100—rapeseed oil
+12.5%
−13.3%
PLN
unmodified—M
system
+16% 4. Conclusions In this study, a comparative analysis on the use of RME and diesel fuel mixtures on
a single-cylinder compression ignition diesel engine has been carried out. The engine
presents an in-line mechanical fuel pumping system. This typology of fuel injection
technology is still widely adopted in small scale engines and in agricultural machine
engines, and, thanks to its intrinsic simplicity, is also useful to better evidence the effects
of biodiesel use. In particular, the study focuses on the effect of an alternative fueling on
the injection timing, the in-chamber pressure, the heat release rate, and the NOx emissions. The results of the present article, and the ones available in the literature, regarding the
fuel pressure trace in the injection system allow for the conclusion that there is no general Energies 2021, 14, 7535 22 of 25 22 of 25 agreement on the effects of biodiesel on the injection pressure even in the same types of
injection systems. This may be due to a noncorrect pressure referencing of the injection
pressure traces. In the PLN injection systems, the pressure increase with biodiesel is indeed
steeper than with diesel fuel, but the biodiesel pressure just before the injection (i.e., the
residual pressure) is lower and, thus, its maximum injection pressure is only slightly greater
to the level recorded with the diesel fuel. Unfortunately, even after carefully consulting
the literature, it is not always possible to verify which kind of injection pump was used
because most of the authors did not record such details. The fuel injection also affects the combustion heat release and the emissions. No
significant variations in the engine torque are found with the variation of the fueling. The
injection advance with the RME also anticipates the start of the combustion, and a higher
heat release and peak in-cylinder pressure are found on the whole rotational speed range
of the engine. As concerns the emissions, the higher nitrogen oxides are often found when
adopting biodiesels. In the literature, there are several interpretations of the biodiesel
NOx effect, and the causes of the increase of this pollutant are mainly attributed to the
higher content of oxygen in the fuel, the higher combustion pressure and temperature (due
to an advance in the SOC), and the higher heat release rate. 4. Conclusions This study also proposes a
modification in the fuel injection advance so to counterbalance the NOx effect without
affecting the other performance figures of the engine. g
p
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Nevertheless, though research on the characterization of biodiesel performance in
diesel engines started a long time ago, some issues have not yet been successfully addressed,
and further research is still needed to better focus on them. In this respect, this study has
tried to supply some considerations and remarks for a specific category of biodiesel-fueled
engines with a mechanical injection system that can, however, be extended also to modern
engines adopting common rail systems. Author Contributions: The authors contributed equally to each part of this work. All authors have
read and agreed to the published version of the manuscript. Funding: This research was developed within the “Bio-Tract-Efficiency” (“Experimental investigation
on the efficiency of agricultural machines powered with different fuels”) internal research project,
grant number TN200F, and the “XENERGY” (“Collaboration project with UNIVPM in the field of
fluid machines and energy systems”) research frame agreement, involving the Libera Università di
Bolzano and the Università Politecnica delle Marche. Institutional Review Board Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The authors would like to thank Giorgio Tombari for his help during the
experimental phase of this study. Acknowledgments: The authors would like to thank Giorgio Tombari for his help during the
experimental phase of this study. Acknowledgments: The authors would like to thank Giorgio Tombari for his help during the
experimental phase of this study. Nomenclature
B100
pure biodiesel
BMEP
brake mean effective pressures
BSFC
brake-specific fuel consumption
BTDC
before top dead center
BTE
brake thermal efficiency
CA
crank angle (degrees)
CI
compression-ignited (engine)
CO
carbon monoxide
CO2
carbon dioxide
EASAC
European Academies’ Science Advisory Council 23 of 25 Energies 2021, 14, 7535 ECU
electronic control unit
EOI
end of injection
EU
European Union
GHG
greenhouse gases
HC
hydrocarbon
HRR
heat release rate
LHVm
lower heating value on a mass basis
ME
methyl esters
MSW
municipal solid waste
NHRR
net heat release rate
NOx
nitrogen oxides
NREL
National Renewable Energy Laboratory
NY
State of New York
OHV
on-head valve
PLN
pump-line-nozzle
PM
particulate matter
RME
rapeseed oil methyl ester
SOC
start of combustion
SOI
start of ignition
USA
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46. Sharma, A.; Murugan, S. Combustion, performance and emission characteristics of a DI diesel engine fuelled with non-petroleum
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Development of a methodology for evaluating the efficiency level of monitoring agroecosystems using big data technologies
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VVRD 2021
BIO Web of Conferences 43, 0
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3012 VVRD 2021
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3012 https://doi.org/10.1051/bioconf/20224303012 Development of a methodology for evaluating
the
efficiency
level
of
monitoring
agroecosystems using big data technologies
Aleksey Petrov1,*, Leonid Andreev2 and Sergey Grigorishin3
1Norilsk State Industrial Institute, 663310, 50 years of October str. 7, Norilsk, Russian Federation
2Northern Trans-Ural State Agricultural Universtity, 625003, 7, Republic st. Tyumen, Russian
Federation
3Tyumen State University, 625003, 6, Volodarskogo st., Tyumen, Russian Federation 3Tyumen State University, 625003, 6, Volodarskogo st., Tyumen, Russian Federation Abstract. The article discusses the theory of monitoring agroecosystems
for the effectiveness of using Big Data technologies. The relationship
between the agricultural areas of the Tyumen region, the Big Data sources
available in them, and the technologies for working with Big Data obtained
from sources are described. The article also developed a methodology that
makes it possible to assess the level of effectiveness of monitoring
agroecosystems using Big Data technologies, based on the result of which
a strategy for development of the region as a whole and its
agroecosystems, in particular, is formed in terms of equipment with
information technologies. The methodology presented in the article is
formed on the basis of an engineering ontology, which in the future is able
to lower the degree of the human factor in global and local monitoring of
agroecosystems for the effectiveness of using Big Data technologies. 1 Introduction The monitoring and assessment of natural ecosystems primarily implies development
and operation of such observation systems, on the basis of which it is possible to assess
their condition. The assessment, in turn, is primarily the result of an analysis of the
quantitative characteristics of ecosystems, which are dynamic, since they have the property
of variability, relative to the impact of external factors (the name of the factors depends on
the type of ecosystems). Agricultural ecosystems are commonly referred to as
agroecosystems. Such monitoring of agroecosystems [1], on the one hand, is of great economic
importance for the region [2], since, based on its result, the agroecosystem manager can
develop a strategy and take measures (crop conservation, pest control, and etc.), on the
other hand, it is of great ecological importance, as agroecosystems are part of larger natural
ecosystems. © 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/). * Corresponding author: darker2012@yandex.ru 2 Methods In the existing realities, monitoring of agroecosystems is not possible without the use of
technologies for processing large amounts of data [3], since the agroecosystem is, first of
all, dynamic, which means that the manager of the agroecosystem is interested in obtaining
information about its state as quickly as possible. This, in turn, means that it is possible to develop a methodology for assessing the
readiness of an agroecosystem to analyze the data obtained about its state using Big Data
technologies, or in other words, a methodology for assessing the level of effectiveness of
monitoring agroecosystems using Big Data technologies. This implies that if ten sensors of
soil acidity are located on some hypothetical agricultural field (agroecosystem), then using
Big Data technologies will be ineffective, since the data obtained can be processed in
simple Microsoft Excel and there is no point in creating software for analyzing such data
based on Python or another programming language. This means that there is a certain minimum required amount of data that must come
from the agricultural field (agroeosystem) for use of Big Data technologies to be expedient. Moreover, even if the required amount of data is available, it is necessary to determine
which of the many currently developed Big Data technologies for agriculture [4] are
suitable for analysis. y
In the article [5], the authors presented a table responsible for the distribution of
technologies for analyzing big data, depending on the field of agriculture. For clarity, we
present Table 1. Table 1. Distribution of Big Data technologies, depending on the field of agriculture. Table 1. Distribution of Big Data technologies, depending on the field of agriculture. Agriculture area
Sources of Big Data
Big Data technologies
Weather and climate
data
Geospatial data;
Meteorological stations;
Freely available historical
information
Other data obtained remotely. Statistical analysis;
Machine notation (K-means
clustering algorithm,
random/deep tree construction
algorithm);
GIS analysis;
Distributed computing model. Livestock breeding
Ground sensors;
Thermal data;
Optical sensors;
Incoming feed sensors;
Data on meat and dairy products. Neural networks;
Scalable vector machines;
Decision trees. Crops
Freely available historical
information
Satellite data;
Ground sensors. K-means clustering algorithm;
Support Vector Machine;
Fourier transform;
Wavelet analysis;
Land resources;
Geospatial data;
Freely available historical
information
Other data obtained remotely;
Aerial survey data;
K-means clustering algorithm;
algorithm for constructing
random/deep trees;
Image processing;
Vegetation index ndvi. * Corresponding author: darker2012@yandex.ru © 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/). VVRD 2021
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3012 https://doi.org/10.1051/bioconf/20224303012 2 Methods Weeds
Freely available historical
information
Data received from drones;
Aerial survey data;
Sensor placed in the fields;
Digital Web Libraries. Neural networks;
Logistic regression;
Image processing;
Soil condition;
Freely available historical
K-means clustering algorithm; 2 2 VVRD 2021
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3012 https://doi.org/10.1051/bioconf/20224303012 information
Ground sensors;
Information posted in the database of
government agencies;
Humidity sensors;
Optical sensors;
Neural networks. Biological resistance
Geospatial data;
Freely available historical
information
Information posted in the database of
government agencies;
Statistical modeling;
Bayesian functions;
Food safety;
Geospatial data;
Freely available historical
information
Other data obtained remotely;
Survey data;
Growth depth sensors;
Neural networks;
Geospatial modeling;
Statistical modeling;
Image processing;
Farms;
Freely available historical
information
Optical sensors;
Information posted in the database of
government agencies;
Meteorological stations;
Social networks;
Big Data Benchmarking;
Web services;
Mobile applications;
Remote sensing;
Geospatial data;
Satellite data;
Geospatial data;
Meteorological stations;
Data received from drones;
Digital Web Libraries. Cloud computing with a
distributed computing model;
Geospatial modeling;
Computer vision;
Artificial intelligence;
Insurance and finance;
Optical sensors;
Information posted in the database of
government agencies;
Digital web libraries;
Information from private banks;
Survey data;
Statistical modeling;
Predictive analytics;
Cloud technologies. As you can see, for each area of agriculture, a source of information was selected for
analysis using Big data technology and directly, the methods of analysis themselves. Of
course, the areas of agriculture in Ireland, in any case, their categorization is different from
the categorization of similar areas of the Tyumen region. However, the "general" idea can
still be traced and "transferred" it to the realities of the Tyumen region. It would be
advisable to check the adequacy of the selected methods of analysis based on the applied
problem. 3 Results Currently, in the Tyumen region, there is an acute issue of Big Data study in agriculture. Therefore, using the column "Sources of Big Data", one can assess the level of
preparedness of a particular area of agriculture in the region for such studies. Also, based
on the direct availability of "Sources of Big Data", it is possible to build an ontological
graph of interaction with Big Data technologies, which in turn will lead to a methodology
for assessing agricultural areas on the effectiveness of using Big Data technologies and 3 3 VVRD 2021
BIO Web of Conferences 43, 0
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3012 https://doi.org/10.1051/bioconf/20224303012 ommendations in which particular area of agriculture which sources to develop. For this,
present Table 2. recommendations in which particular area of agriculture which sources to develop. For this,
we present Table 2. present Table 2. Table 2. Availability of Big Data sources regarding agriculture in the Tyumen region. 3 Results Agriculture area
Sources of Big Data
Source availability
Weather and climate
data
Geospatial data
Absent
Meteorological stations
Present
Freely available historical information
Present
Other data obtained remotely
Absent
Livestock breeding
Ground sensors
Present
Thermal data
Absent
Optical sensors
Absent
Incoming feed sensors
Present
Data on meat and dairy products
Present
Crops
Freely available historical information
Present
Satellite data
Present
Ground sensors
Present
Land resources
Geospatial data
Absent
Freely available historical information
Present
Other data obtained remotely
Present
Aerial survey data
Absent
Weeds
Freely available historical information
Absent
Data received from drones
Absent
Aerial survey data;
Absent
Sensor placed in the fields
Absent
Digital Web Libraries
Absent
Soil condition
Freely available historical information;
Present
Ground sensors;
Present
Information posted in the database of
government agencies;
Present
Humidity sensors;
Present
Optical sensors;
Absent
Biological resistance
Geospatial data;
Present
Freely available historical information
Absent
Information posted in the database of
government agencies;
Present
Food safety;
Geospatial data;
Absent
Freely available historical information
Absent
Other data obtained remotely
Present
Survey data
Absent
Growth depth sensors
Absent
Farms
Freely available historical information
Present
Optical sensors
Absent
Information posted in the database of
government agencies
Present
Meteorological stations
Present
Social networks
Present
Remote sensing
Geospatial data
Absent
Satellite data
Absent
Geospatial data
Absent 4 VVRD 2021
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3012 https://doi.org/10.1051/bioconf/20224303012 Meteorological stations
Absent
Data received from drones
Absent
Digital Web Libraries
Absent
Insurance and finance
Optical sensors
Absent
Information posted in the database of
government agencies;
Present
Digital web libraries
Absent
Information from private banks
Absent
Survey data
Absent By adding the column "presence of a source", you can quantitatively visualize the filling
of areas and display in Table 3. the Tyumen region. Agriculture area
Source availability
Weather and climate data
2/4
Livestock breeding
2/5
Crops
3/3
Land resources
2/4
Weeds
0/5
Soil condition
4/5
Biological resistance
2/3
Food safety
1/5
Farms
4/5
Remote sensing
0/5
Insurance and finance
1/5 So it can be seen that the most prepared area of agriculture is Crops, Soil Condition, and
Farms. Based on them, it will be possible to make initial ontological graphs for creating a
methodology based on engineering ontology. 4 Discussion nsider the process of creating an ontological graph based on the "Crops" area. Consider the process of creating an ontological graph based on the "Crops" area. Consider the process of creating an ontological graph based on the Crops area. Table 4. Analysis of the "Crops" area. Crops
Freely available historical
information
K-means clustering algorithm
Support Vector Machine
Fourier transform
Wavelet analysis
Satellite data
Ground sensors To begin with, you need to understand to which subdomain "Crops" which Big Data
technology to implement most effectively. To begin with, you need to understand to which subdomain "Crops" which Big Data
technology to implement most effectively. The K-means clustering algorithm technology consists in dividing a certain volume of
observations (points) into an arbitrary number of clusters, limited by a certain standard [6]. The key feature is the presence of a centroid for each cluster and the separation of points in 5 VVRD 2021
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3012 https://doi.org/10.1051/bioconf/20224303012 accordance with similarity. In other words, distribution into clusters occurs, depending on
the location of the central point (centroid), which is obtained by averaging the positions of
all points, or vice versa. After distribution into clusters, through the calculation of the
distance, two or more samples are compared with each other. accordance with similarity. In other words, distribution into clusters occurs, depending on
the location of the central point (centroid), which is obtained by averaging the positions of
all points, or vice versa. After distribution into clusters, through the calculation of the
distance, two or more samples are compared with each other. The use of this technology is correct if there are a number of points associated with a
geographic location. That is, from the available "Sources of Big Data" only ground sensors
in tandem with satellite or other methods of obtaining spatial information about ground
sensors can be used. A source such as “Freely available historical information” is too broad
concept; therefore, its rating of use for the analysis of “Crops” by Big Data technologies
will be low. Support vector machine technology [7] consists in learning by precedents that take into
account the cases of linear separability, optima and gaps between classes. It is noteworthy
that analytics using this technology most often occurs in tasks related to linear regression,
which in parallel includes the search for minimization. 4 Discussion The correct application of this technology is possible if there is a time axis, in other
words, to trace the change in information over time. Among the offered "Sources of Big
Data", this can be "Freely available historical information", but on condition that it can be
expressed in quantitative metrics. The technology of the Fourier transform [8] consists in the comparison of one function
into a complex variable, which describes the coefficients in the expansion of the original
function into elementary components. In the area we are considering, such a transformation
is used to process signals and represent them in the form of time series, with the possibility
of displaying them in the form of a frequency spectrum. Correct application is possible in presence of constantly incoming signals, in other
words, in the presence of ground sensors. Wavelet analysis technology [9] consists in the analysis of different types of frequency
components of the data, most often along the "scale - time - level" chain. Most often they
are used to obtain more accurate data after the Fourier transform, or to refine this
transformation, since they give a more accurate binding of the quantitative features of the
parameter, relative to time. Therefore, we can conclude that the wavelet analysis is only an
auxiliary analysis and is used in special cases together with the Fourier transform. Based on the above, an ontological graph of the "Crops" area can be built. For
construction, we use the Protégé environment [10], the resulting graph is shown in Figure 1. Fig. 1.. Ontological graph of the "Crops" area. As you can see in the presented column, "Big Data Sources" were represented in the
form of "Entities" of the Protégé environment, and "Big Data technologies" in the form of
"Individuals", such a solution gives us, first of all, the opportunity to trace "paired" sources
for big data analysis. From an applied perspective, this means the presence of the same
analysis technologies, the same software for data from different sources, but in the same Fig. 1.. Ontological graph of the "Crops" area. Fig. 1.. Ontological graph of the "Crops" area. Fig. 1.. Ontological graph of the "Crops" area. 4 Discussion As you can see in the presented column, "Big Data Sources" were represented in the
form of "Entities" of the Protégé environment, and "Big Data technologies" in the form of
"Individuals", such a solution gives us, first of all, the opportunity to trace "paired" sources
for big data analysis. From an applied perspective, this means the presence of the same
analysis technologies, the same software for data from different sources, but in the same As you can see in the presented column, "Big Data Sources" were represented in the
form of "Entities" of the Protégé environment, and "Big Data technologies" in the form of
"Individuals", such a solution gives us, first of all, the opportunity to trace "paired" sources
for big data analysis. From an applied perspective, this means the presence of the same
analysis technologies, the same software for data from different sources, but in the same 6 VVRD 2021
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3012 https://doi.org/10.1051/bioconf/20224303012 agroecosystem. Thus, it will be easier for big data analysts to navigate when finding
dependencies, obtaining additional information, making forecasts, and more. agroecosystem. Thus, it will be easier for big data analysts to navigate when finding
dependencies, obtaining additional information, making forecasts, and more. In addition, in the ontological graph, you can see that such a source as "Freely available
historical information" is analyzed by only one technique and does not intersect with other
"Sources of Big Data". This, in turn, suggests that with development of Big Data in the
region, there are two ways to go: 1. Stop this kind of source, without the need for its further development and analysis of
big data. g
2. Present data in an accessible form for the analysis of other Big Data technologies. As you can understand, such ontological graphs can be built for any area of agriculture
and matched for any agroecosystem. For clarity, let's expand the existing graph to include
areas such as Soil Condition, Farms, and Weather and Climate Data, thereby creating a
hypothetical agro-ecosystem that includes the listed areas. That is, the regions are chosen in
such a way that, in fact, they are responsible for the analysis of the meta-region and cover
the interests in Big Data of farms engaged in crop production. Thus, we have obtained a search ontological graph. 4 Discussion It is difficult to visualize it in full,
but Protégé has search functions, by searching for the keyword "data" we see what is shown
in Figure 2. Fig. 2. The result of displaying a search ontological graph by the keyword "data". Fig. 2. The result of displaying a search ontological graph by the keyword "data". On this search ontological graph, we see that all "Entities" related to the column
"Sources of Big Data" and one way or another associated with data, as well as the
"Individuals" and areas of agriculture attached to them, were displayed. In view of this, a
kind of "coupling" is obtained, where, on the one hand, we can observe the fullness of Big
Data technologies, and on the other hand, the field of agriculture. Such a visualized
dependence and work with the resulting ontology can give a specialist the following
information: 1. Understanding of the readiness of the agricultural sector for Big Data technologies. derstanding of the readiness of the agricultural sector for Big Data technologies. g
g
g
g
2. Understanding of the fullness of the agricultural field with Big Data sources. 3. What sources of Big Data in what area of agriculture needs to be brought in to
rease the efficiency of using Big Data technologies. 4. What Big Data technologies to use in what sources. 5. What "paired" Big Data sources can be used to capture the largest number of areas of
agriculture for analysis. 7 VVRD 2021
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3012 https://doi.org/10.1051/bioconf/20224303012 6. What Big Data technologies can be used, depending on the type of agricultural
enterprise. As a result, it turns out that the very methodology for assessing the level of
effectiveness of monitoring agroecosystems using Big Data technologies is, first of all, in
the correctness of work with the resulting ontology. In order to translate the methodology
directly into a quantitative metric, that is, to be able to "put down" each agroecosystem its
own "score" from one to ten points, it is necessary to use such Protege functionality as
"Data properties" and "Object properties". "Data properties" in Protégé are literal properties and can be attached to "Entities", so it
becomes possible to represent "Entities" as a list of property names and associated
quantitative metrics. 5 Conclusion As a summary of this article, a number of the following conclusions can be made: The method of monitoring agroecosystems to assess the level of efficiency of using
Big Data technologies allows visualizing the lagging and leading areas to form an effective
development strategy. The agroecosystem manager shall guide not only the selection of Big Data
technologies for analyzing information about the agroecosystem, but also focus on the
available data sources about it. The methodology can be improved through use of engineering ontologies and Protégé
software, which can internally evaluate technologies for agro-ecosystems and search for
hidden knowledge, excluding the human factor. To improve the methodology in the future, it is necessary to compare different
agroecosystems for the effectiveness of using Big Data technologies and analyze the
results. 4 Discussion As a result, each "Entities" can be represented by a strictly defined
numerical value, which can be changed only if additional information is added to the
ontology. "Object properties" in Protégé are a property pointer to "Entities". The pointer, in turn,
has two key properties, namely the address allocated for a specific "Entities" and the
connection with a specific type of "Entities". Having filled in "Data properties" and "Object properties" for each "Entities", we will
be able to quantify each area of agriculture, in other words, present them in the form of a
line histogram, at Figure 3. Fig. 3. Linear histogram for assessing the level of effectiveness of monitoring agroecosystems using
Big Data technologies. Fig. 3. Linear histogram for assessing the level of effectiveness of monitoring agroecosystems using
Big Data technologies. From this diagram, we can immediately observe the lagging areas of agriculture, namely
the areas "Remote sensing" and "Weeds". This is due to the fact that such regions are not
considered separately in the Tyumen region. However, it is still logical that in order to
increase the efficiency of using Big Data technologies, a quantitative increase is necessary,
first of all, “Sources of Big Data”. By itself, such a distribution was seen already at the
stage of adding the column "presence of a source" and was displayed in Table 3. However,
it is the addition of quantitative metrics for “Data properties” and “Object properties” to the
ontology that will allow us to adjust the source metrics for the agroecosystem and see a
more accurate picture of “the effectiveness of using Big Data technologies” at a more
detailed level for a specialist. Of course, when making the right decisions for the
development of Big Data for a particular agroecosystem, the cost of its equipment will be
reduced (adding various kinds of data sources), by understanding which sources to put and
accelerating decision-making for development of the region. 8 VVRD 2021
BIO Web of Conferences 43, 0
(2022)
3012 https://doi.org/10.1051/bioconf/20224303012 References 1. P. Srivastava, R. Singh, R. Bhadouria, Pal D. Bahadur, P. Singh, S. Tripathi, ENPS,
(2021) 2. S.Cao, G.Xie, L.Zhen, Ecological Economics, 69, 7 (2010) 2. S.Cao, G.Xie, L.Zhen, Ecological Economics, 69, 7 (2010) 3. V.P. Yakushev, V.V. Yakushev, V.L. Badenko, D.A. Matveenko, Y.V. Chesnokov,
Sel'skokhozyaistvennaya Biologiya, 55, 3 (2020) 4. L.N. Hudson, T. Newbold, S.Contu, S.L.L. Hill, I. Lysenko, A. De Palma, H.R.P. Phillips, T.I. Alhusseini, F.E. Bedford, D.J. Bennett, H. Booth, V.J. Burton, Ecology
and Evolution, 7, 1 (2017) 5. D. Bose, Big data analytics in Agriculture (2020) 5. D. Bose, Big data analytics in Agriculture (2020) 6. M. Ding, T. Wang, X. Wang, ACM Transactions on Knowledge Discovery from Data
16, 2 (2022) 7. A. Singh, S. Saha, M. Hasanuzzaman, A. Jangra, Expert Systems with Applications,
186 (2021) 8. M.M. Alam, M.M.R. Howlader, Sensing and Bio-Sensing Research, 34 (2021) 9. F. Xu, S. Tan, Expert Systems with Applications, 186 (2021) 10. A. Petrov, A. Popov, M. Chekardovsky, A. Pushkarev, CEUR Workshop Proceedings,
2843 (2021) 9 9
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